经动脉栓塞术联合Sunitinib治疗肝癌的实验研究
摘要
第一部分经动脉栓塞术治疗肝癌前后血管生成相关因子表达的变化
目的:
研究经动脉栓塞术治疗肝癌前后血管生成相关因子表达或功能状态的变化。
材料与方法:
取肝癌经动脉化疗栓塞术(TACE)后行二期手术切除者(二期组)43例和肝癌直接行手术切除者(直接组)20例,手术标本制作病理切片,免疫组化方法检测残癌及癌旁组织的血管内皮生长因子受体-2(VEGFR-2、KDR)、磷酸化VEGFR-2(p VEGFR-2)、血小板衍生性生长因子受体β(PDGFR-β)、缺氧诱导因子1α(HIF-1α)和微血管密度(MVD,CD3]标记),比较两组阳性染色的平均光密度(DM)及MVD计数。
用大鼠肝癌细胞株McA-RH7777及Buffalo大鼠制作大鼠肝癌模型,行肝动脉插管注入碘化油(经动脉栓塞组,TAE组)或注入生理盐水(对照组)。分别在术前6h、术后1h、6h、24h、3d和7d行平扫和动态增强MRI检查,测量肿瘤大小和最大增强斜率(IS)百分比;并分别在术前6h、术后6h、3d和7d取肿瘤组织行免疫组化法(IHC)或(/和)蛋白免疫杂交印迹法(WB)检查MVD(CD31标记)、HIF-1α、VEGFR-2、p VEGFR-2、PDGFR-p和磷酸化PDGFR-β(p PDGFR-β)。
经软件SPSS 11.0作统计分析。
结果:
临床病例检查示二期组肝癌组织均见大片坏死区,有6例肿瘤完全坏死。二期组残癌组织p VEGFR-2和HIF-1α的DM为(0.03376±0.01648)、(0.04705±0.02128),直接组分别为(0.02352±0.00893)、(0.03532±0.01623),两组差异均有显著性(P值均＜0.05);二期组癌旁组织VEGFR-2、p VEGFR-2和HIF-1α的DM分别为(0.03962±0.01680)、(0.03064±0.01138)和(0.03749±0.01517),直接组则分别为(0.03022±0.01481)、(0.02010±0.00827)和(0.02383±0.01390),两组差异均有显著性(P值均＜0.05)。两组残癌及癌旁组织PDGFR-β差异无显著性(P＞0.05)。二期组癌旁组织MVD为(58.3±15.2),直接组为(44.4±10.5),两组差异有显著性(P＜0.001)。
动物实验中,TAE组肿瘤体积稍小于对照组,两组差异无显著性(P＞0.05)。MRI示两组肿瘤内信号基本均匀,仅TAE组肿瘤内见少量小灶状坏死;TAE组和对照组的IS百分比,两组差异有显著性(F=430.2,P＜0.001),两两比较示术后1h、6h、24h及3d与对照组差异均有统计学意义(P＜0.05)。IHC检测两组MVD差异无统计学意义(U=59.0,P＞0.05);两组HIF-1α和p VEGFR-2差异有统计学意义(U=22.0、26.0,P均＜0.05)。VEGFR-2和PDGFR-β两组间差异无统计学意义(U=46.0、48.0,P均＞0.05)。WB检测TAE组p VEGFR-2和p PDGFR-β在术前呈低表达,术后6小时、3天较术前和对照组明显增高,术后7天时表达基本正常或略增高;对照组p VEGFR-2和p PDGFR-β术后表达与术前无明显差异;两组VEGFR-2和PDGFR-β在各个时间点差异不明显。
结论:
1、人肝癌经TACE治疗后肝癌坏死明显,残癌和癌旁组织缺氧程度加重,VEGFR-2、PDGFR-β术前后变化不显著,但功能(磷酸化状态)明显增强;
2、大鼠肝癌经TAE治疗后短期内血供减少,肿瘤组织缺氧程度加重,VEGFR-2、PDGFR-β等受体功能明显增强。
第二部分Sunitinib抑制肝癌细胞及血管内皮细胞的体外实验
目的:
研究Sunitinib对肝癌细胞和血管内皮细胞的抑制作用。
材料和方法:
取人肝癌细胞株Hep 3B、Hep G2、大鼠肝癌细胞株McA-RH7777、正常人肝细胞株L-02和人脐静脉内皮细胞(HUVEC),用WST-1法检测血管内皮生长因子-165(VEGF-165)、血小板衍生性生长因子-BB(PDGF-BB)和Sunitinib分别对它们的促增殖或毒性作用;PI单染法流式细胞术检测Sunitinib对各细胞株的促凋亡作用;细胞划痕试验检测Sunitinib对HUVEC的迁移抑制作用;细胞免疫荧光法(IF)和免疫蛋白杂交印迹法(WB)检测各细胞株VEGFR-2、pVEGFR-2、PDGFR-p和p PDGFR-β的表达,以及不同浓度Sunitinib对它们的抑制作用。经软件SPSS 11.0作统计分析。
结果:
VEGF和PDGF浓度分别在25ng/mL以上,对HUVEC有明显促增殖作用(F=8.953、9.731,P值均等于0.001)。(VEGF+PDGF)对人肝癌细胞HepG2、Hep3B、大鼠肝癌细胞McA-RH7777及人正常肝细胞L-02促增殖作用不明显(HepG2组F=0.385、Hep3B组F=0.322、McA-RH7777组F=2.949、L-02组F=0.673,P值均大于0.05)。Sunitinib对HUVEC有显著的抑制作用(F=41.003,P＜0.001);两两比较示浓度在0.01μM及其以上时,对HUVEC有明显的抑制作用(P＜0.05)。在Hep G2和McA-RH7777中,Sunitinib各浓度间差异有统计学意义(F=3.367、4.518,P＜0.05),其中10μM组与0μM组差异显著(P＜0.05)。划痕试验示Sunitinib组在划痕后,划痕未缩小,至24h、48h时渐增宽。流式细胞术检测HUVEC在Sunitinib浓度0.1μM时,凋亡率为19.2%:在1μM时,凋亡率达36.7%。Hep G2、Hep 3B及McA-RH7777在SU11248浓度1μM时,凋亡率分别为9.8%、9.2%、14.5%;在10μM时,凋亡率分别为15.6%、16.3%、19.5%。HUVEC、HepG2和McA-RH7777均表达VEGFR-2和PDGFR-β,加Sunitinib后磷酸化明显受到抑制,且与剂量呈正相关。
结论:
1.Sunitinib能强烈地抑制血管内皮细胞增殖并促进其凋亡,是sunitinib发挥抗肿瘤作用的主要机制;
2.Sunitinib在高浓度下对肝癌细胞也有一定的抑制作用,可能通过非肿瘤血管依赖性途径起作用。
第三部分高效液相色谱法检测Sunitinib不同给药途径药代动力学差异
目的:
建立高效液相色谱法,检测Sunitinib经口服和肝动脉途径给药在不同时相各器官药物分布差异。
材料和方法:
采用内标法(Chrysin为内标),应用高效液相色谱紫外检测大鼠血浆和肝组织中Sunitinib的浓度,建立标准曲线并验证方法的准确度、精密度和最低定量浓度等。
将大鼠肝癌模型分成Sunitinib口服组(口服组,连续灌胃)与TAE+动脉Sunitinib组(TAE组,单次Sunitinib 10mg/Kg与碘油乳化),口服组分别在首次给药后1、6、24、30、48小时及3、5、7、9、14天取血和肝组织样本;TAE组在TAE术后0.5、1、6、12、24、30、48小时及3、5、7、9、14天取血和肝组织样本,分别检测Sunitinib浓度。
结果:
应用本法检测血和肝组织样本Sunitinib浓度的标准曲线,回归方程分别为Y=2197.3 C-2.2907(血),Y=4723.5 C+33.246(肝);根据质控浓度检测得出血浆的准确度在各个浓度中为(-6.4～-4.4)%,天内精密度为(5.4～10.6)%,天间精密度(3.1～9.5)%;肝组织样品的准确度在各个浓度中为(-8.0～3.1)%,天内精密度为(11.4～13.3)%,天间精密度(1.5～2.4)%。准确度与精密度均≤15%,方法稳定可靠。血浆样品最低定量浓度为20ng/ml,肝组织样品最低定量浓度为25ng/ml。
口服组血和肝组织样本约在首次给药后6-10小时达峰值,6小时时测血药浓度约140ng/mL,肝组织浓度约200ng/g;血中半衰期(T_(1/2))约6-12小时。重复给药后,约于6小时再达峰值,每隔24小时检测,直至第9天时血药浓度在(30-70)ng/mL,肝组织浓度在(100-130)ng/g。连续10天给药后,第14天检测时血药浓度已降至检测下限以下,肝组织浓度在45ng/g左右。
TAE组血和肝组织样本在术后30分钟即达峰值,30分钟时一个血样本浓度超出检测上限;至术后24-30小时,血药浓度在(30-40)ng/mL,48小时以后血样本均处于检测下限以下。肝组织浓度则在术后6小时前均超出检测上限(1500ng/g),以后肝组织浓度缓慢下降(相对于血药浓度),至术后第5天在100ng/g左右,第9天及以后降至检测下限以下。
结论:
1.建立高效液相色谱紫外检测法,检测血及肝组织样本Sunitinib浓度,方法简单稳定,能满足动物实验的设计要求;
2.比较Sunitinib经口服途径和TAE途径的药物代谢动力学差异,为其联合TAE治疗肝癌提供药理学依据。
第四部分Sunitinib联合经肝动脉栓塞术治疗肝癌的实验研究
目的:
观察经动脉栓塞术(TAE)联合运用Sunitinib治疗肝癌的疗效,并探讨新生血管变化及分子水平的机制。
材料和方法:
用大鼠肝癌细胞株McA-RH7777和Buffalo大鼠建立大鼠肝癌模型,随机分成五组:生理盐水组(A组)、TAE组(B组)、TAE+动脉Sunitinib组(C组)、TAE+口服Sunitinib组(D组)、TAE+动脉、口服Sunitinib组(E组)。观察大鼠生存时间、肿瘤体积等:各组分别于术后第1周、第2周、第4周及第8周分别作MRI平扫和动态增强;同时取肿瘤组织,大体观察后作IHC及WB检测MVD(CD31标记)、HIF-1α、VEGFR-2、p VEGFR-2、PDGFR-β和p PDGFR-β。经软件SPSS 11.0作统计分析。
结果:
各组间大鼠生存期差异有统计学意义(X~2=10.8,P＜0.05),E组比A组延长最明显(与A组,X~2=5.9,P＜0.05)。各组肿瘤增大差异明显(F4.149,P＜0.001),与A组相比,C、D组术后肿瘤生长明显减缓(术后一周、术后二周时,P均＜0.05);E组在术后一周、二周时肿瘤无明显生长(与A～D组,P坊＜0.05),个别大鼠肿瘤略有缩小。MRI示C～E组较对照组坏死的范围明显增大。最大增强斜率(initial slope,IS)百分比各组间差异明显(F=3.311,P＜0.001),E组减弱最明显(与A组,P＜-0.001)。IHC示MVD各组间差异明显(F=33.005,P＜0.001),C-E组MVD在术后一周、二周时较术前和对照组减少。HIF-1α各组间差异有统计学意义(X~2=22.5,P＜0.001),C、D、E组表达比A组明显增强(P＜0.05)。VEGFR-2 C～E组在术后一周、二周时均较A组有不同程度的增加,组间差异有统计意义(X~2=23.2,P＜0.001)。C～E组p VEGFR-2在术后一、二周却受到明显的抑制,在术后四周开始增强,组间差异有统计学意义(X~2=9.38,P＜0.05)。PDGFR-β各组间有统计学差异(X~2=13.3,P＜0.05),E组表达最强且持续时间较长。WB示C～E组术后第7天、14天VEGFR-2和PDGFR-β的磷酸化水平受到明显抑制,C组在术后第14天的抑制程度不及D组,而E组在第7天、14天时受体的磷酸化水平抑制程度明显强于C、D组:三组各时间点的VEGFR-2和PDGFR-β表达差异不显著。
结论:
1.Sunitinib与TAE联合治疗肝癌,阻断多种促血管生成因子受体,抑制新生血管生成,提高了TAE的疗效;
2.Sunitinib经动脉途径乳化后给药,在短期(一周)内更有效地抑制肝癌的新生血管形成;
3.TAE术中单剂量Sunitinib经动脉给药,结合术后口服给药,抗肿瘤新生血管生成作用强大、持久,对肝癌治疗效果更佳。
Part 1 Expression of angiogenesis-related factors and receptors after the treatment of hepatocellular carcinoma with trans-catheter arterial embolization(TAE)
Objective
To study the expression of angiogenesis-related factors and receptors after the treatment of hepatocellular carcinoma(HCC) with trans-catheter arterial embolization (TAE).
Materials and methods
43 cases of second-stage surgery of HCC after TACE(second-stage group) and 20 of direct surgery(direct surgery group) were taken and made into paraffin-embedded slices.Vascular endothelial growth factor receptor(VEGFR-2),phosphorylated VEGFR-2(p VEGFR-2),platelet-derived growth factor receptor-β(PDGFR-β), hypoxia induced factor-1α(HIF-1α) and microvessel density(MVD)(labeled by CD31)were detected with the immunohistochemical method.
Rat HCC models,which were established with rat HCC line McA-RH7777 and Buffalo rats,were divided into 2 groups:trans-catheter arterial embolization with lipiodol(TAE group) or with saline(control group).Tumor volume and initial slope(IS) percent were measured with MRI at different times:6 hours before,1 hour after,6 hours after,24 hours after,3 days after and 7 days after surgery.MVD(labeled by CD31),HIF-1α,VEGFR-2,p VEGFR-2,PDGFR-βand p PDGFR-βof tumor were also detected with IHC and/or western blotting(WB).
All data were processed by SPSS 11.0.
Results
In clinical samples,The necrosis of cancerous tissue was obvious in second-stage group with 6 cases of complete necrosis.The density means(DM) of phosphorylated VEGFR-2 and HIF-1αin the cancerous tissue of HCC were(0.03376±0.01648) and(0.04705±0.02128) in second-stage group,and(0.02352±0.00893) and (0.03532±0.01623) in direct-surgery group,which showed statistical difference.The DMs of VEGFR-2,phosphorylated VEGFR-2 and HIF-1αin the peri-cancerous tissue were(0.03962±0.01680),(0.03064±0.01138) and(0.03749±0.01517) respectively in direct-surgery group,and(0.03022±0.01481),(0.02010±0.00827) and(0.02383±0.01390) respectively in direct-surgery group,all of which showed statistical difference.MVD of peri-cancerous tissue was(58.3±15.2) in second-stage group and(44.4±10.5) in direct-surgery group,both of which were different statistically.
In the rat samples,tumor volume of TAE group was slightly smaller than control group,but it showed no significant difference(P＞0.05).Tumors' signal of both groups were almost even in MRI,while some dot-like necrosis were found in TAE group.The difference of initial slope(IS) percent between two groups were statistically significant(F=430.2,P＜0.001),and IS percent of TAE group at 1hours, 6hours,24hours and 3 days were all lower than control group significantly.MVD of TAE group was not significantly different than control group(U=59.0,P＞0.05). Differences of HIF-1αand p VEGFR-2 between two groups were significant statistically(U=22.0,26.0,both P value less than 0.05).VEGFR-2 and PDGFR-βshowed no significant difference between two groups(U=46.0、48.0,both P value more than 0.05).All of the above were detected with IHC.Expression of p VEGFR-2 and p PDGFR-β,detected with WB,were higher at 6 hours,3 days after surgery than before surgery and control group.Expressions of them in control group were not significantly different between before and after surgery.VEGFR-2 and PDGFR-βin both groups showed no significant difference between every two times.
Conclusions
1.Human HCC after TACE has obvious necrosis,but the cancerous and peri-cancerous tissues are more hypoxic and both the expression and function of VEGFR-2 enhanced.
2.Rat HCC after TAE shows less blood supply in short period,and the tumor is more hypoxic,and eventually its function of VEGFR-2 and PDGFR-βenhanced.
Part 2 Sunitinib suppresses hepatocellular carcinoma(HCC) cells and vascular endothelial cell in vitro
Obejective
To study the suppression of HCC cells and vascular endothelial cell(VEC) with sunitinib,a multi-targeted receptor tyrosine kinase(RTK) inhibitor.
Materials and methods
Several cell lines were used in this part including human HCC lines(Hep G2, Hep 3B),rat HCC line(McA-RH7777),human umbilical vein endothelial cell(HUVEC) and human normal liver cell(L-02).WST-1 method was applied to detect the proliferation or toxicity of VEGF-165,PDGF-BB and sunitinib for these cells.The apoptosis of cells with sunitinib was also detected with PI staining method by flow cytometry.Cell scratch test was used to find the movement inhibition of sunitinib for HUVEC.Cell immunofluorescence(IF) and western blotting(WB) were applied to detect the inhibition of VEGFR-2、p VEGFR-2、PDGFR-βand p PDGFR-βwith sunitinib in different concentration.
All data were processed by SPSS 11.0.
Results
VEGF and PDGF promoted the proliferation of HUVEC at 25ng/mL and above(F=8.953、9.731,P=0.001),while they didn't show such effect for HepG2,Hep 3B,McA-RH7777 and L-02.Sunitinib suppressed the proliferation of HUVEC signigicantly at 0.01μM and above(F=41.003,P＜0.001),while it also suppressed Hep G2 and McA-RH7777 significantly at 10μM(F=3.367、4.518,both P value less than 0.05).Scratch test showed HUVEC didn't cross the scratch after incubation with sunitinib.Flow cytometry revealed the apoptosis of HUVEC was 19.2%at 0.1μM of sunitinib,36.7%at 1μM,which of Hep G2,Hep 3B and McA-RH7777 were 9.8%,9.2%and 14.5%at 1μM,and 15.6%,16.3%and 19.5%at 10μM respectively.HUVEC,Hep G2 and McA-RH7777 demonstrated the expression of VEGFR-2、p VEGFR-2、PDGFR-βand p PDGFR-β.After incubation with sunibinib,the phosphorylation of both receptors were suppressed,and furthmore it's correlated with the dose.
Conclusions
1.Sunitinib suppresses the proliferation of HUVEC and promotes its apoptosis, which is the main mechanism of anti-angiogenesis.
2.Sunitinib also shows some kind of inhibition for HCC cells at high concentration,which non-anti-angiogenesis way may play a role in.
Part 3
High performance liquid chromatography(HPLC) detects sunitinib pharmacokinetics(PK) difference between different administration methods
Obejective
To set up a method of HPLC to detect the concentration of sunitinib in blood and liver samples,and study the pharmacokinetics(PK) difference between oral and arterial administration.
Materials and methods
HPLC with ultra-violet(420nm and 268nm) detected the sunitinib concentration in rat blood and liver samples using chrysin as internal standard.Standard curve and its linear scope were set up and their accuracy,precision and minimum limit of quantitation were validated.
Rat HCC models were randomly divided into sunitinib oral administration group(oral group,sunitinib 10mg/kg,10 days continually) and TAE with sunitinib group(TAE group,sunitinib 10mg/kg signally with lipiodol).Blood and liver samples were harvested at 1,6,24,30,48 hours and 3,5,7,9 and 14 days after first dose in oral group,while at 0.5,1,6,12,24,30,48 hours and 3,5,7,9 and 14 days after TAE in TAE group.All samples were detected by the validated methods.
Results
The standard curves were set up for blood and liver samples,which regression equation was y=2197.3×C-2.2907 for blood and y=4723.5×C+33.246 for liver. With the detection of quality concentrations,accuracy at different concentrations was (-6.4～-4.4)%,in-day precision was(5.4～10.6)%,and between-day precision was (3.1～9.5)%in blood samples.Accuracy at different concentrations was(-8.0～3.1) %,in-day precision was(11.4～13.3)%,and between-day precision was(1.5～2.4) %in liver samples.All accuracy and precision were under 15%,which demonstrate the method was stable and reliable.The minimum limit of quantification was 20ng/ml for blood and 25ng/ml for liver.
In oral group,the concentration of sunitinib in blood and liver was peaking within 6～10 hours.The concentration at 6 hours after first dose was about 140ng/ml in blood,and 200ng/ml in liver.Its half-life period(T_(1/2)) was about 6-12 hours in blood.Peak concentration was gotten at about 6 hours after repeated dose.Then detected at every 24 hours after administration,blood concentration was(30-70)ng/ml and liver concentration was(100-130)ng/g at 9 days after the first dose.At 14 days, blood concentration was under the minimum limit and liver concentration was about 45ng/g.
In TAE group,the concentration was peaking at about 30 minutes after TAE in blood and liver.The concentration in blood was(30-40)ng/ml at 24-30 hours after TAE,and under minimum limit at 48 hours and after.The concentration at 6 hours and before in liver were all above the maximum limit of qualification(1500ng/g). Then it decreased slowly comparing to blood concentration,was about 100ng/g at 5 days and under the minimum limit at 9 days and after.
Conclusions
1.The methods of HPLC to detect the concentration of sunitinib in blood and liver samples are stable and reliable,which can satisfy this experiment.
2.The PK difference of sunitinib between oral and arterial administration provides the pharmacological basis for the combination of sunitinib and TAE.
Part 4 Treatment of hepatocellular carcinoma(HCC) with the combination of sunitinib and trans-catheter arterial embolization(TAE) in rat models
Obejective
To investigate the effect of the combination of TAE and sunitinib for HCC,and study the mechanism of angiogenesis and molecular level.
Materials and methods
Rat HCC models,established with McA-RH7777 and Buffalo strait rats,were divided randomly into five groups:saline group(group A,control group),TAE group(group B),TAE+sunitinib iA group(group C),TAE+sunitinib p.o.group(group D) and TAE+sunitinib iA&p.o,group(group E).Rat survival time and tumor volume were evaluated.MRI plain scan and dynamic enhancement were applied for five groups at 1 week,2 weeks,4 weeks and 8 weeks after surgery and at the same time, some rats were sacrificed for the detection of MVD(labeled by CD31),HIF-1α、 VEGFR-2、p VEGFR-2、PDGFR-βand p PDGFR-βwith IHC and/or WB.All data were processed by SPSS 11.0.
Results
Survival time among five groups were different statistically(X~2=10.8,P＜0.05),in which E group was longest(verses A group,X~2=5.9,P＜0.05).Tumor volume was also different statistically(F=4.149,P＜0.001).Among them,tumors in C,D group were retarded(verses A group,both P value less than 0.05),and ones in E group were found little growth(verse A～D group,all P value less than 0.05),some of which even decreased.
MRI demonstrated that the scope of necrosis increased obviously.IS percent among groups was different significantly(F=3.311,P＜0.001),in which E group decreased mostly(verse A group,P＜0.001).MVD in C～E group was reduced at 1 week,2 weeks after surgery with comparison to before surgery and A group,which among groups was different(F=33.005,P＜0.001).HIF-1αin C～E group increased more than A group,which among groups showed significant difference(X~2=22.5,P＜0.001).VEGFR-2 in C～E group at 1 week,2 weeks after surgery also increased at different grade,which difference in groups was significant(X~2=23.2,P＜0.001).But p VEGFR-2 in C～E group at 1 week,2 weeks after surgery was inhibited significantly(X~2=9.38,P＜0.05).PDGFR-βamong groups also had significant difference(X~2=13.3,P＜0.05),among which it in E group expressed strongly and persistently.WB showed that the phosphorylation of VEGFR-2 and PDGFR-βwas inhibited significantly.Among them,the inhibition of phosphorylation in E group at 7 and 14 days was more obvious than C and D group.However,VEGFR-2 and PDGFR-βwere not different significantly.
Conclusions:
1.Sunitinib,with combination of TAE for treatment of HCC,inhibits several angiogenesis-related receptors,suppresses angiogenesis,and enhances the effect of TAE.
2.Sunitinib,through arterial administration after emulsification with lipiodol,can suppress angiogenesis of HCC more effectively in short period.
3.Sunitinib with the combination of arterial administration after emulsification with lipiodol and oral administration continually,can perform more strong and persistent effect of anti-angiogenesis,which is more effective with the combination with TAE for HCC.
目的:
研究经动脉栓塞术治疗肝癌前后血管生成相关因子表达或功能状态的变化。
材料与方法:
取肝癌经动脉化疗栓塞术(TACE)后行二期手术切除者(二期组)43例和肝癌直接行手术切除者(直接组)20例,手术标本制作病理切片,免疫组化方法检测残癌及癌旁组织的血管内皮生长因子受体-2(VEGFR-2、KDR)、磷酸化VEGFR-2(p VEGFR-2)、血小板衍生性生长因子受体β(PDGFR-β)、缺氧诱导因子1α(HIF-1α)和微血管密度(MVD,CD3]标记),比较两组阳性染色的平均光密度(DM)及MVD计数。
用大鼠肝癌细胞株McA-RH7777及Buffalo大鼠制作大鼠肝癌模型,行肝动脉插管注入碘化油(经动脉栓塞组,TAE组)或注入生理盐水(对照组)。分别在术前6h、术后1h、6h、24h、3d和7d行平扫和动态增强MRI检查,测量肿瘤大小和最大增强斜率(IS)百分比;并分别在术前6h、术后6h、3d和7d取肿瘤组织行免疫组化法(IHC)或(/和)蛋白免疫杂交印迹法(WB)检查MVD(CD31标记)、HIF-1α、VEGFR-2、p VEGFR-2、PDGFR-p和磷酸化PDGFR-β(p PDGFR-β)。
经软件SPSS 11.0作统计分析。
结果:
临床病例检查示二期组肝癌组织均见大片坏死区,有6例肿瘤完全坏死。二期组残癌组织p VEGFR-2和HIF-1α的DM为(0.03376±0.01648)、(0.04705±0.02128),直接组分别为(0.02352±0.00893)、(0.03532±0.01623),两组差异均有显著性(P值均＜0.05);二期组癌旁组织VEGFR-2、p VEGFR-2和HIF-1α的DM分别为(0.03962±0.01680)、(0.03064±0.01138)和(0.03749±0.01517),直接组则分别为(0.03022±0.01481)、(0.02010±0.00827)和(0.02383±0.01390),两组差异均有显著性(P值均＜0.05)。两组残癌及癌旁组织PDGFR-β差异无显著性(P＞0.05)。二期组癌旁组织MVD为(58.3±15.2),直接组为(44.4±10.5),两组差异有显著性(P＜0.001)。
动物实验中,TAE组肿瘤体积稍小于对照组,两组差异无显著性(P＞0.05)。MRI示两组肿瘤内信号基本均匀,仅TAE组肿瘤内见少量小灶状坏死;TAE组和对照组的IS百分比,两组差异有显著性(F=430.2,P＜0.001),两两比较示术后1h、6h、24h及3d与对照组差异均有统计学意义(P＜0.05)。IHC检测两组MVD差异无统计学意义(U=59.0,P＞0.05);两组HIF-1α和p VEGFR-2差异有统计学意义(U=22.0、26.0,P均＜0.05)。VEGFR-2和PDGFR-β两组间差异无统计学意义(U=46.0、48.0,P均＞0.05)。WB检测TAE组p VEGFR-2和p PDGFR-β在术前呈低表达,术后6小时、3天较术前和对照组明显增高,术后7天时表达基本正常或略增高;对照组p VEGFR-2和p PDGFR-β术后表达与术前无明显差异;两组VEGFR-2和PDGFR-β在各个时间点差异不明显。
结论:
1、人肝癌经TACE治疗后肝癌坏死明显,残癌和癌旁组织缺氧程度加重,VEGFR-2、PDGFR-β术前后变化不显著,但功能(磷酸化状态)明显增强;
2、大鼠肝癌经TAE治疗后短期内血供减少,肿瘤组织缺氧程度加重,VEGFR-2、PDGFR-β等受体功能明显增强。
第二部分Sunitinib抑制肝癌细胞及血管内皮细胞的体外实验
目的:
研究Sunitinib对肝癌细胞和血管内皮细胞的抑制作用。
材料和方法:
取人肝癌细胞株Hep 3B、Hep G2、大鼠肝癌细胞株McA-RH7777、正常人肝细胞株L-02和人脐静脉内皮细胞(HUVEC),用WST-1法检测血管内皮生长因子-165(VEGF-165)、血小板衍生性生长因子-BB(PDGF-BB)和Sunitinib分别对它们的促增殖或毒性作用;PI单染法流式细胞术检测Sunitinib对各细胞株的促凋亡作用;细胞划痕试验检测Sunitinib对HUVEC的迁移抑制作用;细胞免疫荧光法(IF)和免疫蛋白杂交印迹法(WB)检测各细胞株VEGFR-2、pVEGFR-2、PDGFR-p和p PDGFR-β的表达,以及不同浓度Sunitinib对它们的抑制作用。经软件SPSS 11.0作统计分析。
结果:
VEGF和PDGF浓度分别在25ng/mL以上,对HUVEC有明显促增殖作用(F=8.953、9.731,P值均等于0.001)。(VEGF+PDGF)对人肝癌细胞HepG2、Hep3B、大鼠肝癌细胞McA-RH7777及人正常肝细胞L-02促增殖作用不明显(HepG2组F=0.385、Hep3B组F=0.322、McA-RH7777组F=2.949、L-02组F=0.673,P值均大于0.05)。Sunitinib对HUVEC有显著的抑制作用(F=41.003,P＜0.001);两两比较示浓度在0.01μM及其以上时,对HUVEC有明显的抑制作用(P＜0.05)。在Hep G2和McA-RH7777中,Sunitinib各浓度间差异有统计学意义(F=3.367、4.518,P＜0.05),其中10μM组与0μM组差异显著(P＜0.05)。划痕试验示Sunitinib组在划痕后,划痕未缩小,至24h、48h时渐增宽。流式细胞术检测HUVEC在Sunitinib浓度0.1μM时,凋亡率为19.2%:在1μM时,凋亡率达36.7%。Hep G2、Hep 3B及McA-RH7777在SU11248浓度1μM时,凋亡率分别为9.8%、9.2%、14.5%;在10μM时,凋亡率分别为15.6%、16.3%、19.5%。HUVEC、HepG2和McA-RH7777均表达VEGFR-2和PDGFR-β,加Sunitinib后磷酸化明显受到抑制,且与剂量呈正相关。
结论:
1.Sunitinib能强烈地抑制血管内皮细胞增殖并促进其凋亡,是sunitinib发挥抗肿瘤作用的主要机制;
2.Sunitinib在高浓度下对肝癌细胞也有一定的抑制作用,可能通过非肿瘤血管依赖性途径起作用。
第三部分高效液相色谱法检测Sunitinib不同给药途径药代动力学差异
目的:
建立高效液相色谱法,检测Sunitinib经口服和肝动脉途径给药在不同时相各器官药物分布差异。
材料和方法:
采用内标法(Chrysin为内标),应用高效液相色谱紫外检测大鼠血浆和肝组织中Sunitinib的浓度,建立标准曲线并验证方法的准确度、精密度和最低定量浓度等。
将大鼠肝癌模型分成Sunitinib口服组(口服组,连续灌胃)与TAE+动脉Sunitinib组(TAE组,单次Sunitinib 10mg/Kg与碘油乳化),口服组分别在首次给药后1、6、24、30、48小时及3、5、7、9、14天取血和肝组织样本;TAE组在TAE术后0.5、1、6、12、24、30、48小时及3、5、7、9、14天取血和肝组织样本,分别检测Sunitinib浓度。
结果:
应用本法检测血和肝组织样本Sunitinib浓度的标准曲线,回归方程分别为Y=2197.3 C-2.2907(血),Y=4723.5 C+33.246(肝);根据质控浓度检测得出血浆的准确度在各个浓度中为(-6.4～-4.4)%,天内精密度为(5.4～10.6)%,天间精密度(3.1～9.5)%;肝组织样品的准确度在各个浓度中为(-8.0～3.1)%,天内精密度为(11.4～13.3)%,天间精密度(1.5～2.4)%。准确度与精密度均≤15%,方法稳定可靠。血浆样品最低定量浓度为20ng/ml,肝组织样品最低定量浓度为25ng/ml。
口服组血和肝组织样本约在首次给药后6-10小时达峰值,6小时时测血药浓度约140ng/mL,肝组织浓度约200ng/g;血中半衰期(T_(1/2))约6-12小时。重复给药后,约于6小时再达峰值,每隔24小时检测,直至第9天时血药浓度在(30-70)ng/mL,肝组织浓度在(100-130)ng/g。连续10天给药后,第14天检测时血药浓度已降至检测下限以下,肝组织浓度在45ng/g左右。
TAE组血和肝组织样本在术后30分钟即达峰值,30分钟时一个血样本浓度超出检测上限;至术后24-30小时,血药浓度在(30-40)ng/mL,48小时以后血样本均处于检测下限以下。肝组织浓度则在术后6小时前均超出检测上限(1500ng/g),以后肝组织浓度缓慢下降(相对于血药浓度),至术后第5天在100ng/g左右,第9天及以后降至检测下限以下。
结论:
1.建立高效液相色谱紫外检测法,检测血及肝组织样本Sunitinib浓度,方法简单稳定,能满足动物实验的设计要求;
2.比较Sunitinib经口服途径和TAE途径的药物代谢动力学差异,为其联合TAE治疗肝癌提供药理学依据。
第四部分Sunitinib联合经肝动脉栓塞术治疗肝癌的实验研究
目的:
观察经动脉栓塞术(TAE)联合运用Sunitinib治疗肝癌的疗效,并探讨新生血管变化及分子水平的机制。
材料和方法:
用大鼠肝癌细胞株McA-RH7777和Buffalo大鼠建立大鼠肝癌模型,随机分成五组:生理盐水组(A组)、TAE组(B组)、TAE+动脉Sunitinib组(C组)、TAE+口服Sunitinib组(D组)、TAE+动脉、口服Sunitinib组(E组)。观察大鼠生存时间、肿瘤体积等:各组分别于术后第1周、第2周、第4周及第8周分别作MRI平扫和动态增强;同时取肿瘤组织,大体观察后作IHC及WB检测MVD(CD31标记)、HIF-1α、VEGFR-2、p VEGFR-2、PDGFR-β和p PDGFR-β。经软件SPSS 11.0作统计分析。
结果:
各组间大鼠生存期差异有统计学意义(X~2=10.8,P＜0.05),E组比A组延长最明显(与A组,X~2=5.9,P＜0.05)。各组肿瘤增大差异明显(F4.149,P＜0.001),与A组相比,C、D组术后肿瘤生长明显减缓(术后一周、术后二周时,P均＜0.05);E组在术后一周、二周时肿瘤无明显生长(与A～D组,P坊＜0.05),个别大鼠肿瘤略有缩小。MRI示C～E组较对照组坏死的范围明显增大。最大增强斜率(initial slope,IS)百分比各组间差异明显(F=3.311,P＜0.001),E组减弱最明显(与A组,P＜-0.001)。IHC示MVD各组间差异明显(F=33.005,P＜0.001),C-E组MVD在术后一周、二周时较术前和对照组减少。HIF-1α各组间差异有统计学意义(X~2=22.5,P＜0.001),C、D、E组表达比A组明显增强(P＜0.05)。VEGFR-2 C～E组在术后一周、二周时均较A组有不同程度的增加,组间差异有统计意义(X~2=23.2,P＜0.001)。C～E组p VEGFR-2在术后一、二周却受到明显的抑制,在术后四周开始增强,组间差异有统计学意义(X~2=9.38,P＜0.05)。PDGFR-β各组间有统计学差异(X~2=13.3,P＜0.05),E组表达最强且持续时间较长。WB示C～E组术后第7天、14天VEGFR-2和PDGFR-β的磷酸化水平受到明显抑制,C组在术后第14天的抑制程度不及D组,而E组在第7天、14天时受体的磷酸化水平抑制程度明显强于C、D组:三组各时间点的VEGFR-2和PDGFR-β表达差异不显著。
结论:
1.Sunitinib与TAE联合治疗肝癌,阻断多种促血管生成因子受体,抑制新生血管生成,提高了TAE的疗效;
2.Sunitinib经动脉途径乳化后给药,在短期(一周)内更有效地抑制肝癌的新生血管形成;
3.TAE术中单剂量Sunitinib经动脉给药,结合术后口服给药,抗肿瘤新生血管生成作用强大、持久,对肝癌治疗效果更佳。
Part 1 Expression of angiogenesis-related factors and receptors after the treatment of hepatocellular carcinoma with trans-catheter arterial embolization(TAE)
Objective
To study the expression of angiogenesis-related factors and receptors after the treatment of hepatocellular carcinoma(HCC) with trans-catheter arterial embolization (TAE).
Materials and methods
43 cases of second-stage surgery of HCC after TACE(second-stage group) and 20 of direct surgery(direct surgery group) were taken and made into paraffin-embedded slices.Vascular endothelial growth factor receptor(VEGFR-2),phosphorylated VEGFR-2(p VEGFR-2),platelet-derived growth factor receptor-β(PDGFR-β), hypoxia induced factor-1α(HIF-1α) and microvessel density(MVD)(labeled by CD31)were detected with the immunohistochemical method.
Rat HCC models,which were established with rat HCC line McA-RH7777 and Buffalo rats,were divided into 2 groups:trans-catheter arterial embolization with lipiodol(TAE group) or with saline(control group).Tumor volume and initial slope(IS) percent were measured with MRI at different times:6 hours before,1 hour after,6 hours after,24 hours after,3 days after and 7 days after surgery.MVD(labeled by CD31),HIF-1α,VEGFR-2,p VEGFR-2,PDGFR-βand p PDGFR-βof tumor were also detected with IHC and/or western blotting(WB).
All data were processed by SPSS 11.0.
Results
In clinical samples,The necrosis of cancerous tissue was obvious in second-stage group with 6 cases of complete necrosis.The density means(DM) of phosphorylated VEGFR-2 and HIF-1αin the cancerous tissue of HCC were(0.03376±0.01648) and(0.04705±0.02128) in second-stage group,and(0.02352±0.00893) and (0.03532±0.01623) in direct-surgery group,which showed statistical difference.The DMs of VEGFR-2,phosphorylated VEGFR-2 and HIF-1αin the peri-cancerous tissue were(0.03962±0.01680),(0.03064±0.01138) and(0.03749±0.01517) respectively in direct-surgery group,and(0.03022±0.01481),(0.02010±0.00827) and(0.02383±0.01390) respectively in direct-surgery group,all of which showed statistical difference.MVD of peri-cancerous tissue was(58.3±15.2) in second-stage group and(44.4±10.5) in direct-surgery group,both of which were different statistically.
In the rat samples,tumor volume of TAE group was slightly smaller than control group,but it showed no significant difference(P＞0.05).Tumors' signal of both groups were almost even in MRI,while some dot-like necrosis were found in TAE group.The difference of initial slope(IS) percent between two groups were statistically significant(F=430.2,P＜0.001),and IS percent of TAE group at 1hours, 6hours,24hours and 3 days were all lower than control group significantly.MVD of TAE group was not significantly different than control group(U=59.0,P＞0.05). Differences of HIF-1αand p VEGFR-2 between two groups were significant statistically(U=22.0,26.0,both P value less than 0.05).VEGFR-2 and PDGFR-βshowed no significant difference between two groups(U=46.0、48.0,both P value more than 0.05).All of the above were detected with IHC.Expression of p VEGFR-2 and p PDGFR-β,detected with WB,were higher at 6 hours,3 days after surgery than before surgery and control group.Expressions of them in control group were not significantly different between before and after surgery.VEGFR-2 and PDGFR-βin both groups showed no significant difference between every two times.
Conclusions
1.Human HCC after TACE has obvious necrosis,but the cancerous and peri-cancerous tissues are more hypoxic and both the expression and function of VEGFR-2 enhanced.
2.Rat HCC after TAE shows less blood supply in short period,and the tumor is more hypoxic,and eventually its function of VEGFR-2 and PDGFR-βenhanced.
Part 2 Sunitinib suppresses hepatocellular carcinoma(HCC) cells and vascular endothelial cell in vitro
Obejective
To study the suppression of HCC cells and vascular endothelial cell(VEC) with sunitinib,a multi-targeted receptor tyrosine kinase(RTK) inhibitor.
Materials and methods
Several cell lines were used in this part including human HCC lines(Hep G2, Hep 3B),rat HCC line(McA-RH7777),human umbilical vein endothelial cell(HUVEC) and human normal liver cell(L-02).WST-1 method was applied to detect the proliferation or toxicity of VEGF-165,PDGF-BB and sunitinib for these cells.The apoptosis of cells with sunitinib was also detected with PI staining method by flow cytometry.Cell scratch test was used to find the movement inhibition of sunitinib for HUVEC.Cell immunofluorescence(IF) and western blotting(WB) were applied to detect the inhibition of VEGFR-2、p VEGFR-2、PDGFR-βand p PDGFR-βwith sunitinib in different concentration.
All data were processed by SPSS 11.0.
Results
VEGF and PDGF promoted the proliferation of HUVEC at 25ng/mL and above(F=8.953、9.731,P=0.001),while they didn't show such effect for HepG2,Hep 3B,McA-RH7777 and L-02.Sunitinib suppressed the proliferation of HUVEC signigicantly at 0.01μM and above(F=41.003,P＜0.001),while it also suppressed Hep G2 and McA-RH7777 significantly at 10μM(F=3.367、4.518,both P value less than 0.05).Scratch test showed HUVEC didn't cross the scratch after incubation with sunitinib.Flow cytometry revealed the apoptosis of HUVEC was 19.2%at 0.1μM of sunitinib,36.7%at 1μM,which of Hep G2,Hep 3B and McA-RH7777 were 9.8%,9.2%and 14.5%at 1μM,and 15.6%,16.3%and 19.5%at 10μM respectively.HUVEC,Hep G2 and McA-RH7777 demonstrated the expression of VEGFR-2、p VEGFR-2、PDGFR-βand p PDGFR-β.After incubation with sunibinib,the phosphorylation of both receptors were suppressed,and furthmore it's correlated with the dose.
Conclusions
1.Sunitinib suppresses the proliferation of HUVEC and promotes its apoptosis, which is the main mechanism of anti-angiogenesis.
2.Sunitinib also shows some kind of inhibition for HCC cells at high concentration,which non-anti-angiogenesis way may play a role in.
Part 3
High performance liquid chromatography(HPLC) detects sunitinib pharmacokinetics(PK) difference between different administration methods
Obejective
To set up a method of HPLC to detect the concentration of sunitinib in blood and liver samples,and study the pharmacokinetics(PK) difference between oral and arterial administration.
Materials and methods
HPLC with ultra-violet(420nm and 268nm) detected the sunitinib concentration in rat blood and liver samples using chrysin as internal standard.Standard curve and its linear scope were set up and their accuracy,precision and minimum limit of quantitation were validated.
Rat HCC models were randomly divided into sunitinib oral administration group(oral group,sunitinib 10mg/kg,10 days continually) and TAE with sunitinib group(TAE group,sunitinib 10mg/kg signally with lipiodol).Blood and liver samples were harvested at 1,6,24,30,48 hours and 3,5,7,9 and 14 days after first dose in oral group,while at 0.5,1,6,12,24,30,48 hours and 3,5,7,9 and 14 days after TAE in TAE group.All samples were detected by the validated methods.
Results
The standard curves were set up for blood and liver samples,which regression equation was y=2197.3×C-2.2907 for blood and y=4723.5×C+33.246 for liver. With the detection of quality concentrations,accuracy at different concentrations was (-6.4～-4.4)%,in-day precision was(5.4～10.6)%,and between-day precision was (3.1～9.5)%in blood samples.Accuracy at different concentrations was(-8.0～3.1) %,in-day precision was(11.4～13.3)%,and between-day precision was(1.5～2.4) %in liver samples.All accuracy and precision were under 15%,which demonstrate the method was stable and reliable.The minimum limit of quantification was 20ng/ml for blood and 25ng/ml for liver.
In oral group,the concentration of sunitinib in blood and liver was peaking within 6～10 hours.The concentration at 6 hours after first dose was about 140ng/ml in blood,and 200ng/ml in liver.Its half-life period(T_(1/2)) was about 6-12 hours in blood.Peak concentration was gotten at about 6 hours after repeated dose.Then detected at every 24 hours after administration,blood concentration was(30-70)ng/ml and liver concentration was(100-130)ng/g at 9 days after the first dose.At 14 days, blood concentration was under the minimum limit and liver concentration was about 45ng/g.
In TAE group,the concentration was peaking at about 30 minutes after TAE in blood and liver.The concentration in blood was(30-40)ng/ml at 24-30 hours after TAE,and under minimum limit at 48 hours and after.The concentration at 6 hours and before in liver were all above the maximum limit of qualification(1500ng/g). Then it decreased slowly comparing to blood concentration,was about 100ng/g at 5 days and under the minimum limit at 9 days and after.
Conclusions
1.The methods of HPLC to detect the concentration of sunitinib in blood and liver samples are stable and reliable,which can satisfy this experiment.
2.The PK difference of sunitinib between oral and arterial administration provides the pharmacological basis for the combination of sunitinib and TAE.
Part 4 Treatment of hepatocellular carcinoma(HCC) with the combination of sunitinib and trans-catheter arterial embolization(TAE) in rat models
Obejective
To investigate the effect of the combination of TAE and sunitinib for HCC,and study the mechanism of angiogenesis and molecular level.
Materials and methods
Rat HCC models,established with McA-RH7777 and Buffalo strait rats,were divided randomly into five groups:saline group(group A,control group),TAE group(group B),TAE+sunitinib iA group(group C),TAE+sunitinib p.o.group(group D) and TAE+sunitinib iA&p.o,group(group E).Rat survival time and tumor volume were evaluated.MRI plain scan and dynamic enhancement were applied for five groups at 1 week,2 weeks,4 weeks and 8 weeks after surgery and at the same time, some rats were sacrificed for the detection of MVD(labeled by CD31),HIF-1α、 VEGFR-2、p VEGFR-2、PDGFR-βand p PDGFR-βwith IHC and/or WB.All data were processed by SPSS 11.0.
Results
Survival time among five groups were different statistically(X~2=10.8,P＜0.05),in which E group was longest(verses A group,X~2=5.9,P＜0.05).Tumor volume was also different statistically(F=4.149,P＜0.001).Among them,tumors in C,D group were retarded(verses A group,both P value less than 0.05),and ones in E group were found little growth(verse A～D group,all P value less than 0.05),some of which even decreased.
MRI demonstrated that the scope of necrosis increased obviously.IS percent among groups was different significantly(F=3.311,P＜0.001),in which E group decreased mostly(verse A group,P＜0.001).MVD in C～E group was reduced at 1 week,2 weeks after surgery with comparison to before surgery and A group,which among groups was different(F=33.005,P＜0.001).HIF-1αin C～E group increased more than A group,which among groups showed significant difference(X~2=22.5,P＜0.001).VEGFR-2 in C～E group at 1 week,2 weeks after surgery also increased at different grade,which difference in groups was significant(X~2=23.2,P＜0.001).But p VEGFR-2 in C～E group at 1 week,2 weeks after surgery was inhibited significantly(X~2=9.38,P＜0.05).PDGFR-βamong groups also had significant difference(X~2=13.3,P＜0.05),among which it in E group expressed strongly and persistently.WB showed that the phosphorylation of VEGFR-2 and PDGFR-βwas inhibited significantly.Among them,the inhibition of phosphorylation in E group at 7 and 14 days was more obvious than C and D group.However,VEGFR-2 and PDGFR-βwere not different significantly.
Conclusions:
1.Sunitinib,with combination of TAE for treatment of HCC,inhibits several angiogenesis-related receptors,suppresses angiogenesis,and enhances the effect of TAE.
2.Sunitinib,through arterial administration after emulsification with lipiodol,can suppress angiogenesis of HCC more effectively in short period.
3.Sunitinib with the combination of arterial administration after emulsification with lipiodol and oral administration continually,can perform more strong and persistent effect of anti-angiogenesis,which is more effective with the combination with TAE for HCC.
引文
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