整合素αvβ3受体显像无创性评估大鼠肝纤维化的可行性研究
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摘要
整合素αvβ3受体显像评估大鼠肝纤维化的可行性研究
肝纤维化(liver fibrosis)是不同病因引起的肝脏慢性损伤后实质再生,同时伴随间质过度增生,潜在可逆的组织修复重建过程。肝纤维化评估对于慢性肝病的诊断和干预评价尤为重要,目前还没一种可靠的肝纤维化无创性评估方法。肝星状细胞(hepatic stellate cell,HSC)活化和表型改变是肝纤维化发生的中心事件,决定着肝纤维化转归。血管新生(angiogenesis)是组织损伤修复的必要条件。HSC活化、细胞外基质(extrocellural matrix,ECM)增多同时伴有新生血管形成是进展性肝纤维化修复重建中突出的病理特征。
整合素(integrin)是黏附分子家族中,由一个α亚基和一个β亚基非共价结合形成的跨细胞膜受体,介导细胞和ECM、细胞与细胞间的黏附和整合细胞内外信息传递。生理情况下整合素为细胞非组成性表达,在整合素家族中各亚型的表达有时序性和分布特异性。整合素αvβ3亚型是最广泛的ECM受体,主要介导间质细胞与纤维连接蛋白、纤维蛋白原、Ⅰ型胶原、玻璃黏结蛋白、层黏蛋白、Ⅷ因子相关抗原(vWF)等ECM的黏附,并和血小板衍生的生长因子(PDGF)、转化生长因子β1(TGFβ-1)、内皮细胞生长因子(VEGF)等细胞因子有信号协同作用,主要传递和细胞增殖、分化、运动、分布、定居、生存或凋亡等有关的细胞信号。正常肝组织仅门静脉周围表达少量整合素αvβ3,肝细胞、库普弗细胞、静止HSC和正常血管内皮细胞不表达该亚型。实验研究了整合素βvβ3在活化HSC和肝纤维化组织中的表达,探讨整合素αvβ3表达、血管新生与肝纤维化间质重塑的关系。
精氨酸-甘氨酸-天门冬氨酸(Arg-Gly-Asp,RGD)三肽模体是ECM中保守序列,也是ECM和整合素受体识别常见位点。整合素αvβ3能识别ECM中特异构象的RGD配体。人工合成含RGD肽链高通量筛选发现,精氨酸-甘氨酸-天门冬氨酸-(右旋)酪氨酸-赖氨酸(RGDyK)、精氨酸-甘氨酸-天门冬氨酸-(右旋)苯丙氨酸-赖氨酸(或RGDfK)等五肽分子也能和整合素αvβ3受体特异结合,对五肽中赖氨酸上活性氨基进行小分子修饰并不影响受体与配基结合活性。实验中通过固相合成整合素αvβ3受体的RGD环肽(cRGD)配基,设计用羧基荧光素(FAM)和锝99同位素(~(99m)TC)等示踪分子标记cRGD作为探针,探讨人工合成的cRGD配基对活化HSC和肝纤维化组织的靶向性结合是课题研究的主要工作。
建立在肝纤维化组织间质重建的分子病理基础上,以~(99m)TC标记的cRGD为示踪分子,通过单光子发射型计算机断层成像(SPECT)探测肝纤维化不同时期肝脏放射性活度的相对变化(肝脏/心脏计数比值),并和常规B型超声、磁共振仪(MRI)比较早期肝纤维化的影像表现。尝试应用分子影像方法,根据肝脏中整合素αvβ3受体结合cRGD配基的变化信息,来反映慢性损伤肝组织重建过程中活化细胞水平及与肝纤维化程度的关系,为建立一种敏感、特异、可靠的肝纤维化无创性受体显像评估方法的可行性提供实验依据。
课题研究分为以下六部分;
第一部分HSC的分离、培养、鉴定和体外活化模型的建立
[目的]建立一种稳定可靠的HSC分离方法,为体外研究提供细胞模型。
[方法]Hank's液在体灌洗大鼠肝脏,离体后用Ⅳ胶原酶、链蛋白酶、DNaseⅠ消化肝脏,11%Nycodenz连续梯度液分离出大鼠HSC,计数细胞得率,0.2%台盼蓝染色计算细胞活率。自发荧光、透射电镜、苏丹Ⅲ染色观察细胞脂滴,Desmin、α-SMA免疫化学和荧光染色对HSC进行鉴定和纯度分析。分离后HSC按1-4×10~6密度接种于未包被塑料培养瓶或皿,含10%胎儿牛血清DMEM培养基,37℃含5%CO_295%空气培养箱中培养,通过延长培养诱导HSC活化。
[结果]分离HSC得率3.5±0.8×10~7/只大鼠,细胞活率＞90%,分离第1天自发荧光和第3天desmin染色阳性细胞＞90%。HSC随着体外培养形态明显改变,分离培养7天后α-SMA阳性细胞＞90%,培养14天或传代后＞95%阳性。
[结论]肝脏离体后消化能达到在体消化同样的效果,应用Nycodenz密度分离介质可获得满意HSC得率和纯度。新分离大鼠HSC培养7天后超过90%激活表型转变为活化HSC。
第二部分整合素αvβ3受体在HSC活化过程中的表达
[目的]探讨HSC表型转变过程中整合素αvβ3受体和α-SMA的定性、定位、定量表达。
[方法]HSC培养的第1、3、5、7和14天分别提取细胞总RNA和总蛋白。总RNA逆转录合成cDNA,分别通过real-time PCR和Western Blot分析整合素αv、β3和α-SMA mRNA和蛋白质在HSC活化过程中的表达。静止期细胞和激活细胞期HSC,分别进行α-SMA和整合素αVβ3细胞免疫荧光染色激光共聚焦观察。
[结果]HSC活化过程中整合素αVβ3表达水平变化有显著性差异(P＜0.01),第14天时在mRNA和蛋白质水平比第1天,分别增加18倍和5.2倍。而比较7天和14天时表达则无明显差异。免疫荧光双染色显示活化HSC中整合素αVβ3极性表达于细胞膜,α-SMA表达于细胞浆并且和整合素αVβ3有共定位。
[结论]整合素αVβ3是激活状态HSC的表型受体,表达上调与HSC活化和活化状态维持有关。
第三部分整合素αvβ3 RGD环肽配基与HSC的结合分析
[目的]研究HSC对羧基荧光素(FAM)标记精氨酸-甘氨酸-天门冬氨酸-(右旋)苯丙氨酸-赖氨酸(FAM-cRGD)环五肽的结合和内化。计算活化HSC对~(125)Ⅰ标记精氨酸-甘氨酸-天门冬氨酸-(右旋)酪氨酸-赖氨酸的亲和力和受体容量。
[方法]采用C末端保护固相法合成精氨酸-甘氨酸-天门冬氨酸-(右旋)苯丙氨酸-赖氨酸环五肽,羧基荧光素合成树脂上和环五肽中赖氨酸上的氨基固相结合为标记的荧光分子探针(FAM-cRGD)。高效液相色谱法(HPLC)和质谱法(MS)检测FAM-cRGD的纯度和分子量。荧光示踪观察HSC对FAM-cRGD探针的摄取和内化。流式细胞仪(FCM)分析HSC孵育FAM-cRGD后细胞平均荧光强度的变化和浓度、时间效应。受体放射性配基结合分析(RBA)测定HSC对~(125)Ⅰ标记精氨酸-甘氨酸-天门冬氨酸-(右旋)酪氨酸-赖氨酸的平衡解离常数(Kd)和最大结合点数(Bmax)。
[结果]荧光示踪显示静止HSC不结合FAM-cRGD探针,活化HSC结合FAM-cRGD并摄入细胞浆,但无细胞核的内化。流式细胞仪分析提示,静止期HSC和10μMFAM-cRGD 37℃孵育45分钟,平均荧光强度和本底比较无明显变化。而活化HSC和10μM FAM-cRGD 37℃孵育45分钟,平均荧光强度较本底增高6.6倍,活化HSC和10μM FAM-cRGD、150μM cRGD 37℃共同孵育45分钟,平均荧光强度抑制超过30%,活化HSC平均荧光强度改变有随FAM-cRGD孵育浓度和孵育时间延长而增加的趋势。受体放射性配基结合分析,通过Scatchard作图法求出活化HSC对cRGD结合的Kd为4.808×10~(-9)mol/L和Bmax为2.112×10~(-10)mol/L。
[结论]活化HSC能与整合素αVβ3受体的cRGD发生受体-配基样结合,活化HSC对cRGD配基有较高的亲和力和受体容量。
第四部分肝纤维化中的整合素αvβ3表达与血管新生
[目的]建立两种大鼠肝肝纤维化模型,探讨肝纤维化组织中整合素αVβ3表达及与血管新生的关系。
[方法]硫代乙酰胺(TAA)腹腔注射和胆总管结扎(BDL)诱导建立两种大鼠肝纤维化模型。建模的第3周末、9周末分别进行肝组织天狼星红染色和计算机图像分析、整合素αVβ3和血小板内皮细胞黏附分子(CD31)免疫化学染色、整合素αVβ3和与α-SMA免疫荧光双重染色及激光共聚焦观察和real-time PCR和Western blot分析肝组织中整合素αVβ3、CD31在mRNA和蛋白表达水平表达的相对定量。
[结果]肝纤维化程度随建模时间延长而加重。两种肝纤维化大鼠和对照大鼠比较,肝组织中整合素αVβ3和CD31在mRNA和蛋白表达水平有显著性差异(TAA组F=28.66,P＜0.01和F=19.62 P＜0.01,BDL组F=32.60 P＜0.01和F=42.36P＜0.01),随肝纤维化进展而有升高的趋势。免疫组织化学和免疫荧光化学染色提示,在肝纤维化扩大的汇管区及纤维隔中有新生血管形成,在间质中整合素αVβ3与α-SMA表达部位一致,从汇管区向肝实质深入。
[结论]肝纤维化进展中整合素αVβ3表达水平增加与HSC活化和血管新生有关,并与间质重建程度平行。
第五部分肝组织整合素αVβ3放射自显影和~(125)Ⅰ-cRGD在体内的分布
[目的]分析肝纤维化组织对~(125)Ⅰ-cRGD配基的结合,和~(125)Ⅰ-cRGD在早期肝纤维大鼠中的分布。
[方法]硫代乙酰胺(TAA)腹腔注射诱导大鼠肝纤维化,在第3周末、9周末分别进行肝组织冰冻切片,切片分别和~(125)Ⅰ-cRGD、~(125)Ⅰ-cRGD加500倍化学剂量cRGD(特异阻断)进行4℃孵育2小时,冲洗切片后接触法暴光2周,进行受体组织放射自显影。6只正常大鼠和6只TAA3周肝纤维化大鼠,各3只分别进行尾静脉注射6μCi ~(125)Ⅰ-cRGD,各3只注射500倍化学剂量cRGD特异阻断后再注射6μCi ~(125)Ⅰ-cRGD、在注射后45min分别取材肝、脾、脑、肾、心、肺和肌肉,测定各脏器的放射性活度,计算各脏器放射性比活度(%ID/g)。
[结果]正常大鼠、TAA3周、TAA9周大鼠切片暴光后的相对灰度分别为8.1±1.6、18.7±2.6和28.7±3.4,用cRGD阻断的切片相对灰度分别为7.7±1.7、12.1±2.5和19.3±3.3,三组间比较差异有显著性(P＜0.01)。在TAA3周和TAA9周中,未用cRGD阻断和cRGD阻断比较差异有显著性P=0.02和P=0.041,阻断的切片中相对灰度降低。正常大鼠和TAA3周的早期肝纤维化大鼠中,~(125)Ⅰ-cRGD体内分布以肾脏组织中最高,其次是肝脏,脑组织最低。正常大鼠中肝脏~(125)Ⅰ-cRGD的分布%ID/g,在未阻断和阻断大鼠分别为0.11±0.03、0.10±0.04,在TAA3周大鼠分别为0.17±0.02和0.12±0.03,4组比较F=5.175,P=0.049差异有显著性,TAA3周大鼠肝脏中的%ID/g较高,在用cRGD阻断时肝脏中放射性比活度有下降的趋势。而比较各组间肾、脾、心、肺、脑、肌肉等脏器放射性比活度则差异无显著性。
[结论]体内外的研究提示肝纤维化组织对~(125)Ⅰ-cRGD配基有较高的结合和摄取,并能被未标记的cRGD所竞争。
第六部分肝脏整合素αvβ3受体显像评估肝纤维化
[目的]以~(99m)Tc-DTPA-cRGD为示踪剂应用SPECT对肝纤维化大鼠肝脏中整合素αvβ3受体进行分子显像,测定正常和肝纤维化大鼠间肝/心的放射性计数的比值。
[方法]硫代乙酰胺(TAA)腹腔诱导大鼠肝纤维化。合成树脂上二亚乙基三基三胺五乙酸(DTPA)固相结合cRGD(DTPA-cRGD),高锝酸盐(~(99m)Tc04-)氯化亚锡还原法标记DTPA-cRGD(~(99m)Tc-DTPA-cRGD),纸层析法测定放射化学纯度。TAA3周早期肝纤维化大鼠进行肝脏B型超声和MRI扫描观察影像表现。在TAA3周和TAA9周,尾静脉注射~(99m)Tc-DTPA-cRGD 6mCi/只,用小动物SPECT进行大鼠腹部扫描,计算注药后45min,感兴趣区(ROI)的肝/心放射性计数比。
[结果]测定~(99m)Tc-DTPA-cRGD的放射化学纯度＞95%,室温下4小时保持稳定。注射~(99m)Tc-DTPA-cRGD后45min,正常组、TAA3周和TAA9周组中感兴趣区的肝/心放射性计数分别为1.73±0.35、2.7±0.44、3.73±0.65差异有显著性(F=13.1,P=0.006)。比较正常大鼠和TAA3周大鼠,TAA3周和TAA9周大鼠组间感兴趣区的肝/心放射性活度比值差异有显著性(P＜0.05)。常规MRI和B型超声检测TAA3周的早期肝纤维化大鼠和正常大鼠在影像上无区别,SPECT扫描提示TAA3周大鼠肝脏对~(99m)Tc-DTPA-cRGD的积聚增加。
[结论]肝纤维化大鼠~(99m)Tc-DTPA-cRGD SPECT显像肝/心放射性计数比值较正常大鼠明显增加与肝组织中整合素αvβ3受体表达上调有关。SPECT和常规影像比较能提示早期肝纤维化中对~(99m)Tc-DTPA-cRGD摄取增强后的影像改变。
Liver fibrosis is a reversible response of wound healing and architecture remodeling after liver cells were attacked by all kinds of insults.Assessment of hepatic fibrosis plays a key role for diagnosis and evaluation in chronic liver injury.At present liver biopsy is widely accepted as a gold standard to document liver fibrosis.However,liver biopsy procedure is limited as an invasive procedure,sampling error, potential morbidity and mortality.Hence,it is vitally important to develop a reliable non-invasive measure to dynamically detect the alteration of hepatic repair in chronic liver disease.The activation of hepatic stellate cell(HSC) is recognized as one of the critical hallmark in liver fibrosis and to decide the fate of injured liver. Recently,increasingly proofs support angiogenesis is precondition of liver tissue repairing in diverse etiologies.The accumulation of extracellular matrix(ECM) and angiogenesis is two significant pathological characteristics of advance liver fibrosis.
Integrin receptor is a cell surface receptor consisting of oneα-subunit and oneβ-subunit,and at least 18 differentαsubunits and 8 differentβsubunits of integrin are currently known.Therefore, various assemblages of subunits result in more than 24 functional subtypes of integrin dimers which differ in distribution,binding ligands and signalling properties.Among them,theαVβ3 subtype is also referred to as the vitronectin receptor,which is an extensive extracellular matrix(ECM) receptor and acts as an auxiliary receptor of connective tissue growth factor(CTGF) and platelet-derived growth factor(PDGF) that mediates cell events involved in proliferation, migration,activation and survival.These actions facilitate wound healing and the remodeling response in injured tissue.Normally,the expression ofαVβ3 integrin is restricted in special cell types and only low levels are produced in adult tissue.It has been shown that expression ofαVβ3 is weakly around the portal vein in a normal liver, with the resting endothelial cells,hepatocytes,Kupffer cells and quiescent HSCs expressing even lessαVβ3 integrin.
Integrin-ligand binding is well-characterized.ECM proteins can bind many integrins via the conservative motif of the arginine-glycine-aspartic acid(RGD) tripeptide,and the flank conformation of RGD determines the selectivity in different integrin subtypes.For example,vitronectin,denatured collagen,laminin, fibrinogen,osteopontin,von Willebrand factor and fibronectin interact withαVβ3 integrin through their special conformation of the RGD sequence.Based on these findings,various RGD-contained peptides have been successfully produced for experimental studies.
The aim of this study was to display the expression ofαVβ3 integrin in vitro and vivo,and to explore the relationship of angiogenesis and architecture remodeling in fibrotic liver.Furthermore,intend to investigate whether ~(99m)Tc labeled artificial Arg-Gly-Asp(RGD) containing cyclic peptides ligand can be applied to assess hepatic fibrosis by receptor imaging using SPECT.This assessing procedure is more effective and sensitive which reflect the molecular pathology mechanism of liver fibrosis.This study will provide a new perspective to explore the feasibility of developing a novel receptor-targeting strategy for non-invasive detecting liver fibrosis.
Part one Isolation and culture of rat primary hepatic stellate cell and establish of activated HSC model in vitro
Objective To build an efficient procedure for isolating hepatic stellate cell and establish activated HSC model.
Methods Primary hepatic stellate cells(HSC) were isolated from normal Sprague-Dawley(SD) rats by infusion and combined digestion of pronase E and collagenaseⅣex situ.Hepatic stellate cells were purified by density centrifugation with 11%Nycodenz.Transmission electron microscope,autofluorescenes,sudanⅢstaining,desmin andα-smooth muscle actin(α-SMA) immunofluorescence staining identified and assayed purity of HSC.Hepatic stellate cells were activated by cultured on uncoated plastic tissue culture dish and cultured in a higher glucose Dulbecco's modified eagles medium(DMEM) supplemented with 10%fetal calf serum under 37℃contained 5%CO2 95%air incubater.
Results The harvest rate of hepatic stellate cells is about 3.5±0.8×10~7 per rat,and the viability is more than 90%.Hepatic stellate cells can be activated by cultured more than 7 days.
Conclusions This reformed method is more efficient to isolate hepatic stellate cell and by culture the hepatic stellate cells can be activated.
Part two Expression ofαVβ3 integrin receptor during activation of hepatic stellate cells
Objective To explore the expression ofαVβ3 integrin receptor in hepatic stellate cell(HSC),and to demonstrate whetherαVβ3 integrin is a phenotypical receptor for activated hepatic stellate cell.
Methods HSCs were isolated from Sprague-Dawley rats.HSCs were activated by prolonged cultured,and identified by autofluorescenes and immunocytochemical staining respectively.Immunofluorescence staining of integrinβ3 and integrinαV combinedβ3,observed both of quiescent HSC and activated HSC.The cells were harvested and real-time PCR quantified mRNA ofαV,β3,α-SMA and Western Blot for protein.
Results HSCs were isolated successfully,and the purity was＞90%.HSC phenotype changed following culture.Integrinβ3 subunits exclusively expressed on activated HSC,and co-located with integrinαv subunits on the membrane of activated HSCs.The expression ofβ3 andα-SMA were low at the first day of isolation,and significant up-regulated following activation.Compared lday with 14day theβ3 mRNA enhanced 18 fold and 5.2 fold on protein.
ConclusionsαVβ3 integrin is a remarkable phenotypical receptor of activated HSC.It provides a promising strategy to target the activated HSC.
Part three Binding detection ofαvβ3 integrin receptor and cyclic RGD ligand in hepatic stellate cells
Objective To detect the capability ofαvβ3 integrin receptor binding with cyclic RGD ligand in hepatic stellate cells.
Methods Using solid-phase synthesis to create theαVβ3 integrin ligands of cyclic arginine-glycine-aspartate-D-phenylalanine-lysine and fluorescently labeled them with 5' -carboxyfluorescein(FAM) as probes (FAM-cRGD) to targetαVβ3 integrin.HSCs were incubated with the probes at a concentration of 10μM,and fluorescence microscopy was used to observe the internalization and distribution of probes.Flow cytometry recorded the fluorescence intensity,concentration and time effect.The equilibrium dissociationconstant(Kd) and maximal binding capacity(Bmax) for HSCs were calculated by radioligand binding assay(RBA).
Results:Fluorescent traces demonstrated that qHSCs did not internalize the probes but aHSC took up the probes into the cytoplasm.The aHSCs incubated with 10μM probes for 45 minutes increased their fluorescence intensity by 6.6-fold as compared to background.Furthermore,the intensity was enhanced in a concentration-and time-dependent manner,and it was inhibited 40%after co-incubation with 100μM unlabeled cRGD.RBA analyzed shown the Kd was 4.81×10~(-9) mol/L and Bmax was 2.112×10~(-10)mol/L.
Conclusions During activation of HSC,anαVβ3 integrin receptor mediated pathway is utilized by aHSCs to bind and internalize ligands of FAM-cRGD.These results support a promising strategy developing therapeutic or diagnostic agents to aim at aHSC.
Part four Relationship betweenαVβ3 integrin expression and angiogenesis in progressive liver fibrosis in rats
Objective To investigate the expression ofαVβ3 integrin and platelet endothelial cell adhesion molecule-1 in progressive liver fibrosis of rats.
Methods Rats two liver fibrosis models were induced by peritoneal injected 10%thioacetamine 175mg.kg~(-1) twice a week(TAA)and common bile duct ligation(BDL) respectively.Treated rats were sacrificed on the third week,ninth week in TAAmodel and on the first week,four week in BDL model respectively.Sirius red stained liver tissue and computer quantified the percent of fibrotic area.Immunohistochemical staining analyzed the expression ofαVβ3 integrin and platelet-endothelial cell adhesion molecule-1(CD31).Immunofluorescence staining and confocal microscopy observed the co-location of bothαVβ3 integrin andα-SMA in liver tissue.The expression ofαVβ3 integrin and CD31 were quantitatively analyzed by real-time PCR and Western blot.
Results The expression ofαVβ3 integrin significantly increased in both models,and parallel with the fibrotic area.The co-location showed corresponded to bothαVβ3 andα-SMA.The expression of CD31 was extended and new vascularization located in the area of portal vein and fibrostic septum in two models.Quantitative analysis suggestedαVβ3 and CD31 mRNA were up-regulated in both TAA and BDL groups(F=28.66,P＜0.01、F=19.62, P＜0.01 and F=32.60 P＜0.01,F=42.36 P＜0.01,respectively) as well on protein that correlated with fibrotic level.
ConclusionsαVβ3 integrin and CD31 were upregulated in liver fibrotic tissue.The overexpression involved in activation of hepatic stellate cells and angiogenesis that correspond with fibrotic remodeling.
Part five Tissue autoradiography ofαVβ3 integrin and distribution of ~(125)I-cRGD in liver fibrosis rats
Objective To determine the autoradiography of fibrotic tissue and the distribution of ~(125)I-cRGD in rats.
Methods Rats liver fibrosis were induced by injected thioacetamide(TAA). Frosted section of liver tissue incubated with ~(125)I-cRGD and cRGD respectively.Ligand binding in tissues was observed by autoradiography. Injected ~(125)I-cRGD and cRGD by tail vein,rats were sacrificed and the liver, spleen,kidney,heart,lung,muscle and brain weighed.Radioactivity percent of the injected dose in per gram(%ID/g) tissue in different organs were counted after 45 minutes.
Results The gray level of autoradiography is significantly difference between normal liver tissue and fibrotic tissue(P＜0.05).Compared with the sections of without block the gray level is reduced significantly in block sections(P＜0.05).Biodistribution results revealed that ~(125)I-cRGD in kidney is the highest in rats,next liver,and the brain is the lowest. Compared with control group,the block group and fibrosis group uptake of%ID/g of liver tissue is 0.11±0.03,0.12±0.03 and 0.17±0.02 respectively,and the difference is significantly(F=5.175,P=0.049). In other organs the difference of uptake is insignificantly.
Conclusions The ligand binding of ~(125)I-cRGD and tissue distribution is higher in fibrotic liver tissue than control rats.
Part six Noninvasive assessment of liver fibrosis usingαVβ3 integrin receptor SPECT imaging in rats
Objective To evaluate liver fibrosis by ~(99m)Tc-DTPA-cRGD as radiotracer in the receptor imaging ofαVβ3 integrin using single photon emission computed tomography(SPECT).
Methods Rats liver fibrosis were induced by injected thioacetamide (TAA).
Using solid-phase synthesis creates theαVβ3 integrin ligands of cyclic arginine-glycine-aspartate-D-phenylalanine-lysine and coupled to the chelator of diethyleletriamepentaacetic acid(DTPA-cRGD).Direct labeled ~(99m)Tc04- with DTPA-cRGD by stannous chloride reduction and Paper chromatography was performed to assay the radiochemical purity of ~(99m)Tc-DTPA-cRGD.On the three weeks and nine weeks of TAA treated the rats were injected 6μCi ~(99m)Tc-DTPA-cRGD by tail vein,and detect the radioactivity using SPECT scaning,after 45 minutes computed the average rate of count per minute(CPM) at the region of interest(ROI) in the target organs and compared with control.
Results The radiochemicalpurity is 95%,and ~(99m)Tc-DTPA-cRGD is stable over 4 h at room temperature with radiolabeling rate more than 90%.The average CPM rate of liver/kidney at ROI in control rats,TAA treated 3 weeks rats and TAA treated 9 weeks rats is 1.73±0.35,2.7±0.44 and 3.73±0.65 respectively,and the difference is significant(F=13.1,P=0.006). Compared with B ultrasonography and MRI the SPECT can distinguish the difference imaging of early stage fibrosis.
Conclusions ~(99m)Tc-DTPA-cRGD as a radiotracer in the receptor imaging ofαVβ3 integrin by SPECT can display early liver fibrosis which possess many merits such as high sensitivity and specificity,and relative high target/non-target ratio.
肝纤维化(liver fibrosis)是不同病因引起的肝脏慢性损伤后实质再生,同时伴随间质过度增生,潜在可逆的组织修复重建过程。肝纤维化评估对于慢性肝病的诊断和干预评价尤为重要,目前还没一种可靠的肝纤维化无创性评估方法。肝星状细胞(hepatic stellate cell,HSC)活化和表型改变是肝纤维化发生的中心事件,决定着肝纤维化转归。血管新生(angiogenesis)是组织损伤修复的必要条件。HSC活化、细胞外基质(extrocellural matrix,ECM)增多同时伴有新生血管形成是进展性肝纤维化修复重建中突出的病理特征。
整合素(integrin)是黏附分子家族中,由一个α亚基和一个β亚基非共价结合形成的跨细胞膜受体,介导细胞和ECM、细胞与细胞间的黏附和整合细胞内外信息传递。生理情况下整合素为细胞非组成性表达,在整合素家族中各亚型的表达有时序性和分布特异性。整合素αvβ3亚型是最广泛的ECM受体,主要介导间质细胞与纤维连接蛋白、纤维蛋白原、Ⅰ型胶原、玻璃黏结蛋白、层黏蛋白、Ⅷ因子相关抗原(vWF)等ECM的黏附,并和血小板衍生的生长因子(PDGF)、转化生长因子β1(TGFβ-1)、内皮细胞生长因子(VEGF)等细胞因子有信号协同作用,主要传递和细胞增殖、分化、运动、分布、定居、生存或凋亡等有关的细胞信号。正常肝组织仅门静脉周围表达少量整合素αvβ3,肝细胞、库普弗细胞、静止HSC和正常血管内皮细胞不表达该亚型。实验研究了整合素βvβ3在活化HSC和肝纤维化组织中的表达,探讨整合素αvβ3表达、血管新生与肝纤维化间质重塑的关系。
精氨酸-甘氨酸-天门冬氨酸(Arg-Gly-Asp,RGD)三肽模体是ECM中保守序列,也是ECM和整合素受体识别常见位点。整合素αvβ3能识别ECM中特异构象的RGD配体。人工合成含RGD肽链高通量筛选发现,精氨酸-甘氨酸-天门冬氨酸-(右旋)酪氨酸-赖氨酸(RGDyK)、精氨酸-甘氨酸-天门冬氨酸-(右旋)苯丙氨酸-赖氨酸(或RGDfK)等五肽分子也能和整合素αvβ3受体特异结合,对五肽中赖氨酸上活性氨基进行小分子修饰并不影响受体与配基结合活性。实验中通过固相合成整合素αvβ3受体的RGD环肽(cRGD)配基,设计用羧基荧光素(FAM)和锝99同位素(~(99m)TC)等示踪分子标记cRGD作为探针,探讨人工合成的cRGD配基对活化HSC和肝纤维化组织的靶向性结合是课题研究的主要工作。
建立在肝纤维化组织间质重建的分子病理基础上,以~(99m)TC标记的cRGD为示踪分子,通过单光子发射型计算机断层成像(SPECT)探测肝纤维化不同时期肝脏放射性活度的相对变化(肝脏/心脏计数比值),并和常规B型超声、磁共振仪(MRI)比较早期肝纤维化的影像表现。尝试应用分子影像方法,根据肝脏中整合素αvβ3受体结合cRGD配基的变化信息,来反映慢性损伤肝组织重建过程中活化细胞水平及与肝纤维化程度的关系,为建立一种敏感、特异、可靠的肝纤维化无创性受体显像评估方法的可行性提供实验依据。
课题研究分为以下六部分;
第一部分HSC的分离、培养、鉴定和体外活化模型的建立
[目的]建立一种稳定可靠的HSC分离方法,为体外研究提供细胞模型。
[方法]Hank's液在体灌洗大鼠肝脏,离体后用Ⅳ胶原酶、链蛋白酶、DNaseⅠ消化肝脏,11%Nycodenz连续梯度液分离出大鼠HSC,计数细胞得率,0.2%台盼蓝染色计算细胞活率。自发荧光、透射电镜、苏丹Ⅲ染色观察细胞脂滴,Desmin、α-SMA免疫化学和荧光染色对HSC进行鉴定和纯度分析。分离后HSC按1-4×10~6密度接种于未包被塑料培养瓶或皿,含10%胎儿牛血清DMEM培养基,37℃含5%CO_295%空气培养箱中培养,通过延长培养诱导HSC活化。
[结果]分离HSC得率3.5±0.8×10~7/只大鼠,细胞活率＞90%,分离第1天自发荧光和第3天desmin染色阳性细胞＞90%。HSC随着体外培养形态明显改变,分离培养7天后α-SMA阳性细胞＞90%,培养14天或传代后＞95%阳性。
[结论]肝脏离体后消化能达到在体消化同样的效果,应用Nycodenz密度分离介质可获得满意HSC得率和纯度。新分离大鼠HSC培养7天后超过90%激活表型转变为活化HSC。
第二部分整合素αvβ3受体在HSC活化过程中的表达
[目的]探讨HSC表型转变过程中整合素αvβ3受体和α-SMA的定性、定位、定量表达。
[方法]HSC培养的第1、3、5、7和14天分别提取细胞总RNA和总蛋白。总RNA逆转录合成cDNA,分别通过real-time PCR和Western Blot分析整合素αv、β3和α-SMA mRNA和蛋白质在HSC活化过程中的表达。静止期细胞和激活细胞期HSC,分别进行α-SMA和整合素αVβ3细胞免疫荧光染色激光共聚焦观察。
[结果]HSC活化过程中整合素αVβ3表达水平变化有显著性差异(P＜0.01),第14天时在mRNA和蛋白质水平比第1天,分别增加18倍和5.2倍。而比较7天和14天时表达则无明显差异。免疫荧光双染色显示活化HSC中整合素αVβ3极性表达于细胞膜,α-SMA表达于细胞浆并且和整合素αVβ3有共定位。
[结论]整合素αVβ3是激活状态HSC的表型受体,表达上调与HSC活化和活化状态维持有关。
第三部分整合素αvβ3 RGD环肽配基与HSC的结合分析
[目的]研究HSC对羧基荧光素(FAM)标记精氨酸-甘氨酸-天门冬氨酸-(右旋)苯丙氨酸-赖氨酸(FAM-cRGD)环五肽的结合和内化。计算活化HSC对~(125)Ⅰ标记精氨酸-甘氨酸-天门冬氨酸-(右旋)酪氨酸-赖氨酸的亲和力和受体容量。
[方法]采用C末端保护固相法合成精氨酸-甘氨酸-天门冬氨酸-(右旋)苯丙氨酸-赖氨酸环五肽,羧基荧光素合成树脂上和环五肽中赖氨酸上的氨基固相结合为标记的荧光分子探针(FAM-cRGD)。高效液相色谱法(HPLC)和质谱法(MS)检测FAM-cRGD的纯度和分子量。荧光示踪观察HSC对FAM-cRGD探针的摄取和内化。流式细胞仪(FCM)分析HSC孵育FAM-cRGD后细胞平均荧光强度的变化和浓度、时间效应。受体放射性配基结合分析(RBA)测定HSC对~(125)Ⅰ标记精氨酸-甘氨酸-天门冬氨酸-(右旋)酪氨酸-赖氨酸的平衡解离常数(Kd)和最大结合点数(Bmax)。
[结果]荧光示踪显示静止HSC不结合FAM-cRGD探针,活化HSC结合FAM-cRGD并摄入细胞浆,但无细胞核的内化。流式细胞仪分析提示,静止期HSC和10μMFAM-cRGD 37℃孵育45分钟,平均荧光强度和本底比较无明显变化。而活化HSC和10μM FAM-cRGD 37℃孵育45分钟,平均荧光强度较本底增高6.6倍,活化HSC和10μM FAM-cRGD、150μM cRGD 37℃共同孵育45分钟,平均荧光强度抑制超过30%,活化HSC平均荧光强度改变有随FAM-cRGD孵育浓度和孵育时间延长而增加的趋势。受体放射性配基结合分析,通过Scatchard作图法求出活化HSC对cRGD结合的Kd为4.808×10~(-9)mol/L和Bmax为2.112×10~(-10)mol/L。
[结论]活化HSC能与整合素αVβ3受体的cRGD发生受体-配基样结合,活化HSC对cRGD配基有较高的亲和力和受体容量。
第四部分肝纤维化中的整合素αvβ3表达与血管新生
[目的]建立两种大鼠肝肝纤维化模型,探讨肝纤维化组织中整合素αVβ3表达及与血管新生的关系。
[方法]硫代乙酰胺(TAA)腹腔注射和胆总管结扎(BDL)诱导建立两种大鼠肝纤维化模型。建模的第3周末、9周末分别进行肝组织天狼星红染色和计算机图像分析、整合素αVβ3和血小板内皮细胞黏附分子(CD31)免疫化学染色、整合素αVβ3和与α-SMA免疫荧光双重染色及激光共聚焦观察和real-time PCR和Western blot分析肝组织中整合素αVβ3、CD31在mRNA和蛋白表达水平表达的相对定量。
[结果]肝纤维化程度随建模时间延长而加重。两种肝纤维化大鼠和对照大鼠比较,肝组织中整合素αVβ3和CD31在mRNA和蛋白表达水平有显著性差异(TAA组F=28.66,P＜0.01和F=19.62 P＜0.01,BDL组F=32.60 P＜0.01和F=42.36P＜0.01),随肝纤维化进展而有升高的趋势。免疫组织化学和免疫荧光化学染色提示,在肝纤维化扩大的汇管区及纤维隔中有新生血管形成,在间质中整合素αVβ3与α-SMA表达部位一致,从汇管区向肝实质深入。
[结论]肝纤维化进展中整合素αVβ3表达水平增加与HSC活化和血管新生有关,并与间质重建程度平行。
第五部分肝组织整合素αVβ3放射自显影和~(125)Ⅰ-cRGD在体内的分布
[目的]分析肝纤维化组织对~(125)Ⅰ-cRGD配基的结合,和~(125)Ⅰ-cRGD在早期肝纤维大鼠中的分布。
[方法]硫代乙酰胺(TAA)腹腔注射诱导大鼠肝纤维化,在第3周末、9周末分别进行肝组织冰冻切片,切片分别和~(125)Ⅰ-cRGD、~(125)Ⅰ-cRGD加500倍化学剂量cRGD(特异阻断)进行4℃孵育2小时,冲洗切片后接触法暴光2周,进行受体组织放射自显影。6只正常大鼠和6只TAA3周肝纤维化大鼠,各3只分别进行尾静脉注射6μCi ~(125)Ⅰ-cRGD,各3只注射500倍化学剂量cRGD特异阻断后再注射6μCi ~(125)Ⅰ-cRGD、在注射后45min分别取材肝、脾、脑、肾、心、肺和肌肉,测定各脏器的放射性活度,计算各脏器放射性比活度(%ID/g)。
[结果]正常大鼠、TAA3周、TAA9周大鼠切片暴光后的相对灰度分别为8.1±1.6、18.7±2.6和28.7±3.4,用cRGD阻断的切片相对灰度分别为7.7±1.7、12.1±2.5和19.3±3.3,三组间比较差异有显著性(P＜0.01)。在TAA3周和TAA9周中,未用cRGD阻断和cRGD阻断比较差异有显著性P=0.02和P=0.041,阻断的切片中相对灰度降低。正常大鼠和TAA3周的早期肝纤维化大鼠中,~(125)Ⅰ-cRGD体内分布以肾脏组织中最高,其次是肝脏,脑组织最低。正常大鼠中肝脏~(125)Ⅰ-cRGD的分布%ID/g,在未阻断和阻断大鼠分别为0.11±0.03、0.10±0.04,在TAA3周大鼠分别为0.17±0.02和0.12±0.03,4组比较F=5.175,P=0.049差异有显著性,TAA3周大鼠肝脏中的%ID/g较高,在用cRGD阻断时肝脏中放射性比活度有下降的趋势。而比较各组间肾、脾、心、肺、脑、肌肉等脏器放射性比活度则差异无显著性。
[结论]体内外的研究提示肝纤维化组织对~(125)Ⅰ-cRGD配基有较高的结合和摄取,并能被未标记的cRGD所竞争。
第六部分肝脏整合素αvβ3受体显像评估肝纤维化
[目的]以~(99m)Tc-DTPA-cRGD为示踪剂应用SPECT对肝纤维化大鼠肝脏中整合素αvβ3受体进行分子显像,测定正常和肝纤维化大鼠间肝/心的放射性计数的比值。
[方法]硫代乙酰胺(TAA)腹腔诱导大鼠肝纤维化。合成树脂上二亚乙基三基三胺五乙酸(DTPA)固相结合cRGD(DTPA-cRGD),高锝酸盐(~(99m)Tc04-)氯化亚锡还原法标记DTPA-cRGD(~(99m)Tc-DTPA-cRGD),纸层析法测定放射化学纯度。TAA3周早期肝纤维化大鼠进行肝脏B型超声和MRI扫描观察影像表现。在TAA3周和TAA9周,尾静脉注射~(99m)Tc-DTPA-cRGD 6mCi/只,用小动物SPECT进行大鼠腹部扫描,计算注药后45min,感兴趣区(ROI)的肝/心放射性计数比。
[结果]测定~(99m)Tc-DTPA-cRGD的放射化学纯度＞95%,室温下4小时保持稳定。注射~(99m)Tc-DTPA-cRGD后45min,正常组、TAA3周和TAA9周组中感兴趣区的肝/心放射性计数分别为1.73±0.35、2.7±0.44、3.73±0.65差异有显著性(F=13.1,P=0.006)。比较正常大鼠和TAA3周大鼠,TAA3周和TAA9周大鼠组间感兴趣区的肝/心放射性活度比值差异有显著性(P＜0.05)。常规MRI和B型超声检测TAA3周的早期肝纤维化大鼠和正常大鼠在影像上无区别,SPECT扫描提示TAA3周大鼠肝脏对~(99m)Tc-DTPA-cRGD的积聚增加。
[结论]肝纤维化大鼠~(99m)Tc-DTPA-cRGD SPECT显像肝/心放射性计数比值较正常大鼠明显增加与肝组织中整合素αvβ3受体表达上调有关。SPECT和常规影像比较能提示早期肝纤维化中对~(99m)Tc-DTPA-cRGD摄取增强后的影像改变。
Liver fibrosis is a reversible response of wound healing and architecture remodeling after liver cells were attacked by all kinds of insults.Assessment of hepatic fibrosis plays a key role for diagnosis and evaluation in chronic liver injury.At present liver biopsy is widely accepted as a gold standard to document liver fibrosis.However,liver biopsy procedure is limited as an invasive procedure,sampling error, potential morbidity and mortality.Hence,it is vitally important to develop a reliable non-invasive measure to dynamically detect the alteration of hepatic repair in chronic liver disease.The activation of hepatic stellate cell(HSC) is recognized as one of the critical hallmark in liver fibrosis and to decide the fate of injured liver. Recently,increasingly proofs support angiogenesis is precondition of liver tissue repairing in diverse etiologies.The accumulation of extracellular matrix(ECM) and angiogenesis is two significant pathological characteristics of advance liver fibrosis.
Integrin receptor is a cell surface receptor consisting of oneα-subunit and oneβ-subunit,and at least 18 differentαsubunits and 8 differentβsubunits of integrin are currently known.Therefore, various assemblages of subunits result in more than 24 functional subtypes of integrin dimers which differ in distribution,binding ligands and signalling properties.Among them,theαVβ3 subtype is also referred to as the vitronectin receptor,which is an extensive extracellular matrix(ECM) receptor and acts as an auxiliary receptor of connective tissue growth factor(CTGF) and platelet-derived growth factor(PDGF) that mediates cell events involved in proliferation, migration,activation and survival.These actions facilitate wound healing and the remodeling response in injured tissue.Normally,the expression ofαVβ3 integrin is restricted in special cell types and only low levels are produced in adult tissue.It has been shown that expression ofαVβ3 is weakly around the portal vein in a normal liver, with the resting endothelial cells,hepatocytes,Kupffer cells and quiescent HSCs expressing even lessαVβ3 integrin.
Integrin-ligand binding is well-characterized.ECM proteins can bind many integrins via the conservative motif of the arginine-glycine-aspartic acid(RGD) tripeptide,and the flank conformation of RGD determines the selectivity in different integrin subtypes.For example,vitronectin,denatured collagen,laminin, fibrinogen,osteopontin,von Willebrand factor and fibronectin interact withαVβ3 integrin through their special conformation of the RGD sequence.Based on these findings,various RGD-contained peptides have been successfully produced for experimental studies.
The aim of this study was to display the expression ofαVβ3 integrin in vitro and vivo,and to explore the relationship of angiogenesis and architecture remodeling in fibrotic liver.Furthermore,intend to investigate whether ~(99m)Tc labeled artificial Arg-Gly-Asp(RGD) containing cyclic peptides ligand can be applied to assess hepatic fibrosis by receptor imaging using SPECT.This assessing procedure is more effective and sensitive which reflect the molecular pathology mechanism of liver fibrosis.This study will provide a new perspective to explore the feasibility of developing a novel receptor-targeting strategy for non-invasive detecting liver fibrosis.
Part one Isolation and culture of rat primary hepatic stellate cell and establish of activated HSC model in vitro
Objective To build an efficient procedure for isolating hepatic stellate cell and establish activated HSC model.
Methods Primary hepatic stellate cells(HSC) were isolated from normal Sprague-Dawley(SD) rats by infusion and combined digestion of pronase E and collagenaseⅣex situ.Hepatic stellate cells were purified by density centrifugation with 11%Nycodenz.Transmission electron microscope,autofluorescenes,sudanⅢstaining,desmin andα-smooth muscle actin(α-SMA) immunofluorescence staining identified and assayed purity of HSC.Hepatic stellate cells were activated by cultured on uncoated plastic tissue culture dish and cultured in a higher glucose Dulbecco's modified eagles medium(DMEM) supplemented with 10%fetal calf serum under 37℃contained 5%CO2 95%air incubater.
Results The harvest rate of hepatic stellate cells is about 3.5±0.8×10~7 per rat,and the viability is more than 90%.Hepatic stellate cells can be activated by cultured more than 7 days.
Conclusions This reformed method is more efficient to isolate hepatic stellate cell and by culture the hepatic stellate cells can be activated.
Part two Expression ofαVβ3 integrin receptor during activation of hepatic stellate cells
Objective To explore the expression ofαVβ3 integrin receptor in hepatic stellate cell(HSC),and to demonstrate whetherαVβ3 integrin is a phenotypical receptor for activated hepatic stellate cell.
Methods HSCs were isolated from Sprague-Dawley rats.HSCs were activated by prolonged cultured,and identified by autofluorescenes and immunocytochemical staining respectively.Immunofluorescence staining of integrinβ3 and integrinαV combinedβ3,observed both of quiescent HSC and activated HSC.The cells were harvested and real-time PCR quantified mRNA ofαV,β3,α-SMA and Western Blot for protein.
Results HSCs were isolated successfully,and the purity was＞90%.HSC phenotype changed following culture.Integrinβ3 subunits exclusively expressed on activated HSC,and co-located with integrinαv subunits on the membrane of activated HSCs.The expression ofβ3 andα-SMA were low at the first day of isolation,and significant up-regulated following activation.Compared lday with 14day theβ3 mRNA enhanced 18 fold and 5.2 fold on protein.
ConclusionsαVβ3 integrin is a remarkable phenotypical receptor of activated HSC.It provides a promising strategy to target the activated HSC.
Part three Binding detection ofαvβ3 integrin receptor and cyclic RGD ligand in hepatic stellate cells
Objective To detect the capability ofαvβ3 integrin receptor binding with cyclic RGD ligand in hepatic stellate cells.
Methods Using solid-phase synthesis to create theαVβ3 integrin ligands of cyclic arginine-glycine-aspartate-D-phenylalanine-lysine and fluorescently labeled them with 5' -carboxyfluorescein(FAM) as probes (FAM-cRGD) to targetαVβ3 integrin.HSCs were incubated with the probes at a concentration of 10μM,and fluorescence microscopy was used to observe the internalization and distribution of probes.Flow cytometry recorded the fluorescence intensity,concentration and time effect.The equilibrium dissociationconstant(Kd) and maximal binding capacity(Bmax) for HSCs were calculated by radioligand binding assay(RBA).
Results:Fluorescent traces demonstrated that qHSCs did not internalize the probes but aHSC took up the probes into the cytoplasm.The aHSCs incubated with 10μM probes for 45 minutes increased their fluorescence intensity by 6.6-fold as compared to background.Furthermore,the intensity was enhanced in a concentration-and time-dependent manner,and it was inhibited 40%after co-incubation with 100μM unlabeled cRGD.RBA analyzed shown the Kd was 4.81×10~(-9) mol/L and Bmax was 2.112×10~(-10)mol/L.
Conclusions During activation of HSC,anαVβ3 integrin receptor mediated pathway is utilized by aHSCs to bind and internalize ligands of FAM-cRGD.These results support a promising strategy developing therapeutic or diagnostic agents to aim at aHSC.
Part four Relationship betweenαVβ3 integrin expression and angiogenesis in progressive liver fibrosis in rats
Objective To investigate the expression ofαVβ3 integrin and platelet endothelial cell adhesion molecule-1 in progressive liver fibrosis of rats.
Methods Rats two liver fibrosis models were induced by peritoneal injected 10%thioacetamine 175mg.kg~(-1) twice a week(TAA)and common bile duct ligation(BDL) respectively.Treated rats were sacrificed on the third week,ninth week in TAAmodel and on the first week,four week in BDL model respectively.Sirius red stained liver tissue and computer quantified the percent of fibrotic area.Immunohistochemical staining analyzed the expression ofαVβ3 integrin and platelet-endothelial cell adhesion molecule-1(CD31).Immunofluorescence staining and confocal microscopy observed the co-location of bothαVβ3 integrin andα-SMA in liver tissue.The expression ofαVβ3 integrin and CD31 were quantitatively analyzed by real-time PCR and Western blot.
Results The expression ofαVβ3 integrin significantly increased in both models,and parallel with the fibrotic area.The co-location showed corresponded to bothαVβ3 andα-SMA.The expression of CD31 was extended and new vascularization located in the area of portal vein and fibrostic septum in two models.Quantitative analysis suggestedαVβ3 and CD31 mRNA were up-regulated in both TAA and BDL groups(F=28.66,P＜0.01、F=19.62, P＜0.01 and F=32.60 P＜0.01,F=42.36 P＜0.01,respectively) as well on protein that correlated with fibrotic level.
ConclusionsαVβ3 integrin and CD31 were upregulated in liver fibrotic tissue.The overexpression involved in activation of hepatic stellate cells and angiogenesis that correspond with fibrotic remodeling.
Part five Tissue autoradiography ofαVβ3 integrin and distribution of ~(125)I-cRGD in liver fibrosis rats
Objective To determine the autoradiography of fibrotic tissue and the distribution of ~(125)I-cRGD in rats.
Methods Rats liver fibrosis were induced by injected thioacetamide(TAA). Frosted section of liver tissue incubated with ~(125)I-cRGD and cRGD respectively.Ligand binding in tissues was observed by autoradiography. Injected ~(125)I-cRGD and cRGD by tail vein,rats were sacrificed and the liver, spleen,kidney,heart,lung,muscle and brain weighed.Radioactivity percent of the injected dose in per gram(%ID/g) tissue in different organs were counted after 45 minutes.
Results The gray level of autoradiography is significantly difference between normal liver tissue and fibrotic tissue(P＜0.05).Compared with the sections of without block the gray level is reduced significantly in block sections(P＜0.05).Biodistribution results revealed that ~(125)I-cRGD in kidney is the highest in rats,next liver,and the brain is the lowest. Compared with control group,the block group and fibrosis group uptake of%ID/g of liver tissue is 0.11±0.03,0.12±0.03 and 0.17±0.02 respectively,and the difference is significantly(F=5.175,P=0.049). In other organs the difference of uptake is insignificantly.
Conclusions The ligand binding of ~(125)I-cRGD and tissue distribution is higher in fibrotic liver tissue than control rats.
Part six Noninvasive assessment of liver fibrosis usingαVβ3 integrin receptor SPECT imaging in rats
Objective To evaluate liver fibrosis by ~(99m)Tc-DTPA-cRGD as radiotracer in the receptor imaging ofαVβ3 integrin using single photon emission computed tomography(SPECT).
Methods Rats liver fibrosis were induced by injected thioacetamide (TAA).
Using solid-phase synthesis creates theαVβ3 integrin ligands of cyclic arginine-glycine-aspartate-D-phenylalanine-lysine and coupled to the chelator of diethyleletriamepentaacetic acid(DTPA-cRGD).Direct labeled ~(99m)Tc04- with DTPA-cRGD by stannous chloride reduction and Paper chromatography was performed to assay the radiochemical purity of ~(99m)Tc-DTPA-cRGD.On the three weeks and nine weeks of TAA treated the rats were injected 6μCi ~(99m)Tc-DTPA-cRGD by tail vein,and detect the radioactivity using SPECT scaning,after 45 minutes computed the average rate of count per minute(CPM) at the region of interest(ROI) in the target organs and compared with control.
Results The radiochemicalpurity is 95%,and ~(99m)Tc-DTPA-cRGD is stable over 4 h at room temperature with radiolabeling rate more than 90%.The average CPM rate of liver/kidney at ROI in control rats,TAA treated 3 weeks rats and TAA treated 9 weeks rats is 1.73±0.35,2.7±0.44 and 3.73±0.65 respectively,and the difference is significant(F=13.1,P=0.006). Compared with B ultrasonography and MRI the SPECT can distinguish the difference imaging of early stage fibrosis.
Conclusions ~(99m)Tc-DTPA-cRGD as a radiotracer in the receptor imaging ofαVβ3 integrin by SPECT can display early liver fibrosis which possess many merits such as high sensitivity and specificity,and relative high target/non-target ratio.
引文
1. Friedman SL. The cellular basis of hepatic fibrosis: mechanisms and treatment strageies. N Engl J Med, 1993,323:1828-35.
2. Hendriks HF, Verhoofstad WA, Brouwer A, et al. Perisinusiodal fat-storing cells are the main vitamin A storage sites in rat liver. Exp.Cell Res, 1985, 160:138-149.
3. Geerts A. History, hereogeneity, developmental biology, and functions of quiescent hepatic stellate cells. Sem Liv.Dis, 2001,21:311-335.
4. Friedman SL. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J. Biol. Chem, 275,2247-2250.
5. Moreira RK. Hepatic stellate cells and liver fibrosis. Arch Pathol Lab Med, 2007, 131:1728-1734.
6. Moreira RK. Hepatic stellate cells and liver fibrosis. Arch Pathol Lab Med, 2007, 131:1728-1734.
7. E. Wisse and D. L Knook Editors. Kupffer cells and other liver sinusoidal cells. Elsevier/North-Holl, 2nd edition 1979 printed in the Netherlands.
8. Brenner DA, Waterboer T, Choi SK, et al. New aspects of hepatic fibrosis. J Hepatol, 2000, 32: 32-38.
9. Friedman SL. Cellular sources of collagen and regulation of collagen production in liver. Semin Liver Dis, 1990,10:20-29.
10. Friedman SL, Roll FJ. Isolation and culture of hepatic lipocytes, Kupffer cells, and sinusoidal endothelial cells by density gradient centrifugation with Stractan. Anal Biochem. 1987,161:207-218.
11. Blomhoff R, Berg T. Isolation and cultivation of rat liver stellate cells. Meth Enzymol, 1990, 190:58-71.
12. Geerts A, Niki T, Hellemans K, et al. Purification of rat hepatic stellate cells by side scatter-activated cell sorting. Hepatology, 1998, 27:590-59.
13. Ballardini G, Groff P, Badiali de Giorgi L, Schuppan D, et al. Ito cell heterogeneity: desmin negative Ito cells in normal rat liver. Hepatology, 1994, 19:440-446.
14. Hynes RO. Integrins: Versatility Modulation, and Signaling in Cell Adhesion. Cell, 1992,69:11-25.
15. Imai K, Sato T, Senoo H. Adhesion between C ells and Extracellular Matrix with Special Reference to Hepatic Stellate Cells Adhesion to Three-dimensional Collagen Fibers. Cell Structure and Function, 2000, 25:329-336.
16. Humphries MJ. Integrin Structure. Biochemical Society, 2000, 28:311-340.
17. Eng FJ, Friedman SL. Fibrogenesis I. New insights into hepatic stellate cell activation: the simple becomes complex. Am J Physiol Gastrointest Liver Physiol, 2000, 279: G7-G11.
18. Friedman SL, Molecular Regulation of Hepatic Fibrosis, an Integrated Cellular Response to Tissue Injury. J. Biol. Chem, 2000, 275;2247-2250.
19. Sohara N, Znoyko I, Levy MT, et al. Reversal of activation of human myofibroblast-like cells by culture on a basement membrane-like substrate, J. Hepatol, 2002,37:125-129.
20. Hynes R 0. Integrins: Bidirectional, Allosteric Signaling Machines. Cell,2002,110;673-687.
21. Plow E F, Haas T A, Zhang L, et al. Ligand Binding to Integrins. J. Biol. Chem, 2000, 275:21785-21788.
22. Znoyko I Trojanowska M Reuben A. Collagen binding alpha(2)beta(1) and alpha(1)beta(1) integrins play contrasting roles in regulation of Ets-l expression in human liver myofibroblasts. Molecular & Cellular Biochemistry, 2006, 282:89-99.
23. BreitkopfK, Roeyen C, Sawitza I, et al. Expression patterns of PDGF-A, -B, -C and -D and the PDGF-receptors a and b in activated rat hepatic stellate cells (HSC). Cytokine, 2005, 31:349-357.
24. Liu C, Gaca MD, Swenson ES, et al. Smads 2 and 3 are differentially activated by transforming growth factor-beta (TGF-beta) in quiescent and activated hepatic stellate cells: constitutive nuclear localization of Smads in activated cells is TGF-beta-independent. J. Biol. Chem, 2003, 278:11721-11728.
25. Borkham-Kamphorst E, Stoll D, Gressner AM, et al. Antisense strategy against PDGF B-chain proves effective in preventing experimental liver fibrogenesis. Biochemical and Biophysical Research Communications, 2004, 321: 413-423.
26. Byzova TV, Goldman CK, Pampori N, et al. A Mechanism for Modulation of Cellular Responses to VEGF: Activation of the Integrins. Molecular Cell, 2000, 6:851-860.
27. Gao R, Brigstock DR. Connective Tissue Growth Factor(CCN2) Induces Adhesion of Rat Activated Hepatic Stellate Cells by Binding of Its C-terminal Domain to Integrin avb3 and Heparan Sulfate Proteglycan. J. Biol. Chem, 2004, 279:8848-8855.
28. Liu ZJ, Snyder R, Soma A, et al. VEGF-A and αVβ3 integrin synergistically rescue angiogenesis via N-Ras and PI3-K signaling in human microvascular endothelial cells. FASEB, 2003, 17:1931-1933.
29. Ridley A J, Schwartz MA, BurridgeK, et al. Cell Migration: Integrating Signals from Front to Back. Science, 2003, 302:1704-1709.
30. Carloni V, Romanelli RG, Pinzani M, et al. Expression and Function of Integrin Receptors for Collagen and Laminin in Culture Human Stellate Cells. Gastroenterology, 1996, 110:1127-1136.
31. Yang CQ, ZeisbergM, Mosterman B, et al. Liver fibrosis: insights into migration of hepatic stellate cells in response to extracellular matrix and growth factors. Gastroenterology, 2003, 124:147-159.
32 Carloni V, Romanelli RG, Pinzani M, et al. Expression and Function of Integrin Receptors for Collagen and Laminin in Culture Human Stellate Cells. Gastroenterology, 1996, 110:1127-1136.
33 Zhou XY, Murphy FR, Gehdu N, et al. Engagement of αvβ3 Integrin Regulates Proliferation and Apoptosis of Hepatic Stellate Cells. J. Biol. Chem, 2004, 279:23996-24006.
34 Wu Y, Nan L, Bardag-Gorce F, et al. The role of laminin-integrin signaling in triggering MB formation. An in vivo and in vitro study. Experimental & Molecular Pathology, 2005, 79:1-8.
35. Iwamoto H, Sakai H, Nawata H, et al. Inhibition of integrin signaling with Arg-Gly-Asp motifs in rat hepatic stellate cells. J. Hepatol, 1998,29:752-759.
36. Patsenker E, Popov Y, Wiesner M, et al. Pharmacological inhibition of the vitronectin receptor abrogates PDGF-BB-induced hepatic stellate cell migration and activation in vitro. Journal of Hepatology, 2007, 46:878-887.
37. Horton MA. The avb3 integrin "Vitronectin Receptor" Int. J. Biochem. Cell Biol, 1997, 29:721-725.
38. Cluzel C, Saltel F, Lussi J, et al. The mechanisms and dynamics of (alpha) v(beta)3 integrin clustering in living cells. J. Biol. Chem, 2005, 171:383-392.
39. Lygoe KA, Norman JT, Marshall JF, et al. αv integrins play an important role inmyofibroblast differentiation. Wound Rep Reg, 2004, 12:461-470.
40. Kunicki TJ, Ely KR, Kunicki TC, et al. The exchange of Arg-Gly-Asp (RGD) and Arg-Tyr-Asp (RYD) binding sequences in a recombinant murine Fab fragment specific for the integrin alpha IIb beta 3 does not alter integrin recognition. J Biol Chem, 1995, 270:16660-16665.
41. Craig D, Gao M, Schulten K, et al. Structural insights into how the MIDAS ion stabilizes integrin binding to an RGD peptide under force. Structure, 2004, 12:2049-2058.
42. Yahalom D, Wittelsberger A, Mierke DF, et al. Identification of the principal binding site for RGD-containing ligands in the alpha(V)beta(3) integrin: a photoaffinity cross-linking study. Biochemistry, 2002, 41:8321-8331.
43. van Well RM, Overkleeft HS, van der Marel GA, et al. Solid-phase synthesis of cyclic RGD-furanoid sugar amino acid peptides as integrin inhibitors. Bioorg Med Chem Lett, 2003, 13:331-334.
44. Sadeghi MM, Krassilnikova S, Zhang JS, et al. Detection of Injury-Induced Vascular Remodeling by Targeting Activated αvβ3 integrin In Vivo. Circulation, 2004,110:84-90.
45. Matsuno H, Stassen JM, Vermylen J, et al. Inhibition of integrin function by a cyclic RGD-containing peptide prevents neointima formation. Circulation, 1994, 90:2203-2206.
46. Chen X, PlasenciaC, Hou Y, et al. Synthesis and biological evaluation of dimeric RGD peptide-paclitaxel conjugate as a model for integrin-targeted drug delivery. J Med Chem, 2005, 48:1098-1106.
47. Wu Y, Zhang X, Xiong Z, et al. microPET imaging of glioma integrin {alpha}v{beta}3 expression using (64)Cu-labeled tetrameric RGD peptide. J Nucl Med, 2005, 46:1707-1718.
48. Mogford JE, Davis GE, Platts SH, et al. Vascular smooth muscle alpha v beta 3 integrin mediates arteriolar vasodilation in response to RGD peptides. Circ Res, 1996, 79:821-826.
49. Smith JW and Cheresh DA. Integrin (alpha v beta 3)-ligand interaction. Identification of a heterodimeric RGD binding site on the vitronectin receptor. J Biol Chem, 1990, 265:2168-2172.
50. Wu Y, Cai W, and Chen X. Near-Infrared Fluorescence Imaging of Tumor Integrin alpha(v)beta (3) Expression with Cy7-Labeled RGD Multimers. Mol Imaging Biol, 2006.
51. Wang W, McMurray JS, Wu Q, et al. Convenient Solid-Phase Synthesis of Diethylenetriaminepenta-Acetic Acid (DTPA)-conjugated Cyclic RGD Peptide Analogues. Cancer Biotherapy & Radiopharmaceuticals, 2005, 20:547-556.
52. Liu, Radiolabeled multimeric cyclic RGD peptides as integrin alpha v beta3 targeted radiotracers for tumor imaging, Mol. Pharm, 2006, 3:472-487.
53. Marinelli L, Lavecchia A, Gottsechalk KE, et al. Docking Studies on αvβ3 Integrin Ligands: Pharmacophore Refinement and Implications for Drug Design. J. Med. Chem, 2003, 46:4393-4404.
54. Goodman SL, Holzemann G, Sulyok GAG, et al. Nanomolar Small Molecule Inhibitors for avp6 avp5 αvβ3 Integrins. J. Med. Chem, 2002, 45:1045-1051.
55. Wang WW, McMurray, John S L, et al. Probing for integrin alpha v beta3 binding of RGD peptides using fluorescence polarization. Bioconjug Chem, 2005, 16: 729-734.
56. Wang W, McMurray JS, Wu Q, et al. Convenient Solid-Phase Synthesis of Diethylenetriaminepenta-Acetic Acid (DTPA)-conjugated Cyclic RGD Peptide Analogues. Cancer Biotherapy & Radiopharmaceuticals, 2005, 20:547-556.
57. Line BR, Mitra A, Nan A, et al. Targeting tumor angiogenesis: Comparison of peptide and polymer-peptide conjugates. J Nucl Med , 2005, 46: 1552-1560.
58. Xiong JP, Stehle T, Diefenbach B, et al. Crystal structure of the Extracellular Segment of Integrin αvβ3. Science, 2001,294:339-345.
59. Xiong JP, Stehle T, Zhang R, et al. Crystal structure of the Extracellular Segment of Integrin αvβ3 in Complex with an Arg-Gly-Asp Ligand. Science, 2002,296:151-154.
60. Salcedo X, Medina J, Sanz-Cameno P, et al. The potential of angiogenesis soluble markers in chronic hepatitis C. Hepatology, 2005, 42:696-701.
61. Sanz-Cameno P, Martin-Vilchez S, Lara-Pezz E, et al. Heaptitis B Virus Promotes Angiopoietin-2 Expression in Liver Tissue. The American Journal of Pathology, 2006, 169:1217-1221.
62. Deli G, Yangm G, Makuuch S, et al. Immunohistochemical assessment of angiogenesis in hepatocellular carcinoma and surrounding cirrhotic liver tissues. World J Gastroenterol, 2005, 11: 960-963.
63. Yoshiji H, Kuriyama S, Yoshii J, et al. Vascular endothelial growth factor and receptor interaction is a prerequisite for murine hepatic fibrogenesis. Gut, 2003, 52:1347-1354.
64. Vogten JM, Smakman N, Voest EE, et al. Intravital Analysis of Microcirculation in the Regenerating Mouse Liver. Journal of Surgical Research, 2003, 113:264-269.
65. Noguchi, Namisaki R, Ikenaka T, et al. Leptin-mediated neovascularization is a prerequisite for progression of nonalcoholic steatohepatitis in rats. Hepatology, 2006, 44:983-91.
66. Tsuneyama K, Sato K, Nakanuma Y, et al. A comparative histological and morphometric study of vascular changes in idiopathic portal hypertension and alcoholic fibrosis/cirrhosis. Histopathology, 2004, 43:55-61.
67. Gaudio E, Pannarale L, Onori P, Riggio O: A scanning electron microscopic study of liver microcirculation disarrangement in experimental rat cirrhosis. Hepatology, 1993, 17:477-485.
68. Gaudio E, Onori P, Pannarale L, et al. Hepatic microcirculation and peribiliary plexus in experimental biliary cirrhosis: A morphological study. Gastroenterology, 2002, 4:1118-1124.
69. Vollmar B, Siegmund S, Menger M D. An intravital fluorescence microscopic study of hepatic microvascular and cellular derangements in developing cirrhosis in rats. Hepatology, 1998, 27:1544-1553.
70. Presta M, Dell' Era P, Mitola S, et al. Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis. Cytokine & Growth Factor Reviews, 2005, 16:159-178.
71. Langer DA, Shab VH. Nitric oxide and portal hypertension: Interface of vasoreactivity and angiogenesis. Journal of Hepatology, 2005, 1-8.
72. Grunewald M, Avraham I, Dor Y, et al. VEGF-induced adult neovascularization: recruitment, retention, and role of accessory. Cell, 2006, 124:175-189.
73. Folkman J. Angiogenesis research: from laboratory to clinic. Forum (Genova), 1999, 9:59-62.
74. Lode HN, Wenkel J, and Gaedicke G. Angiogenesis, views and news. The 93rd annual meeting of the American Association for Cancer Research. April 6-10 2002, Moscone Convention Center, San Francisco, USA. Expert Opin Biol Ther, 2002, 2:671-676.
75. Cramer T, Schuppan D, Bauer M, et al. Hepatocyte growth factor and c-Met expression in rat and human liver fibrosis. Liver International, 2004, 24:335-344.
76. Won-Ii J, Kim W, Jeong SJ, et al. Hypoxia potentiates transforming growth factor-beta expression of hepatocyte during the cirrhotic condition in rat liver. Liver Int, 2004, 24: 658-668.
77. Tugues S, Fernandez-Varo G, Munoz-Luque J, et al. Antiangiogenic treatment with sunitinib ameliorates inflammatory infiltrate, fibrosis, and portal pressure in cirrhotic rats. Hepatology, 2007, 46:1919-1926.
78. Wei CL, Khoo HE, Lee KH, et al. Differential expression and localization of nitric oxide synthases in cirrhotic livers of bile duct-ligated rats. Nitric Oxide, 2002, 7: 91-102.
79. Paul C. Lee, 1 Melina R. Kibbe, Matthew J. et al. Nitric Oxide Induces Angiogenesis and Upregulates avb3 Integrin Expression on Endothelial Cells. Microvascular Research, 2000, 60:269-280.
80. Sieber CC, Sumanovski LT, Stumm M, et al. In vivo angiogenesis in normal and portal hypertensive rats: role of basic fibroblast growth factor and nitric oxide. Journal of Hepatology, 2001, 34:644-650.
81. Siitonen SM, Haapasalo HK, Rantala IS, et al. Comparison of different immunohistochemical methods in the assessment of angiogenesis: lack of prognostic value in a group of 77 selected node-negative breast carcinomas. Mod Pathol, 1995,8:745-752.
82. Untergasser G, Koeck R, Wolf D, et al. CD34+/CD133- circulating endothelial precursor cells (CEP): characterization, senescence and in vivo application. Exp Gerontol, 2006, 41:600-608.
83. van Beijnum JR, van der Linden E, and Griffioen AW. Angiogenic profiling and comparison of immortalized endothelial cells for functional genomics. Exp Cell Res, 2008, 314:264-272.
84. Antoine, M, Borkham-Kamphorst M, Kiefer E, et al. Upregulation of pleiotrophin expression in rat hepatic stellate cells by PDGF and hypoxia: implications for its role in experimental biliary liver fibrogenesis. Biochem Biophys Res Commun, 2005, 337:1153-1164.
85. Corpechot C, Barbu V, Wendum D, Kinnman N, Rey C, Poupon R, et al. Hypoxia-induced VEGF and collagen I expressions are associated with angiogenesis and fibrogenesis in experimental cirrhosis. Hepatology, 2002, 35: 1010-1021.
86. Wang YQ, Luk JM, Ikeda K, et al. Regulatory role of vHL/HIF-1a in hypoxia-induced VEGF production in hepatic stellate cells. Biochemical & Biophysical Research Communications, 2004, 317:358-362.
87. Ankoma-Sey V, Wang Y, Dai Z. Hypoxic stimulation of vascular endothelial growth factor expression in activated rat hepatic stellate cells. Hepatology, 2000, 31:141-148.
88. Abha Sahni, and Charles W. Francis Stimulation of endothelial cell proliferation by FGF-2 in the presence of fibrinogen requires avB3. Blood, 2004, 104:3635-3641.
89. Liu ZJ, Snyder R, Soma A, et al. VEGF-A and α V β 3 integrin synergistically rescue angiogenesis via N-Ras and PI3-K signaling in human microvascular endothelial cells. FASEB, 2003, 17:1931-1933.
90. Amarapurkar AD, Amarapurkar DN, Vibhav S, et al. Angiogenesis in chronic liver disease. Ann Hepatol, 2007, 6:170-173.
91. Corpechot C, Barbu V, Wendum D, et al. Hypoxia-induced VEGF and collagen I expressions are associated with angiogenesis and fibrogenesis in experimental cirrhosis. Hepatology, 2002, 35:1010-1021.
92. Bozova S and Elpek GO. Hypoxia-inducible factor-lalpha expression in experimental cirrhosis: correlation with vascular endothelial growth factor expression and angiogenesis. Apmis, 2007, 115:795-801.
93. Novo E, Cannito S, Zamara E, et al. Proangiogenic cytokines as hypoxia-dependent factors stimulating migration of human hepatic stellate cells. Am J Pathol, 2007,170:1942-1953.
94. Lai, W K, W K Adams, David H. Angiogenesis and chronic inflammation; he potential for novel therapeutic approaches in chronic liver disease. Journal of Hepatology, 2005, 42: 7-11.
95. Ng CP, Hinz B, Swartz MA, et al. Interstitial fluid flow induces myofibroblast differentiation and collagen alignment in vitro. Journal of Cell Science, 2005, 118:4731-4739.
96. Vogten JM, Drixler TA, Velde EA, et al . Angiostatin inhibits experimental liver fibrosis in mice. International Journal of Colorectal Disease, 2004, 19:387-394.
97. Angeli P, Gulini U. Perspectives in receptor research. Farmaco, 1996, 51:97-106.
98. Yokoyama K, Zhang XP, Medved L, et al. Specific binding of integrin alpha v beta 3 to the fibrinogen gamma and alpha E chain C-terminal domains. Biochemistry, 1999,38:5872-5877.
99. Quirion R and Gaudreau P. Strategies in neuropeptide receptor binding research. Neurosci Biobehav Rev, 1985, 9:413-420.
100. Wollemann M. Recent developments in the research of opioid receptor subtype molecular characterization. J Neurochem, 1990, 54:1095-1101.
101. Bitan G, Scheibler L, Greenberg Z, et al. Mapping the integrin alpha V beta 3-ligand interface by photoaffinity cross-linking. Biochemistry, 1999, 38:3414-3420.
102. Thorpe DS, Yeoman H, Chan AW, et al. Combinatorial chemistry reveals a new motif that binds the platelet fibrinogen receptor, gpIIbIIIa. Biochem Biophys Res Commun, 1999, 256:537-541.
103. Rensing S and Schrader T. The first synthetic receptor for the RGD sequence. Org Lett, 2002, 4:2161-2164.
104. Miyamoto T, Arai F, Ohneda O, et al. An adherent condition is required for formation of multinuclear osteoclasts in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor kappa B ligand. Blood, 2000, 96:4335-4343.
105. Carman-Krzan M. Radioligand-receptor binding in membrane receptor research. Prog Med Chem, 1986, 23:41-89.
106. Yokoyama K, Zhang XP, Medved L, et al. Specific binding of integrin alpha v beta 3 to the fibrinogen gamma and alpha E chain C-terminal domains. Biochemistry, 1999, 38:5872-5877.
107. Wong A, Hwang SM, McDevitt P, et al. Studies on alpha v beta 3/ligand interactions using a [3H]SK&F-107260 binding assay. Mol Pharmacol, 1996, 50:529-537.
108. Bitan G, Scheibler L, Mierke DF, et al. Ligand-integrin alpha v beta 3 interaction determined by photoaffinity cross-linking: a challenge to the prevailing model. Biochemistry, 2000, 39:11014-11023.
109. Yahalom D, Wittelsberger A, Mierke DF, et al. Identification of the principal binding site for RGD-containing ligands in the alpha(V)beta(3) integrin: a photoaffinity cross-linking study. Biochemistry, 2002, 41:8321-8331.
110. Scaffidi AK, Petrovic N, Moodley YP, et al. alpha(v)beta(3) Integrin interacts with the transforming growth factor beta (TGFbeta) type II receptor to potentiate the proliferative effects of TGFbetal in living human lung fibroblasts. J Biol Chem, 2004, 279:37726-37733.
111. Chen X, Sievers E, Hou Y, et al. Integrin alpha v beta 3-targeted imaging of lung cancer. Neoplasia, 2005, 7:271-279.
112．贺师鹏 胡雅儿 夏宗勤 等 受体研究技术 北京：北京大学医学出版社,2004.
113. Friedman S. L. Liver fibrosis-from bench to bedside. Journal of Hepatology, 2003, 38:s38-s53.
114. Bataller R, Brenner D A. Liver fibrosis. The Journal of Clinical Investigation, 2005, 115:208-218.
115. Bedossa P, Dargere D, Paradis V. Sampling variability of liver fibrosis in chronic hepatitis C. Hepatology, 2003,38:1449-1457.
116. Afdhal NH. Diagnosing fibrosis in hepatitis C: is the pendulum swinging from biopsy to blood tests ? Hepatology, 2003, 37:972-974.
117. Poynard T, Munteanu M, Imbert-Bismut F, et al. Prospective analysis of discordant results between biochemical markers and biopsy in patients with chronic hepatitis C. Clin Chem, 2004, 50:1344-1355.
118. Rosenthal-Allieria M A, Peritoreb M-L, Tranb A, et al. Analytical variability of the Fibrotest proteins. Clinical Biochemistry, 2005, 38:473-478.
119. Ziol M, Handra-Luca A, Kettaneh A, et al. Noninvasive assessment of liver fibrosis by measurement of stiffness in patients with chronic hepatitis C. Hepatology, 2005, 41:48-54.
120. Castera L, Vergniol J, Foucher J, et al. Prospective comparison of transient elastography, Fibrotest, APRI, and liver biopsy for the assessment of fibrosis in chronic hepatitis C. Gastroenterology, 2005, 128:343-350.
121. Rockey D C, Bissell M, Noninvasive measures of liver fibrosis. Hepatology, 2006, 43;S113-S120.
122. Michalet X, Pinaud FF, Bentolia LA, Tsay JM, et al. Quantum dots for live cells, in vivo imaging, and diagnostics. Science, 2005, 307:538-544.
123. Carmeliet P. Angiogenesis in health and disease. Nature medicine, 2003,9:653-660.
124. Kalluri R, Sukhatme VP. Fibrosis and angiogenesis. Current Opinion in Nephrology and Hypertension, 2000, 9:413-418.
125. Friedman SL. Cytokins and fibrogenesis. Semin Liver Dis, 1999, 19:129-140.
126. Pichler, Kneilling BJ, Haubner M, et al. Imaging of delayed-type hypersensitivity reaction by PET and 18F-galacto-RGD. Journal of nuclear medicine, 2005, 46:184-189.
127. Janssen ML, Oyen WJ, Dijkgraaf I, et al. Tumor Targeting with Radiolabeled α Vβ3 Integrin Binding Petides in a Nude Mouse Model. Cancer Research, 2002,62:6146-6151.
128. Mulder, Willem J M, Willem J M, et al. MR molecular imaging and fluorescence microscopy for identification of activated tumor endothelium using a bimodal lipidic nanoparticle. FASEB J, 2005 , Oct 4.
129. Beer, Ambros J, Ambros J, et al. Biodistribution and pharmacokinetics of the alphavbeta3-selective tracer 18F-galacto-RGD in cancer patients. J Nucl Med, 2005, 46:1333-1341.
130. Schmieder AH. Molecular MR imaging of melanoma angiogenesis with alphanubeta3-targeted paramagnetic nanoparticles. Magnetic Resonance in Medicine, 2005,53(3):621-627.
131. Achilefu, Samuel, Samuel, et al. Synergistic effects of light-emitting probes and peptides for targeting and monitoring integrin expression. Proc Natl Acad Sci U S A, 2005, 102:7976-7981.
132. arteriogenesis by contrast ultrasound molecular imaging of integrin expression. Circulation, 2005,111:3248-3254.
133. Su ZF, Liu G, Gupta S, et al. In vitro and in vivo evaluation of a Technetium-99m-labeled cyclic RGD peptide as a specific marker of alpha(V) beta(3) integrin for tumor imaging. Bioconjugate Chemistry, 2002,13:561-70.
134. Iguchi T, Sato S, Kouno Y, et al. Comparison of Tc-99m-GSA scintigraphy with hepatic f ibrosis and regeneration in patients with hepatectomy. Annals of Nuclear Medicine, 2003, 17:227-233.
135. Malhi H, Bhargava K K, Afriyie M 0, et al. 99mTc-Mebrofenin Scintigraphy for Evaluating Liver Disease in a Rat Model of Wilson' s Disease. Journal of Nuclear Medicine, 2002, 43:246-252.
1.Imai K,Sato T,Senoo H.Adhesion between Cells and Extracellular Matrixwith Special Reference to Hepatic Stellate Cells Adhesion to Three-dimensional Collagen Fibers.Cell Structure and Function,2000,25:329-336.
2.Friedman S L,Molecular Regulation of Hepatic Fibrosis,an Integrated Cellular Response to Tissue Injury.The Journal of Biological Chemistry,2000,275:2247-2250.
3.Hynes R O.Integrins:Bidirectional,Allosteric Signaling Machines.Cell,2002,110;673-687.
4.Jiang GY,Glannone G,Critchley D R,et al.Two-piconewton slip bond between fibronectin and the cytoskeleton depends on talin.Nature,2003,424:334-337.
5.Plow E F,Haas T A,Zhang L,et al.Ligand Binding to Integrins.The Journal of Biological Chemistry,2000,275:21785-21788.
6.Lee JW,Juliano R.Mitogenic Signal Transduction by intrgrin and growth Factor Receptor-mediated Pathway.Molecullars and cells,2004,17:188-202.
7.Hynes R O.Integrins:Bidirectional,Allosteric Signaling Machines.Cell,2002,110:673-687.
8.Xiong JP,Stehle T,Zhang R,et al.Crystal Structure of the Extracellular Segment of Integrin a ν β 3 in Complex with an Arg-Gly-Asp Ligand.Science,2002,296:151-154.
9.Beljaars L,Molema G,Schuppan D,et al,Successful Targeting to Rat Hepatic Stellate Cells Using Albumin Modified with Cyclic Peptides That Recognize the Collagen Type VI Receptor.The Journal of Biological Chemistry,2000,275:12743-12751.
10.Kotoh K,Nakamuta M,Kohjima M,et al.Arg-Gly-Asp(RGD) peptide ameliorates carbon tetrachloride-induced liver fibrosis via inhibition of collagen production and acceleration of collagenase activity.Int J Mol Med,2004,14:1049-i053.
11.Carloni V,Romanelli RG,Pinzani M,et al.Expression and Function of Integrin Receptors for Collagen and Laminin in Culture Human Stellate Cells.Gastroenterology,1996,110:1127-1136.
12. Zhou XY, Murphy FR, Gehdu N, et al. Engagement of αvβ3 Integrin Regulates Proliferation and Apoptosis of Hepatic Stellate Cells. The Journal of Biological Chemistry, 2004, 279:23996-24006.
13. Wu Y, Nan L, Bardag-Gorce F, et al. The role of laminin-integrin signaling in triggering MB formation. An in vivo and in vitro study. Experimental & Molecular Pathology, 2005, 79:1-8.
14. Milliano MT, Luxon BA. Initial signaling of the fibronectin receptor (a5bl integrin) in hepatic stellate cells is independent of tyrosine phosphorylation. Journal of Hepatology, 2003,39:32-37.
15. Znoyko, Reuben I, Trojanowska A, et al. Collagen binding alpha(2)beta(1) and alpha(1)beta(1) integrins play contrasting roles in regulation of Ets-l expression in human liver myofibroblasts. Mol Cell Biochem, 2005, 282:89-99.
16. Ng CP, Hinz B, Swartz MA, et al. Interstitial fluid flow induces myofibroblast differentiation and collagen alignment in vitro. Journal of Cell Science, 2005, 118:4731-4739.
17. Znoyko I Trojanowska M Reuben A. Collagen binding alpha(2)beta(1) and alpha(1)beta(1) integrins play contrasting roles in regulation of Ets-1 expression in human liver myofibroblasts. Molecular & Cellular Biochemistry, 2006, 282:89-99.
18. Liu C, Gaca MD, Swenson ES, et al. Smads 2 and 3 are differentially activated by transforming growth factor-beta (TGF-beta) in quiescent and activated hepatic stellate cells: constitutive nuclear localization of Smads in activated cells is TGF-beta-independent. The Journal of Biological Chemistry, 2003, 278:11721-11728.
19. Lygoe KA, Norman JT, Marshall JF, et al. αv integrins play an important role in myofibroblast differentiation. WOUND REP REG, 2004, 12:461-470.
20. BreitkopfK, Roeyen C, Sawitza I, et al. Expression patterns of PDGF-A, -B, -C and -D and the PDGF-receptors a and b in activated rat hepatic stellate cells (HSC). Cytokine, 2005,31:349-357.
21. Borkham-Kamphorst E, Stoll D, Gressner AM, et al. Antisense strategy against PDGF B-chain proves effective in preventing experimental liver fibrogenesis. Biochemical and Biophysical Research Communications, 2004, 321: 413-423.
22. Byzova TV, Goldman CK, Pampori N, et al. A Mechanism for Modulation of Cellular Responses to VEGF: Activation of the Integrins. Molecular Cell, 2000, 6:851-860.
23. Yoshiji H, Kuriyama S, Yoshii J, et al. Vascular endothelial growth factor and receptor interaction is a prerequisite for murine hepatic fibrogenesis. GUT, 2003, 52:1347-1352.
24. Milliano MT, Luxon B A. Initial signaling of the fibronectin receptor (a5bl integrin) in hepatic stellate cells is independent of tyrosine phosphorylation. Journal of Hepatology, 2003,39:32-37.
25. Perbal B. CCN proteins: multifunctional signalling regulators. Lancet, 2004, 363:62-64.
26. Gao R, Brigstock DR. Connective Tissue Growth Factor(CCN2) Induces Adhesion of Rat Activated Hepatic Stellate Cells by Binding of Its C-terminal Domain to Integrin avb3 and Heparan Sulfate Proteglycan. The Journal of Biological Chemistry, 2004, 279:8848-8855.
27. Campbell S, Otis M, Cote M, et al. Connection between Integrins and Cell Activation in RatAdrenal Glomerulosa Cells: A Role for Arg-Gly-Asp Peptide in the Activation of the p42/p44~(mapk) Pathway and Intracellular Calcium. Endocrinology, 2002, 144:1486-1495.
28. Ridley AJ, Schwartz MA, Burridge K, et al. Cell Migration: Integrating Signals from Front to Back. Science, 2003, 302:1704-1709.
29. Katsumi A, Naoe T, Matsushita T, et al. Integrin Activation and Matrix Binding Mediaye Cellular Responses to Mechanical Stretch. The Journal of Biological Chemistry, 2005, 280:16546-16549.
30. Patsenker E, Popov Y, Wiesner M, et al. Pharmacological inhibition of the vitronectin receptor abrogates PDGF-BB-induced hepatic stellate cell migration and activation in vitro. Journal of Hepatology, 2007, 46:878-887.
31. Yang CQ, Zeisberg M, Mosterman B, et al. Liver fibrosis: insights into migration of hepatic stellate cells in response to extracellular matrix and growth factors. Gastroenterology, 2003, 124:147-159.
32. Pozo MA, Alderson NB, Kiosses WB, et al. Integrins Regulate Rac Targeting by Internalization of Membrane Domains. Science, 2004, 303:839-842.
33. Ridley A J, Schwartz MA, Burridge K, et al. Cell Migration: Integrating Signals from Front to Back. Science, 2003, 302:1704-1709.
34. Elsharkawy A M, Oakley F, Mann D A. The role and regulation of hepatic stellate cell apoptosis in reversal of liver fibrosis. Apoptosis, 2005,10:927-939.
35. Kotoh K, Nakamuta M, Kohjima M, et al. Arg-Gly-Asp (RGD) peptide ameliorates carbon tetrachloride-induced liver fibrosis via inhibition of collagen production and acceleration of collagenase activity. International Journal of Molecular Medicine, 2004, 14:1049-1053.
1.Salcedo X,Medina J,Sanz-Cameno P,et al.The potential of angiogenesis soluble markers in chronic hepatitis C.Hepatology,2005,42:696-701.
2.Sanz-Cameno P,Martin-Vilchez S,Lara-Pezz E,et al.Heaptitis B Virus Promotes Angiopoietin-2 Expression in Liver Tissue.The American Journal of Pathology,2006,169:1217-1221.
3.Deli G,Yangm G,Makuuch S,et al.Immunohistochemical assessment of angiogenesis in hepatocellular carcinoma and surrounding cirrhotic liver tissues.World J Gastroenterol.2005,11:960-963.
4.Yoshiji H,Kuriyama S,Yoshii J,et al.Vascular endothelial growth factor and receptor interaction is a prerequisite for murine hepatic fibrogenesis.Gut,2003,52:1347-1354.
5.Vollmar B,Siegmund S,Menger M D.An intravital fluorescence microscopic study of hepatic microvascular and cellular derangements in developing cirrhosis in rats.Hepatology,1998,27:1544-1553.
6.Noguchi,Namisaki R,Ikenaka T,et al.heptin-mediated neovascularization is a prerequisite for progression of nonalcoholic steatohepatitis in rats.Hepatology,2006,44:983-91.
7.Tsuneyama K,Sato K,Nakanuma Y,et al.A comparative histological and morphometric study of vascular changes in idiopathic portal hypertension and alcoholic fibrosis/cirrhosis.Histopathology,2004,43:55-61.
8.Presta M,Dell' Era P,Mitola S,et al.Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis.Cytokine & Growth Factor Reviews,2005,16:159-178.
9.Langer DA,Shab VH.Nitric oxide and portal hypertension:Interface of vasoreactivity and angiogenesis.Journal of Hepatology,2005,1-8.
10.Grunewald M,Avraham I,Dor Y,et al.VEGF-induced adult neovascularization:recruitment,retention,and role of accessory.Cell,2006,124:175-189.
11.Cramer T,Schuppan D,Bauer M,et al.Hepatocyte growth factor and c-Met expression in rat and human liver fibrosis. Liver International, 2004, 24:335-344.
12. Won-Ii J, Kim W, Jeong SJ, et al. Hypoxia potentiates transforming growth factor-beta expression of hepatocyte during the cirrhotic condition in rat liver. Liver Int, 2004, 24: 658-668.
13. Schlueter C, Weber H, Meyer B, et al. Angiogenetic signaling through hypoxia HMGB1: An angiogenetic switch molecule. Am J Pathol, 2005, 166:1259-1263.
14. Antoine, M, Borkham-Kamphorst M, Kiefer E, et al. Upregulation of pleiotrophin expression in rat hepatic stellate cells by PDGF and hypoxia: implications for its role in experimental biliary liver fibrogenesis. Biochem Biophys Res Commun, 2005, 337:1153-1164.
15. Corpechot C, Barbu V, Wendum D, Kinnman N, Rey C, Poupon R, et al. Hypoxia-induced VEGF and collagen I expressions are associated with angiogenesis and fibrogenesis in experimental cirrhosis. Hepatology, 2002, 35: 1010-1021.
16. Wang YQ, Luk JM, Ikeda K, et al. Regulatory role of vHL/HIF-1a in hypoxia-induced VEGF production in hepatic stellate cells. Biochemical & Biophysical Research Communications, 2004, 317:358-362.
17. Ankoma-Sey V, Wang Y, Dai Z. Hypoxic stimulation of vascular endothelial growth factor expression in activated rat hepatic stellate cells. Hepatology, 2000, 31:141-148.
18. Abha Sahni, and Charles W. Francis Stimulation of endothelial cell proliferation by FGF-2 in the presence of fibrinogen requires αvβ3. Blood, 2004, 104:3635-3641.
19. Liu ZJ, Snyder R, Soma A, et al. VEGF-A and α Vβ 3 integrin synergistically rescue angiogenesis via N-Ras and PI3-K signaling in human microvascular endothelial cells. FASEB, 2003, 17:1931-1933.
20. Zhou XY, Murphy FR, Gehdu N, et al. Engagement of αvβ3 Integrin Regulates Proliferation and Apoptosis of Hepatic Stellate Cells. The Journal of Biological Chemistry, 2004, 279:23996-24006.
21. Wei CL, Khoo HE, Lee KH, et al. Differential expression and localization of nitric oxide synthases in cirrhotic livers of bile duct-ligated rats. Nitric Oxide, 2002, 7: 91-102.
22. Paul C. Lee,l Melina R. Kibbe, Matthew J. et al. Nitric Oxide Induces Angiogenesis and Upregulates avb3 Integrin Expression on Endothelial Cells. Microvascular Research, 2000, 60:269-280.
23. Sieber CC, Sumanovski LT, Stumm M, et al. In vivo angiogenesis in normal and portal hypertensive rats: role of basic fibroblast growth factor and nitric oxide. Journal of Hepatology, 2001, 34:644-650.
24. Yoshiji, Hitoshi, Hitoshi, et al. Angiotensin-I-converting enzyme inhibitors may be an alternative anti-angiogenic strategy in the treatment of liver fibrosis and hepatocellular carcinoma. Possible role of vascular endothelial growth factor. Tumour Biol, 2002, 23:348-356.
25. Vogten JM, Smakman N, Voest EE, et al. Intravital Analysis of Microcirculation in the Regenerating Mouse Liver. Journal of Surgical Research, 2003, 113:264-269.
26. Antoine M, Borkham-Kamphorst M, Kiefe Er, et al. Upregulation of pleiotrophin expression in rat hepatic stellate cells by PDGF and hypoxia: implications for its role in experimental biliary liver fibrogenesis. Biochem Biophys Res Commun, 2005, 337: 1153-1164.
27. Onori P, Morim S, Franchitto A, et al. Hepatic microvascular features in experimental cirrhosis: a structural and morphometrical study in CC14-treated rats. Journal of Hepatology, 2000, 33:555-563.
28. Langer DA, Shab VH. Nitric oxide and portal hypertension: Interface of vasoreactivity and angiogenesis. Journal of Hepatology, 2005, 1-8.
29. Fernandez M, Mejias M, Angermayr B, et al. Inhibition of VEGF receptor-2 decreases the development of hyperdynamic splanchnic circulation and portal-systemic collateral vessels in portal hypertensive rats. Journal of Hepatology, 2005, 43:98-103.
30. Moreau R. VEGF-induced angiogenesis drives collateral circulation in portal hypertension. Journal of Hepatology, 2005, 43:6-8.
31. Fernandez M, Vizzutti F, Garcia-Pagan JC, et al. Anti-VEGF receptor-2 monoclonal antibody prevents portal-systemic collateral vessel formation in portal hypertensive mice. Gastroenterology, 2004, 126:886-894.
32. Mitsuhashi N, Shiraizu H, Ohtsuka M, et al. Angiopoietins and Tie-2 expression in angiogenesis and proliferation of human hepatocellular carcinoma. Hepatology, 2003, 37: 1105-1113.
33. Yoshiji H, Kuriyama S, Noguchi R, et al. Combination of vitamin K2 and the angiotensin-converting enzyme inhibitor, perindopril, attenuates the liver enzyme-altered preneoplastic lesions in rats via angiogenesis suppression. Journal of Hepatology, 2005, 42: 687-693.
34. Lai, W K, W K Adams, David H. Angiogenesis and chronic inflammation; the potential for novel therapeutic approaches in chronic liver disease. Journal of Hepatology, 2005, 42: 7-11.
35. Medinal J, Sanz-Camenol P, Garcl'a-Buey L, et al. Evidence of angiogenesis in primary biliary cirrhosis: an immunohistochemical descriptive study. Journal of Hepatology, 2005, 42:124-131
36. Issbrücke K, Marti HH, Hippenstiel S, et al. p38 MAP Kinase, a molecular switch between VEGF induced angiogenesis and vascular hyperpermeability. FASEB, 2002, 16:1928-1930.
37. Ng CP, Hinz B, Swartz MA, et al. Interstitial fluid flow induces myofibroblast differentiation and collagen alignment in vitro. Journal of Cell Science, 2005, 118:4731-4739.
38. Vogten JM, Drixler TA, Velde EA, et al . Angiostatin inhibits experimental liver fibrosis in mice. International Journal of Colorectal Disease, 2004, 19:387-394.
2. Hendriks HF, Verhoofstad WA, Brouwer A, et al. Perisinusiodal fat-storing cells are the main vitamin A storage sites in rat liver. Exp.Cell Res, 1985, 160:138-149.
3. Geerts A. History, hereogeneity, developmental biology, and functions of quiescent hepatic stellate cells. Sem Liv.Dis, 2001,21:311-335.
4. Friedman SL. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J. Biol. Chem, 275,2247-2250.
5. Moreira RK. Hepatic stellate cells and liver fibrosis. Arch Pathol Lab Med, 2007, 131:1728-1734.
6. Moreira RK. Hepatic stellate cells and liver fibrosis. Arch Pathol Lab Med, 2007, 131:1728-1734.
7. E. Wisse and D. L Knook Editors. Kupffer cells and other liver sinusoidal cells. Elsevier/North-Holl, 2nd edition 1979 printed in the Netherlands.
8. Brenner DA, Waterboer T, Choi SK, et al. New aspects of hepatic fibrosis. J Hepatol, 2000, 32: 32-38.
9. Friedman SL. Cellular sources of collagen and regulation of collagen production in liver. Semin Liver Dis, 1990,10:20-29.
10. Friedman SL, Roll FJ. Isolation and culture of hepatic lipocytes, Kupffer cells, and sinusoidal endothelial cells by density gradient centrifugation with Stractan. Anal Biochem. 1987,161:207-218.
11. Blomhoff R, Berg T. Isolation and cultivation of rat liver stellate cells. Meth Enzymol, 1990, 190:58-71.
12. Geerts A, Niki T, Hellemans K, et al. Purification of rat hepatic stellate cells by side scatter-activated cell sorting. Hepatology, 1998, 27:590-59.
13. Ballardini G, Groff P, Badiali de Giorgi L, Schuppan D, et al. Ito cell heterogeneity: desmin negative Ito cells in normal rat liver. Hepatology, 1994, 19:440-446.
14. Hynes RO. Integrins: Versatility Modulation, and Signaling in Cell Adhesion. Cell, 1992,69:11-25.
15. Imai K, Sato T, Senoo H. Adhesion between C ells and Extracellular Matrix with Special Reference to Hepatic Stellate Cells Adhesion to Three-dimensional Collagen Fibers. Cell Structure and Function, 2000, 25:329-336.
16. Humphries MJ. Integrin Structure. Biochemical Society, 2000, 28:311-340.
17. Eng FJ, Friedman SL. Fibrogenesis I. New insights into hepatic stellate cell activation: the simple becomes complex. Am J Physiol Gastrointest Liver Physiol, 2000, 279: G7-G11.
18. Friedman SL, Molecular Regulation of Hepatic Fibrosis, an Integrated Cellular Response to Tissue Injury. J. Biol. Chem, 2000, 275;2247-2250.
19. Sohara N, Znoyko I, Levy MT, et al. Reversal of activation of human myofibroblast-like cells by culture on a basement membrane-like substrate, J. Hepatol, 2002,37:125-129.
20. Hynes R 0. Integrins: Bidirectional, Allosteric Signaling Machines. Cell,2002,110;673-687.
21. Plow E F, Haas T A, Zhang L, et al. Ligand Binding to Integrins. J. Biol. Chem, 2000, 275:21785-21788.
22. Znoyko I Trojanowska M Reuben A. Collagen binding alpha(2)beta(1) and alpha(1)beta(1) integrins play contrasting roles in regulation of Ets-l expression in human liver myofibroblasts. Molecular & Cellular Biochemistry, 2006, 282:89-99.
23. BreitkopfK, Roeyen C, Sawitza I, et al. Expression patterns of PDGF-A, -B, -C and -D and the PDGF-receptors a and b in activated rat hepatic stellate cells (HSC). Cytokine, 2005, 31:349-357.
24. Liu C, Gaca MD, Swenson ES, et al. Smads 2 and 3 are differentially activated by transforming growth factor-beta (TGF-beta) in quiescent and activated hepatic stellate cells: constitutive nuclear localization of Smads in activated cells is TGF-beta-independent. J. Biol. Chem, 2003, 278:11721-11728.
25. Borkham-Kamphorst E, Stoll D, Gressner AM, et al. Antisense strategy against PDGF B-chain proves effective in preventing experimental liver fibrogenesis. Biochemical and Biophysical Research Communications, 2004, 321: 413-423.
26. Byzova TV, Goldman CK, Pampori N, et al. A Mechanism for Modulation of Cellular Responses to VEGF: Activation of the Integrins. Molecular Cell, 2000, 6:851-860.
27. Gao R, Brigstock DR. Connective Tissue Growth Factor(CCN2) Induces Adhesion of Rat Activated Hepatic Stellate Cells by Binding of Its C-terminal Domain to Integrin avb3 and Heparan Sulfate Proteglycan. J. Biol. Chem, 2004, 279:8848-8855.
28. Liu ZJ, Snyder R, Soma A, et al. VEGF-A and αVβ3 integrin synergistically rescue angiogenesis via N-Ras and PI3-K signaling in human microvascular endothelial cells. FASEB, 2003, 17:1931-1933.
29. Ridley A J, Schwartz MA, BurridgeK, et al. Cell Migration: Integrating Signals from Front to Back. Science, 2003, 302:1704-1709.
30. Carloni V, Romanelli RG, Pinzani M, et al. Expression and Function of Integrin Receptors for Collagen and Laminin in Culture Human Stellate Cells. Gastroenterology, 1996, 110:1127-1136.
31. Yang CQ, ZeisbergM, Mosterman B, et al. Liver fibrosis: insights into migration of hepatic stellate cells in response to extracellular matrix and growth factors. Gastroenterology, 2003, 124:147-159.
32 Carloni V, Romanelli RG, Pinzani M, et al. Expression and Function of Integrin Receptors for Collagen and Laminin in Culture Human Stellate Cells. Gastroenterology, 1996, 110:1127-1136.
33 Zhou XY, Murphy FR, Gehdu N, et al. Engagement of αvβ3 Integrin Regulates Proliferation and Apoptosis of Hepatic Stellate Cells. J. Biol. Chem, 2004, 279:23996-24006.
34 Wu Y, Nan L, Bardag-Gorce F, et al. The role of laminin-integrin signaling in triggering MB formation. An in vivo and in vitro study. Experimental & Molecular Pathology, 2005, 79:1-8.
35. Iwamoto H, Sakai H, Nawata H, et al. Inhibition of integrin signaling with Arg-Gly-Asp motifs in rat hepatic stellate cells. J. Hepatol, 1998,29:752-759.
36. Patsenker E, Popov Y, Wiesner M, et al. Pharmacological inhibition of the vitronectin receptor abrogates PDGF-BB-induced hepatic stellate cell migration and activation in vitro. Journal of Hepatology, 2007, 46:878-887.
37. Horton MA. The avb3 integrin "Vitronectin Receptor" Int. J. Biochem. Cell Biol, 1997, 29:721-725.
38. Cluzel C, Saltel F, Lussi J, et al. The mechanisms and dynamics of (alpha) v(beta)3 integrin clustering in living cells. J. Biol. Chem, 2005, 171:383-392.
39. Lygoe KA, Norman JT, Marshall JF, et al. αv integrins play an important role inmyofibroblast differentiation. Wound Rep Reg, 2004, 12:461-470.
40. Kunicki TJ, Ely KR, Kunicki TC, et al. The exchange of Arg-Gly-Asp (RGD) and Arg-Tyr-Asp (RYD) binding sequences in a recombinant murine Fab fragment specific for the integrin alpha IIb beta 3 does not alter integrin recognition. J Biol Chem, 1995, 270:16660-16665.
41. Craig D, Gao M, Schulten K, et al. Structural insights into how the MIDAS ion stabilizes integrin binding to an RGD peptide under force. Structure, 2004, 12:2049-2058.
42. Yahalom D, Wittelsberger A, Mierke DF, et al. Identification of the principal binding site for RGD-containing ligands in the alpha(V)beta(3) integrin: a photoaffinity cross-linking study. Biochemistry, 2002, 41:8321-8331.
43. van Well RM, Overkleeft HS, van der Marel GA, et al. Solid-phase synthesis of cyclic RGD-furanoid sugar amino acid peptides as integrin inhibitors. Bioorg Med Chem Lett, 2003, 13:331-334.
44. Sadeghi MM, Krassilnikova S, Zhang JS, et al. Detection of Injury-Induced Vascular Remodeling by Targeting Activated αvβ3 integrin In Vivo. Circulation, 2004,110:84-90.
45. Matsuno H, Stassen JM, Vermylen J, et al. Inhibition of integrin function by a cyclic RGD-containing peptide prevents neointima formation. Circulation, 1994, 90:2203-2206.
46. Chen X, PlasenciaC, Hou Y, et al. Synthesis and biological evaluation of dimeric RGD peptide-paclitaxel conjugate as a model for integrin-targeted drug delivery. J Med Chem, 2005, 48:1098-1106.
47. Wu Y, Zhang X, Xiong Z, et al. microPET imaging of glioma integrin {alpha}v{beta}3 expression using (64)Cu-labeled tetrameric RGD peptide. J Nucl Med, 2005, 46:1707-1718.
48. Mogford JE, Davis GE, Platts SH, et al. Vascular smooth muscle alpha v beta 3 integrin mediates arteriolar vasodilation in response to RGD peptides. Circ Res, 1996, 79:821-826.
49. Smith JW and Cheresh DA. Integrin (alpha v beta 3)-ligand interaction. Identification of a heterodimeric RGD binding site on the vitronectin receptor. J Biol Chem, 1990, 265:2168-2172.
50. Wu Y, Cai W, and Chen X. Near-Infrared Fluorescence Imaging of Tumor Integrin alpha(v)beta (3) Expression with Cy7-Labeled RGD Multimers. Mol Imaging Biol, 2006.
51. Wang W, McMurray JS, Wu Q, et al. Convenient Solid-Phase Synthesis of Diethylenetriaminepenta-Acetic Acid (DTPA)-conjugated Cyclic RGD Peptide Analogues. Cancer Biotherapy & Radiopharmaceuticals, 2005, 20:547-556.
52. Liu, Radiolabeled multimeric cyclic RGD peptides as integrin alpha v beta3 targeted radiotracers for tumor imaging, Mol. Pharm, 2006, 3:472-487.
53. Marinelli L, Lavecchia A, Gottsechalk KE, et al. Docking Studies on αvβ3 Integrin Ligands: Pharmacophore Refinement and Implications for Drug Design. J. Med. Chem, 2003, 46:4393-4404.
54. Goodman SL, Holzemann G, Sulyok GAG, et al. Nanomolar Small Molecule Inhibitors for avp6 avp5 αvβ3 Integrins. J. Med. Chem, 2002, 45:1045-1051.
55. Wang WW, McMurray, John S L, et al. Probing for integrin alpha v beta3 binding of RGD peptides using fluorescence polarization. Bioconjug Chem, 2005, 16: 729-734.
56. Wang W, McMurray JS, Wu Q, et al. Convenient Solid-Phase Synthesis of Diethylenetriaminepenta-Acetic Acid (DTPA)-conjugated Cyclic RGD Peptide Analogues. Cancer Biotherapy & Radiopharmaceuticals, 2005, 20:547-556.
57. Line BR, Mitra A, Nan A, et al. Targeting tumor angiogenesis: Comparison of peptide and polymer-peptide conjugates. J Nucl Med , 2005, 46: 1552-1560.
58. Xiong JP, Stehle T, Diefenbach B, et al. Crystal structure of the Extracellular Segment of Integrin αvβ3. Science, 2001,294:339-345.
59. Xiong JP, Stehle T, Zhang R, et al. Crystal structure of the Extracellular Segment of Integrin αvβ3 in Complex with an Arg-Gly-Asp Ligand. Science, 2002,296:151-154.
60. Salcedo X, Medina J, Sanz-Cameno P, et al. The potential of angiogenesis soluble markers in chronic hepatitis C. Hepatology, 2005, 42:696-701.
61. Sanz-Cameno P, Martin-Vilchez S, Lara-Pezz E, et al. Heaptitis B Virus Promotes Angiopoietin-2 Expression in Liver Tissue. The American Journal of Pathology, 2006, 169:1217-1221.
62. Deli G, Yangm G, Makuuch S, et al. Immunohistochemical assessment of angiogenesis in hepatocellular carcinoma and surrounding cirrhotic liver tissues. World J Gastroenterol, 2005, 11: 960-963.
63. Yoshiji H, Kuriyama S, Yoshii J, et al. Vascular endothelial growth factor and receptor interaction is a prerequisite for murine hepatic fibrogenesis. Gut, 2003, 52:1347-1354.
64. Vogten JM, Smakman N, Voest EE, et al. Intravital Analysis of Microcirculation in the Regenerating Mouse Liver. Journal of Surgical Research, 2003, 113:264-269.
65. Noguchi, Namisaki R, Ikenaka T, et al. Leptin-mediated neovascularization is a prerequisite for progression of nonalcoholic steatohepatitis in rats. Hepatology, 2006, 44:983-91.
66. Tsuneyama K, Sato K, Nakanuma Y, et al. A comparative histological and morphometric study of vascular changes in idiopathic portal hypertension and alcoholic fibrosis/cirrhosis. Histopathology, 2004, 43:55-61.
67. Gaudio E, Pannarale L, Onori P, Riggio O: A scanning electron microscopic study of liver microcirculation disarrangement in experimental rat cirrhosis. Hepatology, 1993, 17:477-485.
68. Gaudio E, Onori P, Pannarale L, et al. Hepatic microcirculation and peribiliary plexus in experimental biliary cirrhosis: A morphological study. Gastroenterology, 2002, 4:1118-1124.
69. Vollmar B, Siegmund S, Menger M D. An intravital fluorescence microscopic study of hepatic microvascular and cellular derangements in developing cirrhosis in rats. Hepatology, 1998, 27:1544-1553.
70. Presta M, Dell' Era P, Mitola S, et al. Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis. Cytokine & Growth Factor Reviews, 2005, 16:159-178.
71. Langer DA, Shab VH. Nitric oxide and portal hypertension: Interface of vasoreactivity and angiogenesis. Journal of Hepatology, 2005, 1-8.
72. Grunewald M, Avraham I, Dor Y, et al. VEGF-induced adult neovascularization: recruitment, retention, and role of accessory. Cell, 2006, 124:175-189.
73. Folkman J. Angiogenesis research: from laboratory to clinic. Forum (Genova), 1999, 9:59-62.
74. Lode HN, Wenkel J, and Gaedicke G. Angiogenesis, views and news. The 93rd annual meeting of the American Association for Cancer Research. April 6-10 2002, Moscone Convention Center, San Francisco, USA. Expert Opin Biol Ther, 2002, 2:671-676.
75. Cramer T, Schuppan D, Bauer M, et al. Hepatocyte growth factor and c-Met expression in rat and human liver fibrosis. Liver International, 2004, 24:335-344.
76. Won-Ii J, Kim W, Jeong SJ, et al. Hypoxia potentiates transforming growth factor-beta expression of hepatocyte during the cirrhotic condition in rat liver. Liver Int, 2004, 24: 658-668.
77. Tugues S, Fernandez-Varo G, Munoz-Luque J, et al. Antiangiogenic treatment with sunitinib ameliorates inflammatory infiltrate, fibrosis, and portal pressure in cirrhotic rats. Hepatology, 2007, 46:1919-1926.
78. Wei CL, Khoo HE, Lee KH, et al. Differential expression and localization of nitric oxide synthases in cirrhotic livers of bile duct-ligated rats. Nitric Oxide, 2002, 7: 91-102.
79. Paul C. Lee, 1 Melina R. Kibbe, Matthew J. et al. Nitric Oxide Induces Angiogenesis and Upregulates avb3 Integrin Expression on Endothelial Cells. Microvascular Research, 2000, 60:269-280.
80. Sieber CC, Sumanovski LT, Stumm M, et al. In vivo angiogenesis in normal and portal hypertensive rats: role of basic fibroblast growth factor and nitric oxide. Journal of Hepatology, 2001, 34:644-650.
81. Siitonen SM, Haapasalo HK, Rantala IS, et al. Comparison of different immunohistochemical methods in the assessment of angiogenesis: lack of prognostic value in a group of 77 selected node-negative breast carcinomas. Mod Pathol, 1995,8:745-752.
82. Untergasser G, Koeck R, Wolf D, et al. CD34+/CD133- circulating endothelial precursor cells (CEP): characterization, senescence and in vivo application. Exp Gerontol, 2006, 41:600-608.
83. van Beijnum JR, van der Linden E, and Griffioen AW. Angiogenic profiling and comparison of immortalized endothelial cells for functional genomics. Exp Cell Res, 2008, 314:264-272.
84. Antoine, M, Borkham-Kamphorst M, Kiefer E, et al. Upregulation of pleiotrophin expression in rat hepatic stellate cells by PDGF and hypoxia: implications for its role in experimental biliary liver fibrogenesis. Biochem Biophys Res Commun, 2005, 337:1153-1164.
85. Corpechot C, Barbu V, Wendum D, Kinnman N, Rey C, Poupon R, et al. Hypoxia-induced VEGF and collagen I expressions are associated with angiogenesis and fibrogenesis in experimental cirrhosis. Hepatology, 2002, 35: 1010-1021.
86. Wang YQ, Luk JM, Ikeda K, et al. Regulatory role of vHL/HIF-1a in hypoxia-induced VEGF production in hepatic stellate cells. Biochemical & Biophysical Research Communications, 2004, 317:358-362.
87. Ankoma-Sey V, Wang Y, Dai Z. Hypoxic stimulation of vascular endothelial growth factor expression in activated rat hepatic stellate cells. Hepatology, 2000, 31:141-148.
88. Abha Sahni, and Charles W. Francis Stimulation of endothelial cell proliferation by FGF-2 in the presence of fibrinogen requires avB3. Blood, 2004, 104:3635-3641.
89. Liu ZJ, Snyder R, Soma A, et al. VEGF-A and α V β 3 integrin synergistically rescue angiogenesis via N-Ras and PI3-K signaling in human microvascular endothelial cells. FASEB, 2003, 17:1931-1933.
90. Amarapurkar AD, Amarapurkar DN, Vibhav S, et al. Angiogenesis in chronic liver disease. Ann Hepatol, 2007, 6:170-173.
91. Corpechot C, Barbu V, Wendum D, et al. Hypoxia-induced VEGF and collagen I expressions are associated with angiogenesis and fibrogenesis in experimental cirrhosis. Hepatology, 2002, 35:1010-1021.
92. Bozova S and Elpek GO. Hypoxia-inducible factor-lalpha expression in experimental cirrhosis: correlation with vascular endothelial growth factor expression and angiogenesis. Apmis, 2007, 115:795-801.
93. Novo E, Cannito S, Zamara E, et al. Proangiogenic cytokines as hypoxia-dependent factors stimulating migration of human hepatic stellate cells. Am J Pathol, 2007,170:1942-1953.
94. Lai, W K, W K Adams, David H. Angiogenesis and chronic inflammation; he potential for novel therapeutic approaches in chronic liver disease. Journal of Hepatology, 2005, 42: 7-11.
95. Ng CP, Hinz B, Swartz MA, et al. Interstitial fluid flow induces myofibroblast differentiation and collagen alignment in vitro. Journal of Cell Science, 2005, 118:4731-4739.
96. Vogten JM, Drixler TA, Velde EA, et al . Angiostatin inhibits experimental liver fibrosis in mice. International Journal of Colorectal Disease, 2004, 19:387-394.
97. Angeli P, Gulini U. Perspectives in receptor research. Farmaco, 1996, 51:97-106.
98. Yokoyama K, Zhang XP, Medved L, et al. Specific binding of integrin alpha v beta 3 to the fibrinogen gamma and alpha E chain C-terminal domains. Biochemistry, 1999,38:5872-5877.
99. Quirion R and Gaudreau P. Strategies in neuropeptide receptor binding research. Neurosci Biobehav Rev, 1985, 9:413-420.
100. Wollemann M. Recent developments in the research of opioid receptor subtype molecular characterization. J Neurochem, 1990, 54:1095-1101.
101. Bitan G, Scheibler L, Greenberg Z, et al. Mapping the integrin alpha V beta 3-ligand interface by photoaffinity cross-linking. Biochemistry, 1999, 38:3414-3420.
102. Thorpe DS, Yeoman H, Chan AW, et al. Combinatorial chemistry reveals a new motif that binds the platelet fibrinogen receptor, gpIIbIIIa. Biochem Biophys Res Commun, 1999, 256:537-541.
103. Rensing S and Schrader T. The first synthetic receptor for the RGD sequence. Org Lett, 2002, 4:2161-2164.
104. Miyamoto T, Arai F, Ohneda O, et al. An adherent condition is required for formation of multinuclear osteoclasts in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor kappa B ligand. Blood, 2000, 96:4335-4343.
105. Carman-Krzan M. Radioligand-receptor binding in membrane receptor research. Prog Med Chem, 1986, 23:41-89.
106. Yokoyama K, Zhang XP, Medved L, et al. Specific binding of integrin alpha v beta 3 to the fibrinogen gamma and alpha E chain C-terminal domains. Biochemistry, 1999, 38:5872-5877.
107. Wong A, Hwang SM, McDevitt P, et al. Studies on alpha v beta 3/ligand interactions using a [3H]SK&F-107260 binding assay. Mol Pharmacol, 1996, 50:529-537.
108. Bitan G, Scheibler L, Mierke DF, et al. Ligand-integrin alpha v beta 3 interaction determined by photoaffinity cross-linking: a challenge to the prevailing model. Biochemistry, 2000, 39:11014-11023.
109. Yahalom D, Wittelsberger A, Mierke DF, et al. Identification of the principal binding site for RGD-containing ligands in the alpha(V)beta(3) integrin: a photoaffinity cross-linking study. Biochemistry, 2002, 41:8321-8331.
110. Scaffidi AK, Petrovic N, Moodley YP, et al. alpha(v)beta(3) Integrin interacts with the transforming growth factor beta (TGFbeta) type II receptor to potentiate the proliferative effects of TGFbetal in living human lung fibroblasts. J Biol Chem, 2004, 279:37726-37733.
111. Chen X, Sievers E, Hou Y, et al. Integrin alpha v beta 3-targeted imaging of lung cancer. Neoplasia, 2005, 7:271-279.
112．贺师鹏 胡雅儿 夏宗勤 等 受体研究技术 北京：北京大学医学出版社,2004.
113. Friedman S. L. Liver fibrosis-from bench to bedside. Journal of Hepatology, 2003, 38:s38-s53.
114. Bataller R, Brenner D A. Liver fibrosis. The Journal of Clinical Investigation, 2005, 115:208-218.
115. Bedossa P, Dargere D, Paradis V. Sampling variability of liver fibrosis in chronic hepatitis C. Hepatology, 2003,38:1449-1457.
116. Afdhal NH. Diagnosing fibrosis in hepatitis C: is the pendulum swinging from biopsy to blood tests ? Hepatology, 2003, 37:972-974.
117. Poynard T, Munteanu M, Imbert-Bismut F, et al. Prospective analysis of discordant results between biochemical markers and biopsy in patients with chronic hepatitis C. Clin Chem, 2004, 50:1344-1355.
118. Rosenthal-Allieria M A, Peritoreb M-L, Tranb A, et al. Analytical variability of the Fibrotest proteins. Clinical Biochemistry, 2005, 38:473-478.
119. Ziol M, Handra-Luca A, Kettaneh A, et al. Noninvasive assessment of liver fibrosis by measurement of stiffness in patients with chronic hepatitis C. Hepatology, 2005, 41:48-54.
120. Castera L, Vergniol J, Foucher J, et al. Prospective comparison of transient elastography, Fibrotest, APRI, and liver biopsy for the assessment of fibrosis in chronic hepatitis C. Gastroenterology, 2005, 128:343-350.
121. Rockey D C, Bissell M, Noninvasive measures of liver fibrosis. Hepatology, 2006, 43;S113-S120.
122. Michalet X, Pinaud FF, Bentolia LA, Tsay JM, et al. Quantum dots for live cells, in vivo imaging, and diagnostics. Science, 2005, 307:538-544.
123. Carmeliet P. Angiogenesis in health and disease. Nature medicine, 2003,9:653-660.
124. Kalluri R, Sukhatme VP. Fibrosis and angiogenesis. Current Opinion in Nephrology and Hypertension, 2000, 9:413-418.
125. Friedman SL. Cytokins and fibrogenesis. Semin Liver Dis, 1999, 19:129-140.
126. Pichler, Kneilling BJ, Haubner M, et al. Imaging of delayed-type hypersensitivity reaction by PET and 18F-galacto-RGD. Journal of nuclear medicine, 2005, 46:184-189.
127. Janssen ML, Oyen WJ, Dijkgraaf I, et al. Tumor Targeting with Radiolabeled α Vβ3 Integrin Binding Petides in a Nude Mouse Model. Cancer Research, 2002,62:6146-6151.
128. Mulder, Willem J M, Willem J M, et al. MR molecular imaging and fluorescence microscopy for identification of activated tumor endothelium using a bimodal lipidic nanoparticle. FASEB J, 2005 , Oct 4.
129. Beer, Ambros J, Ambros J, et al. Biodistribution and pharmacokinetics of the alphavbeta3-selective tracer 18F-galacto-RGD in cancer patients. J Nucl Med, 2005, 46:1333-1341.
130. Schmieder AH. Molecular MR imaging of melanoma angiogenesis with alphanubeta3-targeted paramagnetic nanoparticles. Magnetic Resonance in Medicine, 2005,53(3):621-627.
131. Achilefu, Samuel, Samuel, et al. Synergistic effects of light-emitting probes and peptides for targeting and monitoring integrin expression. Proc Natl Acad Sci U S A, 2005, 102:7976-7981.
132. arteriogenesis by contrast ultrasound molecular imaging of integrin expression. Circulation, 2005,111:3248-3254.
133. Su ZF, Liu G, Gupta S, et al. In vitro and in vivo evaluation of a Technetium-99m-labeled cyclic RGD peptide as a specific marker of alpha(V) beta(3) integrin for tumor imaging. Bioconjugate Chemistry, 2002,13:561-70.
134. Iguchi T, Sato S, Kouno Y, et al. Comparison of Tc-99m-GSA scintigraphy with hepatic f ibrosis and regeneration in patients with hepatectomy. Annals of Nuclear Medicine, 2003, 17:227-233.
135. Malhi H, Bhargava K K, Afriyie M 0, et al. 99mTc-Mebrofenin Scintigraphy for Evaluating Liver Disease in a Rat Model of Wilson' s Disease. Journal of Nuclear Medicine, 2002, 43:246-252.
1.Imai K,Sato T,Senoo H.Adhesion between Cells and Extracellular Matrixwith Special Reference to Hepatic Stellate Cells Adhesion to Three-dimensional Collagen Fibers.Cell Structure and Function,2000,25:329-336.
2.Friedman S L,Molecular Regulation of Hepatic Fibrosis,an Integrated Cellular Response to Tissue Injury.The Journal of Biological Chemistry,2000,275:2247-2250.
3.Hynes R O.Integrins:Bidirectional,Allosteric Signaling Machines.Cell,2002,110;673-687.
4.Jiang GY,Glannone G,Critchley D R,et al.Two-piconewton slip bond between fibronectin and the cytoskeleton depends on talin.Nature,2003,424:334-337.
5.Plow E F,Haas T A,Zhang L,et al.Ligand Binding to Integrins.The Journal of Biological Chemistry,2000,275:21785-21788.
6.Lee JW,Juliano R.Mitogenic Signal Transduction by intrgrin and growth Factor Receptor-mediated Pathway.Molecullars and cells,2004,17:188-202.
7.Hynes R O.Integrins:Bidirectional,Allosteric Signaling Machines.Cell,2002,110:673-687.
8.Xiong JP,Stehle T,Zhang R,et al.Crystal Structure of the Extracellular Segment of Integrin a ν β 3 in Complex with an Arg-Gly-Asp Ligand.Science,2002,296:151-154.
9.Beljaars L,Molema G,Schuppan D,et al,Successful Targeting to Rat Hepatic Stellate Cells Using Albumin Modified with Cyclic Peptides That Recognize the Collagen Type VI Receptor.The Journal of Biological Chemistry,2000,275:12743-12751.
10.Kotoh K,Nakamuta M,Kohjima M,et al.Arg-Gly-Asp(RGD) peptide ameliorates carbon tetrachloride-induced liver fibrosis via inhibition of collagen production and acceleration of collagenase activity.Int J Mol Med,2004,14:1049-i053.
11.Carloni V,Romanelli RG,Pinzani M,et al.Expression and Function of Integrin Receptors for Collagen and Laminin in Culture Human Stellate Cells.Gastroenterology,1996,110:1127-1136.
12. Zhou XY, Murphy FR, Gehdu N, et al. Engagement of αvβ3 Integrin Regulates Proliferation and Apoptosis of Hepatic Stellate Cells. The Journal of Biological Chemistry, 2004, 279:23996-24006.
13. Wu Y, Nan L, Bardag-Gorce F, et al. The role of laminin-integrin signaling in triggering MB formation. An in vivo and in vitro study. Experimental & Molecular Pathology, 2005, 79:1-8.
14. Milliano MT, Luxon BA. Initial signaling of the fibronectin receptor (a5bl integrin) in hepatic stellate cells is independent of tyrosine phosphorylation. Journal of Hepatology, 2003,39:32-37.
15. Znoyko, Reuben I, Trojanowska A, et al. Collagen binding alpha(2)beta(1) and alpha(1)beta(1) integrins play contrasting roles in regulation of Ets-l expression in human liver myofibroblasts. Mol Cell Biochem, 2005, 282:89-99.
16. Ng CP, Hinz B, Swartz MA, et al. Interstitial fluid flow induces myofibroblast differentiation and collagen alignment in vitro. Journal of Cell Science, 2005, 118:4731-4739.
17. Znoyko I Trojanowska M Reuben A. Collagen binding alpha(2)beta(1) and alpha(1)beta(1) integrins play contrasting roles in regulation of Ets-1 expression in human liver myofibroblasts. Molecular & Cellular Biochemistry, 2006, 282:89-99.
18. Liu C, Gaca MD, Swenson ES, et al. Smads 2 and 3 are differentially activated by transforming growth factor-beta (TGF-beta) in quiescent and activated hepatic stellate cells: constitutive nuclear localization of Smads in activated cells is TGF-beta-independent. The Journal of Biological Chemistry, 2003, 278:11721-11728.
19. Lygoe KA, Norman JT, Marshall JF, et al. αv integrins play an important role in myofibroblast differentiation. WOUND REP REG, 2004, 12:461-470.
20. BreitkopfK, Roeyen C, Sawitza I, et al. Expression patterns of PDGF-A, -B, -C and -D and the PDGF-receptors a and b in activated rat hepatic stellate cells (HSC). Cytokine, 2005,31:349-357.
21. Borkham-Kamphorst E, Stoll D, Gressner AM, et al. Antisense strategy against PDGF B-chain proves effective in preventing experimental liver fibrogenesis. Biochemical and Biophysical Research Communications, 2004, 321: 413-423.
22. Byzova TV, Goldman CK, Pampori N, et al. A Mechanism for Modulation of Cellular Responses to VEGF: Activation of the Integrins. Molecular Cell, 2000, 6:851-860.
23. Yoshiji H, Kuriyama S, Yoshii J, et al. Vascular endothelial growth factor and receptor interaction is a prerequisite for murine hepatic fibrogenesis. GUT, 2003, 52:1347-1352.
24. Milliano MT, Luxon B A. Initial signaling of the fibronectin receptor (a5bl integrin) in hepatic stellate cells is independent of tyrosine phosphorylation. Journal of Hepatology, 2003,39:32-37.
25. Perbal B. CCN proteins: multifunctional signalling regulators. Lancet, 2004, 363:62-64.
26. Gao R, Brigstock DR. Connective Tissue Growth Factor(CCN2) Induces Adhesion of Rat Activated Hepatic Stellate Cells by Binding of Its C-terminal Domain to Integrin avb3 and Heparan Sulfate Proteglycan. The Journal of Biological Chemistry, 2004, 279:8848-8855.
27. Campbell S, Otis M, Cote M, et al. Connection between Integrins and Cell Activation in RatAdrenal Glomerulosa Cells: A Role for Arg-Gly-Asp Peptide in the Activation of the p42/p44~(mapk) Pathway and Intracellular Calcium. Endocrinology, 2002, 144:1486-1495.
28. Ridley AJ, Schwartz MA, Burridge K, et al. Cell Migration: Integrating Signals from Front to Back. Science, 2003, 302:1704-1709.
29. Katsumi A, Naoe T, Matsushita T, et al. Integrin Activation and Matrix Binding Mediaye Cellular Responses to Mechanical Stretch. The Journal of Biological Chemistry, 2005, 280:16546-16549.
30. Patsenker E, Popov Y, Wiesner M, et al. Pharmacological inhibition of the vitronectin receptor abrogates PDGF-BB-induced hepatic stellate cell migration and activation in vitro. Journal of Hepatology, 2007, 46:878-887.
31. Yang CQ, Zeisberg M, Mosterman B, et al. Liver fibrosis: insights into migration of hepatic stellate cells in response to extracellular matrix and growth factors. Gastroenterology, 2003, 124:147-159.
32. Pozo MA, Alderson NB, Kiosses WB, et al. Integrins Regulate Rac Targeting by Internalization of Membrane Domains. Science, 2004, 303:839-842.
33. Ridley A J, Schwartz MA, Burridge K, et al. Cell Migration: Integrating Signals from Front to Back. Science, 2003, 302:1704-1709.
34. Elsharkawy A M, Oakley F, Mann D A. The role and regulation of hepatic stellate cell apoptosis in reversal of liver fibrosis. Apoptosis, 2005,10:927-939.
35. Kotoh K, Nakamuta M, Kohjima M, et al. Arg-Gly-Asp (RGD) peptide ameliorates carbon tetrachloride-induced liver fibrosis via inhibition of collagen production and acceleration of collagenase activity. International Journal of Molecular Medicine, 2004, 14:1049-1053.
1.Salcedo X,Medina J,Sanz-Cameno P,et al.The potential of angiogenesis soluble markers in chronic hepatitis C.Hepatology,2005,42:696-701.
2.Sanz-Cameno P,Martin-Vilchez S,Lara-Pezz E,et al.Heaptitis B Virus Promotes Angiopoietin-2 Expression in Liver Tissue.The American Journal of Pathology,2006,169:1217-1221.
3.Deli G,Yangm G,Makuuch S,et al.Immunohistochemical assessment of angiogenesis in hepatocellular carcinoma and surrounding cirrhotic liver tissues.World J Gastroenterol.2005,11:960-963.
4.Yoshiji H,Kuriyama S,Yoshii J,et al.Vascular endothelial growth factor and receptor interaction is a prerequisite for murine hepatic fibrogenesis.Gut,2003,52:1347-1354.
5.Vollmar B,Siegmund S,Menger M D.An intravital fluorescence microscopic study of hepatic microvascular and cellular derangements in developing cirrhosis in rats.Hepatology,1998,27:1544-1553.
6.Noguchi,Namisaki R,Ikenaka T,et al.heptin-mediated neovascularization is a prerequisite for progression of nonalcoholic steatohepatitis in rats.Hepatology,2006,44:983-91.
7.Tsuneyama K,Sato K,Nakanuma Y,et al.A comparative histological and morphometric study of vascular changes in idiopathic portal hypertension and alcoholic fibrosis/cirrhosis.Histopathology,2004,43:55-61.
8.Presta M,Dell' Era P,Mitola S,et al.Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis.Cytokine & Growth Factor Reviews,2005,16:159-178.
9.Langer DA,Shab VH.Nitric oxide and portal hypertension:Interface of vasoreactivity and angiogenesis.Journal of Hepatology,2005,1-8.
10.Grunewald M,Avraham I,Dor Y,et al.VEGF-induced adult neovascularization:recruitment,retention,and role of accessory.Cell,2006,124:175-189.
11.Cramer T,Schuppan D,Bauer M,et al.Hepatocyte growth factor and c-Met expression in rat and human liver fibrosis. Liver International, 2004, 24:335-344.
12. Won-Ii J, Kim W, Jeong SJ, et al. Hypoxia potentiates transforming growth factor-beta expression of hepatocyte during the cirrhotic condition in rat liver. Liver Int, 2004, 24: 658-668.
13. Schlueter C, Weber H, Meyer B, et al. Angiogenetic signaling through hypoxia HMGB1: An angiogenetic switch molecule. Am J Pathol, 2005, 166:1259-1263.
14. Antoine, M, Borkham-Kamphorst M, Kiefer E, et al. Upregulation of pleiotrophin expression in rat hepatic stellate cells by PDGF and hypoxia: implications for its role in experimental biliary liver fibrogenesis. Biochem Biophys Res Commun, 2005, 337:1153-1164.
15. Corpechot C, Barbu V, Wendum D, Kinnman N, Rey C, Poupon R, et al. Hypoxia-induced VEGF and collagen I expressions are associated with angiogenesis and fibrogenesis in experimental cirrhosis. Hepatology, 2002, 35: 1010-1021.
16. Wang YQ, Luk JM, Ikeda K, et al. Regulatory role of vHL/HIF-1a in hypoxia-induced VEGF production in hepatic stellate cells. Biochemical & Biophysical Research Communications, 2004, 317:358-362.
17. Ankoma-Sey V, Wang Y, Dai Z. Hypoxic stimulation of vascular endothelial growth factor expression in activated rat hepatic stellate cells. Hepatology, 2000, 31:141-148.
18. Abha Sahni, and Charles W. Francis Stimulation of endothelial cell proliferation by FGF-2 in the presence of fibrinogen requires αvβ3. Blood, 2004, 104:3635-3641.
19. Liu ZJ, Snyder R, Soma A, et al. VEGF-A and α Vβ 3 integrin synergistically rescue angiogenesis via N-Ras and PI3-K signaling in human microvascular endothelial cells. FASEB, 2003, 17:1931-1933.
20. Zhou XY, Murphy FR, Gehdu N, et al. Engagement of αvβ3 Integrin Regulates Proliferation and Apoptosis of Hepatic Stellate Cells. The Journal of Biological Chemistry, 2004, 279:23996-24006.
21. Wei CL, Khoo HE, Lee KH, et al. Differential expression and localization of nitric oxide synthases in cirrhotic livers of bile duct-ligated rats. Nitric Oxide, 2002, 7: 91-102.
22. Paul C. Lee,l Melina R. Kibbe, Matthew J. et al. Nitric Oxide Induces Angiogenesis and Upregulates avb3 Integrin Expression on Endothelial Cells. Microvascular Research, 2000, 60:269-280.
23. Sieber CC, Sumanovski LT, Stumm M, et al. In vivo angiogenesis in normal and portal hypertensive rats: role of basic fibroblast growth factor and nitric oxide. Journal of Hepatology, 2001, 34:644-650.
24. Yoshiji, Hitoshi, Hitoshi, et al. Angiotensin-I-converting enzyme inhibitors may be an alternative anti-angiogenic strategy in the treatment of liver fibrosis and hepatocellular carcinoma. Possible role of vascular endothelial growth factor. Tumour Biol, 2002, 23:348-356.
25. Vogten JM, Smakman N, Voest EE, et al. Intravital Analysis of Microcirculation in the Regenerating Mouse Liver. Journal of Surgical Research, 2003, 113:264-269.
26. Antoine M, Borkham-Kamphorst M, Kiefe Er, et al. Upregulation of pleiotrophin expression in rat hepatic stellate cells by PDGF and hypoxia: implications for its role in experimental biliary liver fibrogenesis. Biochem Biophys Res Commun, 2005, 337: 1153-1164.
27. Onori P, Morim S, Franchitto A, et al. Hepatic microvascular features in experimental cirrhosis: a structural and morphometrical study in CC14-treated rats. Journal of Hepatology, 2000, 33:555-563.
28. Langer DA, Shab VH. Nitric oxide and portal hypertension: Interface of vasoreactivity and angiogenesis. Journal of Hepatology, 2005, 1-8.
29. Fernandez M, Mejias M, Angermayr B, et al. Inhibition of VEGF receptor-2 decreases the development of hyperdynamic splanchnic circulation and portal-systemic collateral vessels in portal hypertensive rats. Journal of Hepatology, 2005, 43:98-103.
30. Moreau R. VEGF-induced angiogenesis drives collateral circulation in portal hypertension. Journal of Hepatology, 2005, 43:6-8.
31. Fernandez M, Vizzutti F, Garcia-Pagan JC, et al. Anti-VEGF receptor-2 monoclonal antibody prevents portal-systemic collateral vessel formation in portal hypertensive mice. Gastroenterology, 2004, 126:886-894.
32. Mitsuhashi N, Shiraizu H, Ohtsuka M, et al. Angiopoietins and Tie-2 expression in angiogenesis and proliferation of human hepatocellular carcinoma. Hepatology, 2003, 37: 1105-1113.
33. Yoshiji H, Kuriyama S, Noguchi R, et al. Combination of vitamin K2 and the angiotensin-converting enzyme inhibitor, perindopril, attenuates the liver enzyme-altered preneoplastic lesions in rats via angiogenesis suppression. Journal of Hepatology, 2005, 42: 687-693.
34. Lai, W K, W K Adams, David H. Angiogenesis and chronic inflammation; the potential for novel therapeutic approaches in chronic liver disease. Journal of Hepatology, 2005, 42: 7-11.
35. Medinal J, Sanz-Camenol P, Garcl'a-Buey L, et al. Evidence of angiogenesis in primary biliary cirrhosis: an immunohistochemical descriptive study. Journal of Hepatology, 2005, 42:124-131
36. Issbrücke K, Marti HH, Hippenstiel S, et al. p38 MAP Kinase, a molecular switch between VEGF induced angiogenesis and vascular hyperpermeability. FASEB, 2002, 16:1928-1930.
37. Ng CP, Hinz B, Swartz MA, et al. Interstitial fluid flow induces myofibroblast differentiation and collagen alignment in vitro. Journal of Cell Science, 2005, 118:4731-4739.
38. Vogten JM, Drixler TA, Velde EA, et al . Angiostatin inhibits experimental liver fibrosis in mice. International Journal of Colorectal Disease, 2004, 19:387-394.
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