双参通冠方促血管新生作用的物质基础及机制研究
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摘要
冠心病,又称缺血性心脏病,是由于冠状动脉粥样硬化造成血管痉挛,引起血管狭窄或阻塞,产生冠脉循环障碍,造成心肌缺血、缺氧或坏死的一种心脏病。目前,国际上公认的三大治疗途径包括药物治疗、介入治疗及外科治疗。由于大多数药物对病变严重的心肌缺血治疗效果有限,而介入治疗和外科治疗又不能阻止病人的冠状动脉其他部位发生再狭窄,所以这三种治疗只能暂时缓解冠心病症状,而不能根治冠心病。20世纪末,一些学者提出了治疗性血管新生的思路,即通过某些药物的治疗增加功能性的冠状动脉分支或侧支循环,达到恢复缺血心肌血供,改善患者症状和预后的目的,有人形象地称它为“药物促进人体的自身搭桥”。
研究发现,血管新生能帮助肌体建立有效的侧支循环,从根本上缓解心肌缺血症状,改善病情。目前国外大多研究利用基因方法,即直接补充VEGF或FGF等细胞因子的方法或蛋白方法及细胞移植等方法来促进冠心病患者的血管新生然而这些方法不但价格昂贵,而且目前还只是停留在研究阶段,尚无法应用于临床。
完整的内皮功能在心血管疾病的发生发展过程中扮演重要角色,其功能受损导致许多血管性疾病的发生。然而,成熟的内皮细胞是终端分化细胞,其再生能力有限。大量的研究表明EPCs (endothelial progenitor cells)在受损血管内皮的自我修复中发挥重要作用,它不仅参与胚胎时期血管生成,还与损伤的血管内皮的愈合和血管新生(neovascularization)密切相关,尤其是对于前者具有重要的促进作用。它主要存在于骨髓中,在某些生理、病理状态下,可从骨髓释放到外周血,随血液循环迁移到局部血管损伤处,分化为成熟的血管内皮细胞,从而修复损伤内皮、促进血管再内皮化。寻找能够改善内皮功能促进其修复,刺激血管新生的物质受到越来越多研究者的关注
中医治疗缺血性疾病的研究起源久远,并且取得了显著的成效。因此,从中药中寻找具有促进血管新生作用的药物具有重要的经济及社会意义。双参通冠方(SSTG)是由提取的人参总皂苷、丹参总酚酸、元胡总生物碱三种有效组分配伍而成,本实验室前期的药效学研究表明,该方能明显减轻急性心肌缺血犬和大鼠的心肌损伤程度,对缺血心肌有很好的保护作用。但是,之前缺少对其在促进血管新生作用、作用机理及物质基础方面的研究。
本课题采用鸡胚绒毛尿囊膜(chick embryo chorioallantoic membrane,CAM),体外培养内皮祖细胞数量、功能及冠状动脉结扎大鼠模型,观察SSTG促血管生成作用及其物质基础,探寻其作用机制,为其治疗缺血性心脏病的临床应用提供实验依据。
本课题分为三个部分：
一、双参通冠方(SSTG)对鸡胚绒毛尿囊膜(CAM)血管新生的影响
1. SSTG及有效组分含药血清对鸡胚绒毛尿囊膜血管新生的影响
目的：探讨SSTG及单味药提取物含药血清对鸡胚绒毛尿囊膜血管新生的影响。方法：将无菌8日龄鸡胚制备CAM模型,随机分成5组：对照组,SSTG、丹参总酚酸、人参总皂苷及元胡总生物碱组。将蛋壳用75%乙醇消毒后,用镊子小心开1.5cm×1.5cm的窗口,暴露CAM,将受试物品加到无菌的滤纸上,待风干后,小心置于CAM上血管较少的部位,将窗口封上,放入孵箱,于37℃,60%相对湿度继续孵育3天后,对CAM固定后,采集数据进行分析。结果：SSGT(3.25±0.80)、丹参总酚酸(3.11±0.83)及人参总皂苷组(2.97±0.47)血管密度比明显高于对照组(2.45±0.64)(P<0.05,P<0.05,P<0.05)。结论：SSTG、丹参及人参可明显促进鸡胚绒毛尿囊膜血管新生。
2.丹参总酚酸及丹酚酸A、B对鸡胚绒毛尿囊膜血管新生的影响
目的：探讨丹参总酚酸及丹酚酸A、B对鸡胚绒毛尿囊膜血管新生的影响。方法：同前。结果：总酚酸100mg/L (2.97±0.39)、300mg/L (3.07±0.36)给药组血管密度比明显高于对照组(2.47±0.35)(P<0.05,P<0.05)；丹酚酸A 5.56mg/L(4.42±1.16)、16.67 mg/L(5.51±1.15)、50 mg/L(4.60±0.71)给药组血管密度比明显高于对照组(3.07±0.85)(P<0.05,P<0.001,P<0.01)；丹酚酸B50mg/L(2.18±0.31)、150 mg/L(2.43±0.32)给药组血管密度比明显高于对照组(1.86±0.30)(P<0.05,P<0.01)。结论：丹参总酚酸、丹酚酸A及丹酚酸B可明显促进鸡胚绒毛尿囊膜血管新生
二、丹参总酚酸及丹酚酸A对内皮祖细胞(EPCs)功能的影响
1.内皮祖细胞的培养及鉴定
目的：建立新生鼠脾脏来源的内皮祖细胞的培养方法并对其进行鉴定。方法：密度梯度离心法分离SD新生鼠脾脏单个核细胞,EGM-2完全培养基贴壁法,37℃,5%CO2,饱和湿度的恒温培养箱中原代培养。细胞长至近融合时传代,分别于第9天进行细胞鉴定。内皮祖细胞鉴定：免疫细胞化学的CD34和VEGFR-2染色；内吞DiI标记的乙酰低密度脂蛋白(DiI-acLDL)和结合FITC标记的荆豆凝集素-1(FITC-UEA-1)的激光共聚焦显微镜观察及超微结构鉴定。结果：培养细胞的形态学改变：脾脏单个核细胞接种24h后部分细胞开始贴壁、变大,倒置显微镜下贴壁细胞透亮度增强,并逐渐伸出伪足样突起,呈小杆状或梭形。培养第3天贴壁细胞开始增殖,呈“集落”样生长,外周梭形细胞较多；共聚焦显微镜观察显示细胞DiI-acLDL和FITC-UEA-1呈红绿免疫双荧光阳性；培养第9天细胞VEGFR-2和CD34免疫细胞化学染色呈阳性；电镜下观察可见内皮细胞特异性的W-P小体存在；功能鉴定显示EPCs体外培养形成血管结构。结论：新生鼠的脾脏单个核细胞可以培养为内皮祖细胞并能够形成血管结构。
2.丹参总酚酸对正常及H2O2损伤内皮祖细胞功能的影响
目的：探讨丹参总酚酸对内皮祖细胞数量及功能的影响以及对H2O2损伤内皮祖细胞功能。方法：密度梯度离心法获取新生鼠脾脏单个核细胞,细胞长至融合状态后,收集贴壁细胞并加入丹参总酚酸(0.3,3和30mg/L)干预24h,分别观察其对EPCs的增殖、迁移、粘附、体外成管能力的影响。同时,应用200u mol/LH2O2对EPCs进行损伤,观察丹参总酚酸的保护作用。结果：丹参总酚酸促进EPC扩增,3及30mg/L丹参总酚酸作用24h对EPCs数量有显著性影响(P<0.05,P<0.05),同时也显著改善了EPCs的粘附、迁移及体外成管能力。对200μmol/LH2O2损伤EPCs的粘附及成管能力有明显的改善作用。结论：丹参总酚酸可增加EPCs数量并改善其功能并对H2O2损伤的EPCs的功能有改善作用。
3.丹酚酸A对正常及H2O2损伤内皮祖细胞功能的影响及初步机制研究
目的：探讨丹酚酸A对内皮祖细胞数量及功能的影响以及对H2O2损伤内皮祖细胞功能。方法：密度梯度离心法获取新生鼠脾脏单个核细胞,细胞长至融合状态后,收集贴壁细胞并加入丹酚酸A(10-9,10-8和10-7mol/L)干预24h,分别观察其对EPCs的增殖、迁移、体外成管能力的影响,并采用ELISA试剂盒检测加入丹酚酸A对培养上清中SDF-1及MMP-9含量的影响,采用NO试剂盒检测细胞上清中NO量的变化。同时,应用200μmol/LH2O2对EPCs进行损伤,观察丹酚酸A的保护作用。结果：丹酚酸A促进EPCs扩增,10-9,10-8和10-7mol/L酚酸A作用24h对EPCs数量有显著性影响(P<0.05,P<0.05),同时也显著改善了EPCs的迁移、成管能力；显著增加条件培养上清中SDF-1、MMP-9及NO的量。对200μmol/LH2O2损伤EPCs的粘附及成管能力有明显的改善作用。结论：丹酚酸A可增加EPC数量并改善其功能,其机制可能与增加SDF-1、MMP-9表达及NO的量有关,并对H2O2损伤的EPCs的功能有改善作用。
三、丹酚酸A对冠脉结扎大鼠血管新生的影响及机制研究
目的：探讨丹酚酸A对冠脉结扎大鼠血管新生的影响及其机制探讨。方法：结扎Wistar大鼠冠状动脉左旋降支建立心肌缺血大鼠模型,随机分为模型组,丹酚酸A 2.5、5、10mg/kg给药组,阳性对照药组及假手术组。于手术后第二天尾静脉注射给药,每天1次,7天后做以下实验：1)检测心肌缺血面积(NBT染色法)；2)HE染色观察缺血心肌的病理改变；3)免疫组化方法检测心肌梗死周围区VEGF/VEFGR-2的表达以及CD34标记的微血管密度；4) ELISA法检测血清中SDF-1及MMP-9表达量的变化。结果：与假手术组比较,模型组大鼠缺血心肌部位心肌细胞明显损伤(P<0.05),丹酚酸A 10、5剂量组梗死面积／心脏面积比值明显降低(P<0.01,P<0.05)；与假手术组比较,模型组梗死周围区CD34+细胞(P<0.05)明显增多,VEGF (P<0.001)和VEGFR-2 (P<0.001)表达明显增强,与模型组比较,丹酚酸A 10、5、2.5mg/kg剂量组梗死周围区微血管密度明显增多(P<0.001,P<0.001,P<0.001),丹酚酸A10、5mg/kg梗死周围区VEGF(P<0.001,P<0.05)和VEGFR-2 (P<0.001, P<0.001)表达显著升高；与假手术组比较,模型组SDF-1 (P<0.05)及MMP-9 (P<0.05)表达上升；与模型组比较,丹酚酸A10、5、2.5 mg/kg剂量组显著促进SDF-1 (P<0.01, P<0.05)及MMP-9(P<0.001,P<0.001,P<0.05)的表达。结论：丹酚酸A可降低心肌缺血大鼠梗死面积,其可能机制是通过上调VEGF和VEGFR-2及增加SDF-1、MMP-9的表达促进了内源性的血管新生。
综上所述,首先CAM模型的结果显示：双参通冠方具有促进血管新生的作用,其有效组分丹参总酚酸及人参总皂苷也具有促CAM血管新生的作用,我们选择了丹参作为继续研究的对象,结果显示丹酚酸A比丹酚酸B有更好的促进CAM血管新生的作用；丹酚酸A促进体外培养EPCs的数量的增加及功能的改善；整体心肌缺血模型的结果显示,丹酚酸A有明显降低缺血心肌梗死面积,促进缺血后心肌MVD增加的作用,其促进血管新生的可能作用机理为通过上调VEGF和VEGFR-2及增加SDF-1、MMP-9的表达促进EPCs的动员、分化与归巢,促进了内源性的血管新生。因此,我们得出结论：丹酚酸A为双参通冠方促血管新生的有效物质基础之一。
本论文的创新点：
1、首次研究了双参通冠方的促血管新生作用,为用于冠心病的治疗提供实验依据；
2、首次研究了丹酚酸A的促血管新生作用,并对其作用机制作了初步研究。
Cardiovascular disease is the global leading cause of death in the whole world. Currently available approaches for treating patients with ischemic heart disease include medical therapy or coronary revascularization by percutaneous coronary angioplasty (PCA) or coronary artery by pass grafting (CABG). However, a significant number of these patients are not candidates for coronary revascularization procedures or achieve incomplete revascularization with these procedures. Consequently, many of these patients have persistent symptoms of myocardial ischemia despite intensive medical therapy. Preliminary clinical experiences suggest that therapeutic angiogenesis may provide additional blood flow to incompletely revascularized areas. More recently, several studies suggest that implanted bone marrow cells may induce angiogenesis in ischemic myocardium.
Currently there are 3 major approches for therupetic angiogenesis, that is gene therapy, protein therapy and cellbased transplation. Howerve, theses methods are not only expessive but also on the stduy phase, and can not be used in clinical.
Endothelial dysfunction has been implicated in the pathogenesis of many diseases affecting the cardiovascular system. Experimental and clinical studies have shown that endothelial dysfunction may play a cusial role in the development and progression of vascular diseases, such as hypertension ro restenosis, as well as their complications, including myocardial infarction. Mature endothelial cells (ECs), however, have limited regenerative capacity. A growing body of evidence indicates that endothelial progenitor cells (EPCs) promote endothelial repair and contribute to ischemia-induced neovascularization. Increasing evidence suggests that circulating progenitor cells contribute to postnatal neovascularization except in embro. These cells are derived form bone-marrow, in some physiological and pathological status, they are released from bone-marrow into circulation, home to sites of ischemia, adopt an endothelial phenotype, and contribute to new blood vessel formation. Hence, the discovery of candidate molecules able to augment EC funtion to stimulate myocardial angiogenesis has stirred a growing interest in using these molecules for therapeutic application.
Shuangshentongguan (SSTG) is made up of ginsenosides, salvianolic acids and corydalines. Our previous pharmacological studies showed that SSTG could relieve myocardial damage of dog and/or acute myocardial ischemia of rat, show protective effects on myocardium infartion. But, there is little information about its'effect on theraputic angiogenesis and its matierial basis.
So, we designed the experiments to test SSTG effects on angiogenesis, it is realised by 3 models, they are as follows:chick embryo chorioallantoic membrane, endothelial progenitor cell and myocardial ischemia in rats induced by ligation of the left anterior descending (LAD) coronary artery.
Chapter 1:Effects of SSTG on angiogenesis of the chick embryo chorioallantoic membrane
Part 1:Effects of SSTG on angiogenesis of the chick embryo chorioallantoic membrane
Objective:To investigate the effect of SSTG on angiogenesis of the chick embryo chorioallantoic membrane (CAM). Methods:Fifty 8-day-old fertilized eggs were obtained from a commercial source (Beijing Merial Vital Laboratory Animal Technology Co., Ltd). On day 9 of the incubation, the fertile eggs were randomly allocated into five groups. Fertilized eggs are first disinfected with 75%alcohol, a window opening is punctured at the blunt end of the egg facing upwards using sterilized forceps. The eggshell above the air cell was removed and the shell membrane attached to it, was cut off for further examination. Normal development was verified and embryos with malformations or dead embryos were excluded. CAMs (10 eggs per group) were treated as described as follows:1) overlaying them with 20-mm2 sterilized filter discs that had been loaded with 10μ1 of serum without any test materials (negative control),10μl of serum containing SSTG, ginsenosides, salvianolic acids and corydalines. The window was sealed with tape and the eggs were returned to the incubator. After incubation for an additional 72 hours, all samples were immediately fixed in methanol and acetone (1:1) for at least 15min, then they were photographed using a stereomicroscope equipped with a camera and image analysis system. The angiogenic response was evaluated by counting the vessel density converging toward the filter discs..Result:The angiogenesis area in SSTG, ginsenosides, salvianolic acids groups was obviously increased compared with control group (P<0.05, P<0.05, P<0.05). Conclusion:The result showed that SSTG, ginsenosides, salvianolic acids can obviously stimulate angiogenesis of the chick embryo CAM.
Part 2:Effects of salvianolic acids, salvianolic acid A and salvianolic acid B on angiogenesis of the chick embryo chorioallantoic membrane
Objective:To investigate the effect of salvianolic acids, salvianolic acid A and salvianolic acid B on angiogenesis of the chick embryo chorioallantoic membrane (CAM). Methods:the same as above-mentioned. Result:The angiogenesis area in salvianolic acids 100 mg/L (2.97±0.39)、300 mg/L (3.07±0.36) groups was obviously increased compared with control group (2.47±0.35) (P<0.05, P<0.05); the angiogenesis area in salvianolic acid A 5.56 mg/L (4.42±1.16)、16.67 mg/L (5.51±1.15)、50 mg/L (4.60±0.71) was obviously increased compared with control group (3.07±0.85) (P<0.05, P<0.001, P<0.01); the angiogenesis area in salvianolic acid B 50mg/L (2.18±0.31)、150 mg/L (2.43±0.32) was obviously increased compared with control group (1.86±0.30) (P<0.05, P<0.01). Conclusion:The result showed that salvianolic acids, salvianolic acid A and salvianolic acid B can obviously stimulate angiogenesis of the chick embryo CAM.
Chapter 2:Effects of Salvianolic acids (SAs) and Salvianolic acid A on the numbers and function of endothelial progenitor cells
Part 1:cultivation and identification of endothelial progenitor cells
Objective:To investigate the methods of isolation, cultivation and identification of endothelial progenitor cells (EPCs) from rat spleen, as well as their ability of differentiating into endothelial cells. Methods:The mononuclear cells were isolated f rom SD rat spleen and were cultured in vitro via adhesion selection methods. The expression of CD34 and VEGFR-2 were assessed by immunocytochemistry after 9d cultivation. And the adherent cells were stained with DiI complexed acetylated low-density lipoprotein (DiI-acLDL) and fluorescein Ulex Europaeus agglutinin-1 (FITC-UEA-1), and then viewed by laser scanning confocal microscope (LSCM) to confirm EPCs lineage. EPCs phenotype was also tested by ultrastructural analyses. And EPCs were assessed by lightmicroscopy for their capacity of independent tubulogenesis. Results:The adherent cells stretched and exhibited the clone-like morphology in about 20d of cultivation. Immunocytochemistry stain showed that the adherent cells were positive for CD34 and VEGFR-2 from 9d after culture. The cells could take up DiI-acLDL, and bind to FITC-UEA-1, showed double positive fluorescence under LSCM. EPCs phenotype was also confirmed by the presence of Weibel-Palade body in cytoplasm by ultrastructural analyses. Branched interconnecting EC-specific tubules formed with EPCs. Conclusion:EPCs are enriched in rat spleen and may exhibited some of the characteristics of endothelial cells (ECs) after 9d inducing culture in vitro.
Part 2:Effects of Salvianolic acids (SAs) on the numbers and function of endothelial progenitor cells in normal and H2O2 injured station
Objective:Salvia miltiorrhiza (SM, also known as DanShen) is one of the well-known widely used Chinese herbal medicines in clinical. In this study we aimed to demonstrate the effects of Salvianolic acids (SAs), the main active components of aqueous extract of SM on the numbers and function of endothelial progenitor cells in normal and H2O2 injured station. Methods:To do this, new-born rat spleen mononuclear cells were isolated by density gradient and EPCs were expanded. EPCs proliferation, migration and in vitro vasculogenesis activity were assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, transwell chamber assay, and in vitro vasculogenesis kit, respectively. EPCs adhesion assay was performed by replating those on fibronectin-coated dishes, and then counting adherent cells. Results:Incubation of EPCs with SAs increased the numbers of EPCs and promoted EPCs migratory, adhesive, and in vitro vasculogenesis capacity, could also augment function of EPCs injured by H2O2. Conclusion:The results of the present study suggest that SAs may be developed as a new therapeutic remedy for various ischemic diseases such as coronary artery disease.
Part 3:Effects of Salvianolic acid A on the numbers and function of endothelial progenitor cells in normal and H2O2 injured station
Objective:Salvianolic acid A (SA)is one of the well-known widely used Chinese herbal medicines in clinical. In this study we aimed to demonstrate the effects of SA on the numbers and function of endothelial progenitor cells in normal and H2O2 injured station. Methods:same as above-mentioned. Results:Incubation of EPCs with SA increased the numbers of EPCs and promoted EPCs migratory, adhesive, and in vitro vasculogenesis capacity. Besides, SA could augment function of EPCs injured by H2O2 Conclusion:The results of the present study suggest that SA may be developed as a new therapeutic remedy for various ischemic diseases such as coronary artery disease.
Chapter 3:Effects of Salvianolic acid A on angiogenesis after myocardial ischemia in rats and underlying mechanism.
Objective:To investigate the effects of SA on angiogenesis after myocardial ischemia in rats and underlying mechanism. Methods:myocardial ischemia was induced by occluding the left anterior descending (LAD) coronary artery of the hearts of wistar rats. After operation, they were randomly departed into 6 groups: vehicle-treated, SA 2.5、5、10mg/kg groups, positive group and sham-operated group. On 2 day of operation, ischemiac rats received tail intravenous injection once a day. At 7 day post ischemia, these experiments were done:1) to test of area of ischemiac myocardium (NBT staining); 2) to observe pathological chang in myocardium by HE staining; 3) to evaluate the localization and expression of VEGF and VEFGR-2 and CD34-labeled microvascular density (MVD) of the ischemic boundary zone by immunohistochemistry methods; 4) to test SDF-1 and MMP-9 expression in serum by ELISA. Results:compared to sham-operated rats CD34 positive cells(P<0.05), the expression of VEGF (P<0.001) and VEGFR-2 (P<0.001) significantly augmented, exposure to SA with 10、5mg/kg markedly promoted the expression of VEGF (P<0.001, P<0.05) and VEGFR-2 (P<0.001, P<0.001) and CD34-labeled MVD (P <0.001,P<0.001,P<0.001) of the ischemic boundary zone compared to vehicle-treated rats; compared to sham-operated rats, the expression of SDF-1 (P<0.05) and MMP-9 (P<0.05) significantly augmented, exposure to SA with 10、5、2.5mg/kg markedly promoted the expression of SDF-1 (P<0.01, P<0.05) and MMP-9 (P<0.001, P<0.001, P<0.05). Conclusion:the results demonstrated that SA could decrease size of infarciton in myocardial ischemic rats, the underlying mechanisms might be to promote angiogenesis mediated by upregulating expression of VEGF and VEGFR-2, and increasing of SDF-1 and MMP-9.
Taken all together, Salvianolic acid A is one of the effectvie material basis of the promotion of angiogenesis by SSTG the underlying mechanisms might be to promote angiogenesis mediated by upregulating expression of VEGF and VEGFR-2, and increasing of SDF-1 and MMP-9.
研究发现,血管新生能帮助肌体建立有效的侧支循环,从根本上缓解心肌缺血症状,改善病情。目前国外大多研究利用基因方法,即直接补充VEGF或FGF等细胞因子的方法或蛋白方法及细胞移植等方法来促进冠心病患者的血管新生然而这些方法不但价格昂贵,而且目前还只是停留在研究阶段,尚无法应用于临床。
完整的内皮功能在心血管疾病的发生发展过程中扮演重要角色,其功能受损导致许多血管性疾病的发生。然而,成熟的内皮细胞是终端分化细胞,其再生能力有限。大量的研究表明EPCs (endothelial progenitor cells)在受损血管内皮的自我修复中发挥重要作用,它不仅参与胚胎时期血管生成,还与损伤的血管内皮的愈合和血管新生(neovascularization)密切相关,尤其是对于前者具有重要的促进作用。它主要存在于骨髓中,在某些生理、病理状态下,可从骨髓释放到外周血,随血液循环迁移到局部血管损伤处,分化为成熟的血管内皮细胞,从而修复损伤内皮、促进血管再内皮化。寻找能够改善内皮功能促进其修复,刺激血管新生的物质受到越来越多研究者的关注
中医治疗缺血性疾病的研究起源久远,并且取得了显著的成效。因此,从中药中寻找具有促进血管新生作用的药物具有重要的经济及社会意义。双参通冠方(SSTG)是由提取的人参总皂苷、丹参总酚酸、元胡总生物碱三种有效组分配伍而成,本实验室前期的药效学研究表明,该方能明显减轻急性心肌缺血犬和大鼠的心肌损伤程度,对缺血心肌有很好的保护作用。但是,之前缺少对其在促进血管新生作用、作用机理及物质基础方面的研究。
本课题采用鸡胚绒毛尿囊膜(chick embryo chorioallantoic membrane,CAM),体外培养内皮祖细胞数量、功能及冠状动脉结扎大鼠模型,观察SSTG促血管生成作用及其物质基础,探寻其作用机制,为其治疗缺血性心脏病的临床应用提供实验依据。
本课题分为三个部分：
一、双参通冠方(SSTG)对鸡胚绒毛尿囊膜(CAM)血管新生的影响
1. SSTG及有效组分含药血清对鸡胚绒毛尿囊膜血管新生的影响
目的：探讨SSTG及单味药提取物含药血清对鸡胚绒毛尿囊膜血管新生的影响。方法：将无菌8日龄鸡胚制备CAM模型,随机分成5组：对照组,SSTG、丹参总酚酸、人参总皂苷及元胡总生物碱组。将蛋壳用75%乙醇消毒后,用镊子小心开1.5cm×1.5cm的窗口,暴露CAM,将受试物品加到无菌的滤纸上,待风干后,小心置于CAM上血管较少的部位,将窗口封上,放入孵箱,于37℃,60%相对湿度继续孵育3天后,对CAM固定后,采集数据进行分析。结果：SSGT(3.25±0.80)、丹参总酚酸(3.11±0.83)及人参总皂苷组(2.97±0.47)血管密度比明显高于对照组(2.45±0.64)(P<0.05,P<0.05,P<0.05)。结论：SSTG、丹参及人参可明显促进鸡胚绒毛尿囊膜血管新生。
2.丹参总酚酸及丹酚酸A、B对鸡胚绒毛尿囊膜血管新生的影响
目的：探讨丹参总酚酸及丹酚酸A、B对鸡胚绒毛尿囊膜血管新生的影响。方法：同前。结果：总酚酸100mg/L (2.97±0.39)、300mg/L (3.07±0.36)给药组血管密度比明显高于对照组(2.47±0.35)(P<0.05,P<0.05)；丹酚酸A 5.56mg/L(4.42±1.16)、16.67 mg/L(5.51±1.15)、50 mg/L(4.60±0.71)给药组血管密度比明显高于对照组(3.07±0.85)(P<0.05,P<0.001,P<0.01)；丹酚酸B50mg/L(2.18±0.31)、150 mg/L(2.43±0.32)给药组血管密度比明显高于对照组(1.86±0.30)(P<0.05,P<0.01)。结论：丹参总酚酸、丹酚酸A及丹酚酸B可明显促进鸡胚绒毛尿囊膜血管新生
二、丹参总酚酸及丹酚酸A对内皮祖细胞(EPCs)功能的影响
1.内皮祖细胞的培养及鉴定
目的：建立新生鼠脾脏来源的内皮祖细胞的培养方法并对其进行鉴定。方法：密度梯度离心法分离SD新生鼠脾脏单个核细胞,EGM-2完全培养基贴壁法,37℃,5%CO2,饱和湿度的恒温培养箱中原代培养。细胞长至近融合时传代,分别于第9天进行细胞鉴定。内皮祖细胞鉴定：免疫细胞化学的CD34和VEGFR-2染色；内吞DiI标记的乙酰低密度脂蛋白(DiI-acLDL)和结合FITC标记的荆豆凝集素-1(FITC-UEA-1)的激光共聚焦显微镜观察及超微结构鉴定。结果：培养细胞的形态学改变：脾脏单个核细胞接种24h后部分细胞开始贴壁、变大,倒置显微镜下贴壁细胞透亮度增强,并逐渐伸出伪足样突起,呈小杆状或梭形。培养第3天贴壁细胞开始增殖,呈“集落”样生长,外周梭形细胞较多；共聚焦显微镜观察显示细胞DiI-acLDL和FITC-UEA-1呈红绿免疫双荧光阳性；培养第9天细胞VEGFR-2和CD34免疫细胞化学染色呈阳性；电镜下观察可见内皮细胞特异性的W-P小体存在；功能鉴定显示EPCs体外培养形成血管结构。结论：新生鼠的脾脏单个核细胞可以培养为内皮祖细胞并能够形成血管结构。
2.丹参总酚酸对正常及H2O2损伤内皮祖细胞功能的影响
目的：探讨丹参总酚酸对内皮祖细胞数量及功能的影响以及对H2O2损伤内皮祖细胞功能。方法：密度梯度离心法获取新生鼠脾脏单个核细胞,细胞长至融合状态后,收集贴壁细胞并加入丹参总酚酸(0.3,3和30mg/L)干预24h,分别观察其对EPCs的增殖、迁移、粘附、体外成管能力的影响。同时,应用200u mol/LH2O2对EPCs进行损伤,观察丹参总酚酸的保护作用。结果：丹参总酚酸促进EPC扩增,3及30mg/L丹参总酚酸作用24h对EPCs数量有显著性影响(P<0.05,P<0.05),同时也显著改善了EPCs的粘附、迁移及体外成管能力。对200μmol/LH2O2损伤EPCs的粘附及成管能力有明显的改善作用。结论：丹参总酚酸可增加EPCs数量并改善其功能并对H2O2损伤的EPCs的功能有改善作用。
3.丹酚酸A对正常及H2O2损伤内皮祖细胞功能的影响及初步机制研究
目的：探讨丹酚酸A对内皮祖细胞数量及功能的影响以及对H2O2损伤内皮祖细胞功能。方法：密度梯度离心法获取新生鼠脾脏单个核细胞,细胞长至融合状态后,收集贴壁细胞并加入丹酚酸A(10-9,10-8和10-7mol/L)干预24h,分别观察其对EPCs的增殖、迁移、体外成管能力的影响,并采用ELISA试剂盒检测加入丹酚酸A对培养上清中SDF-1及MMP-9含量的影响,采用NO试剂盒检测细胞上清中NO量的变化。同时,应用200μmol/LH2O2对EPCs进行损伤,观察丹酚酸A的保护作用。结果：丹酚酸A促进EPCs扩增,10-9,10-8和10-7mol/L酚酸A作用24h对EPCs数量有显著性影响(P<0.05,P<0.05),同时也显著改善了EPCs的迁移、成管能力；显著增加条件培养上清中SDF-1、MMP-9及NO的量。对200μmol/LH2O2损伤EPCs的粘附及成管能力有明显的改善作用。结论：丹酚酸A可增加EPC数量并改善其功能,其机制可能与增加SDF-1、MMP-9表达及NO的量有关,并对H2O2损伤的EPCs的功能有改善作用。
三、丹酚酸A对冠脉结扎大鼠血管新生的影响及机制研究
目的：探讨丹酚酸A对冠脉结扎大鼠血管新生的影响及其机制探讨。方法：结扎Wistar大鼠冠状动脉左旋降支建立心肌缺血大鼠模型,随机分为模型组,丹酚酸A 2.5、5、10mg/kg给药组,阳性对照药组及假手术组。于手术后第二天尾静脉注射给药,每天1次,7天后做以下实验：1)检测心肌缺血面积(NBT染色法)；2)HE染色观察缺血心肌的病理改变；3)免疫组化方法检测心肌梗死周围区VEGF/VEFGR-2的表达以及CD34标记的微血管密度；4) ELISA法检测血清中SDF-1及MMP-9表达量的变化。结果：与假手术组比较,模型组大鼠缺血心肌部位心肌细胞明显损伤(P<0.05),丹酚酸A 10、5剂量组梗死面积／心脏面积比值明显降低(P<0.01,P<0.05)；与假手术组比较,模型组梗死周围区CD34+细胞(P<0.05)明显增多,VEGF (P<0.001)和VEGFR-2 (P<0.001)表达明显增强,与模型组比较,丹酚酸A 10、5、2.5mg/kg剂量组梗死周围区微血管密度明显增多(P<0.001,P<0.001,P<0.001),丹酚酸A10、5mg/kg梗死周围区VEGF(P<0.001,P<0.05)和VEGFR-2 (P<0.001, P<0.001)表达显著升高；与假手术组比较,模型组SDF-1 (P<0.05)及MMP-9 (P<0.05)表达上升；与模型组比较,丹酚酸A10、5、2.5 mg/kg剂量组显著促进SDF-1 (P<0.01, P<0.05)及MMP-9(P<0.001,P<0.001,P<0.05)的表达。结论：丹酚酸A可降低心肌缺血大鼠梗死面积,其可能机制是通过上调VEGF和VEGFR-2及增加SDF-1、MMP-9的表达促进了内源性的血管新生。
综上所述,首先CAM模型的结果显示：双参通冠方具有促进血管新生的作用,其有效组分丹参总酚酸及人参总皂苷也具有促CAM血管新生的作用,我们选择了丹参作为继续研究的对象,结果显示丹酚酸A比丹酚酸B有更好的促进CAM血管新生的作用；丹酚酸A促进体外培养EPCs的数量的增加及功能的改善；整体心肌缺血模型的结果显示,丹酚酸A有明显降低缺血心肌梗死面积,促进缺血后心肌MVD增加的作用,其促进血管新生的可能作用机理为通过上调VEGF和VEGFR-2及增加SDF-1、MMP-9的表达促进EPCs的动员、分化与归巢,促进了内源性的血管新生。因此,我们得出结论：丹酚酸A为双参通冠方促血管新生的有效物质基础之一。
本论文的创新点：
1、首次研究了双参通冠方的促血管新生作用,为用于冠心病的治疗提供实验依据；
2、首次研究了丹酚酸A的促血管新生作用,并对其作用机制作了初步研究。
Cardiovascular disease is the global leading cause of death in the whole world. Currently available approaches for treating patients with ischemic heart disease include medical therapy or coronary revascularization by percutaneous coronary angioplasty (PCA) or coronary artery by pass grafting (CABG). However, a significant number of these patients are not candidates for coronary revascularization procedures or achieve incomplete revascularization with these procedures. Consequently, many of these patients have persistent symptoms of myocardial ischemia despite intensive medical therapy. Preliminary clinical experiences suggest that therapeutic angiogenesis may provide additional blood flow to incompletely revascularized areas. More recently, several studies suggest that implanted bone marrow cells may induce angiogenesis in ischemic myocardium.
Currently there are 3 major approches for therupetic angiogenesis, that is gene therapy, protein therapy and cellbased transplation. Howerve, theses methods are not only expessive but also on the stduy phase, and can not be used in clinical.
Endothelial dysfunction has been implicated in the pathogenesis of many diseases affecting the cardiovascular system. Experimental and clinical studies have shown that endothelial dysfunction may play a cusial role in the development and progression of vascular diseases, such as hypertension ro restenosis, as well as their complications, including myocardial infarction. Mature endothelial cells (ECs), however, have limited regenerative capacity. A growing body of evidence indicates that endothelial progenitor cells (EPCs) promote endothelial repair and contribute to ischemia-induced neovascularization. Increasing evidence suggests that circulating progenitor cells contribute to postnatal neovascularization except in embro. These cells are derived form bone-marrow, in some physiological and pathological status, they are released from bone-marrow into circulation, home to sites of ischemia, adopt an endothelial phenotype, and contribute to new blood vessel formation. Hence, the discovery of candidate molecules able to augment EC funtion to stimulate myocardial angiogenesis has stirred a growing interest in using these molecules for therapeutic application.
Shuangshentongguan (SSTG) is made up of ginsenosides, salvianolic acids and corydalines. Our previous pharmacological studies showed that SSTG could relieve myocardial damage of dog and/or acute myocardial ischemia of rat, show protective effects on myocardium infartion. But, there is little information about its'effect on theraputic angiogenesis and its matierial basis.
So, we designed the experiments to test SSTG effects on angiogenesis, it is realised by 3 models, they are as follows:chick embryo chorioallantoic membrane, endothelial progenitor cell and myocardial ischemia in rats induced by ligation of the left anterior descending (LAD) coronary artery.
Chapter 1:Effects of SSTG on angiogenesis of the chick embryo chorioallantoic membrane
Part 1:Effects of SSTG on angiogenesis of the chick embryo chorioallantoic membrane
Objective:To investigate the effect of SSTG on angiogenesis of the chick embryo chorioallantoic membrane (CAM). Methods:Fifty 8-day-old fertilized eggs were obtained from a commercial source (Beijing Merial Vital Laboratory Animal Technology Co., Ltd). On day 9 of the incubation, the fertile eggs were randomly allocated into five groups. Fertilized eggs are first disinfected with 75%alcohol, a window opening is punctured at the blunt end of the egg facing upwards using sterilized forceps. The eggshell above the air cell was removed and the shell membrane attached to it, was cut off for further examination. Normal development was verified and embryos with malformations or dead embryos were excluded. CAMs (10 eggs per group) were treated as described as follows:1) overlaying them with 20-mm2 sterilized filter discs that had been loaded with 10μ1 of serum without any test materials (negative control),10μl of serum containing SSTG, ginsenosides, salvianolic acids and corydalines. The window was sealed with tape and the eggs were returned to the incubator. After incubation for an additional 72 hours, all samples were immediately fixed in methanol and acetone (1:1) for at least 15min, then they were photographed using a stereomicroscope equipped with a camera and image analysis system. The angiogenic response was evaluated by counting the vessel density converging toward the filter discs..Result:The angiogenesis area in SSTG, ginsenosides, salvianolic acids groups was obviously increased compared with control group (P<0.05, P<0.05, P<0.05). Conclusion:The result showed that SSTG, ginsenosides, salvianolic acids can obviously stimulate angiogenesis of the chick embryo CAM.
Part 2:Effects of salvianolic acids, salvianolic acid A and salvianolic acid B on angiogenesis of the chick embryo chorioallantoic membrane
Objective:To investigate the effect of salvianolic acids, salvianolic acid A and salvianolic acid B on angiogenesis of the chick embryo chorioallantoic membrane (CAM). Methods:the same as above-mentioned. Result:The angiogenesis area in salvianolic acids 100 mg/L (2.97±0.39)、300 mg/L (3.07±0.36) groups was obviously increased compared with control group (2.47±0.35) (P<0.05, P<0.05); the angiogenesis area in salvianolic acid A 5.56 mg/L (4.42±1.16)、16.67 mg/L (5.51±1.15)、50 mg/L (4.60±0.71) was obviously increased compared with control group (3.07±0.85) (P<0.05, P<0.001, P<0.01); the angiogenesis area in salvianolic acid B 50mg/L (2.18±0.31)、150 mg/L (2.43±0.32) was obviously increased compared with control group (1.86±0.30) (P<0.05, P<0.01). Conclusion:The result showed that salvianolic acids, salvianolic acid A and salvianolic acid B can obviously stimulate angiogenesis of the chick embryo CAM.
Chapter 2:Effects of Salvianolic acids (SAs) and Salvianolic acid A on the numbers and function of endothelial progenitor cells
Part 1:cultivation and identification of endothelial progenitor cells
Objective:To investigate the methods of isolation, cultivation and identification of endothelial progenitor cells (EPCs) from rat spleen, as well as their ability of differentiating into endothelial cells. Methods:The mononuclear cells were isolated f rom SD rat spleen and were cultured in vitro via adhesion selection methods. The expression of CD34 and VEGFR-2 were assessed by immunocytochemistry after 9d cultivation. And the adherent cells were stained with DiI complexed acetylated low-density lipoprotein (DiI-acLDL) and fluorescein Ulex Europaeus agglutinin-1 (FITC-UEA-1), and then viewed by laser scanning confocal microscope (LSCM) to confirm EPCs lineage. EPCs phenotype was also tested by ultrastructural analyses. And EPCs were assessed by lightmicroscopy for their capacity of independent tubulogenesis. Results:The adherent cells stretched and exhibited the clone-like morphology in about 20d of cultivation. Immunocytochemistry stain showed that the adherent cells were positive for CD34 and VEGFR-2 from 9d after culture. The cells could take up DiI-acLDL, and bind to FITC-UEA-1, showed double positive fluorescence under LSCM. EPCs phenotype was also confirmed by the presence of Weibel-Palade body in cytoplasm by ultrastructural analyses. Branched interconnecting EC-specific tubules formed with EPCs. Conclusion:EPCs are enriched in rat spleen and may exhibited some of the characteristics of endothelial cells (ECs) after 9d inducing culture in vitro.
Part 2:Effects of Salvianolic acids (SAs) on the numbers and function of endothelial progenitor cells in normal and H2O2 injured station
Objective:Salvia miltiorrhiza (SM, also known as DanShen) is one of the well-known widely used Chinese herbal medicines in clinical. In this study we aimed to demonstrate the effects of Salvianolic acids (SAs), the main active components of aqueous extract of SM on the numbers and function of endothelial progenitor cells in normal and H2O2 injured station. Methods:To do this, new-born rat spleen mononuclear cells were isolated by density gradient and EPCs were expanded. EPCs proliferation, migration and in vitro vasculogenesis activity were assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, transwell chamber assay, and in vitro vasculogenesis kit, respectively. EPCs adhesion assay was performed by replating those on fibronectin-coated dishes, and then counting adherent cells. Results:Incubation of EPCs with SAs increased the numbers of EPCs and promoted EPCs migratory, adhesive, and in vitro vasculogenesis capacity, could also augment function of EPCs injured by H2O2. Conclusion:The results of the present study suggest that SAs may be developed as a new therapeutic remedy for various ischemic diseases such as coronary artery disease.
Part 3:Effects of Salvianolic acid A on the numbers and function of endothelial progenitor cells in normal and H2O2 injured station
Objective:Salvianolic acid A (SA)is one of the well-known widely used Chinese herbal medicines in clinical. In this study we aimed to demonstrate the effects of SA on the numbers and function of endothelial progenitor cells in normal and H2O2 injured station. Methods:same as above-mentioned. Results:Incubation of EPCs with SA increased the numbers of EPCs and promoted EPCs migratory, adhesive, and in vitro vasculogenesis capacity. Besides, SA could augment function of EPCs injured by H2O2 Conclusion:The results of the present study suggest that SA may be developed as a new therapeutic remedy for various ischemic diseases such as coronary artery disease.
Chapter 3:Effects of Salvianolic acid A on angiogenesis after myocardial ischemia in rats and underlying mechanism.
Objective:To investigate the effects of SA on angiogenesis after myocardial ischemia in rats and underlying mechanism. Methods:myocardial ischemia was induced by occluding the left anterior descending (LAD) coronary artery of the hearts of wistar rats. After operation, they were randomly departed into 6 groups: vehicle-treated, SA 2.5、5、10mg/kg groups, positive group and sham-operated group. On 2 day of operation, ischemiac rats received tail intravenous injection once a day. At 7 day post ischemia, these experiments were done:1) to test of area of ischemiac myocardium (NBT staining); 2) to observe pathological chang in myocardium by HE staining; 3) to evaluate the localization and expression of VEGF and VEFGR-2 and CD34-labeled microvascular density (MVD) of the ischemic boundary zone by immunohistochemistry methods; 4) to test SDF-1 and MMP-9 expression in serum by ELISA. Results:compared to sham-operated rats CD34 positive cells(P<0.05), the expression of VEGF (P<0.001) and VEGFR-2 (P<0.001) significantly augmented, exposure to SA with 10、5mg/kg markedly promoted the expression of VEGF (P<0.001, P<0.05) and VEGFR-2 (P<0.001, P<0.001) and CD34-labeled MVD (P <0.001,P<0.001,P<0.001) of the ischemic boundary zone compared to vehicle-treated rats; compared to sham-operated rats, the expression of SDF-1 (P<0.05) and MMP-9 (P<0.05) significantly augmented, exposure to SA with 10、5、2.5mg/kg markedly promoted the expression of SDF-1 (P<0.01, P<0.05) and MMP-9 (P<0.001, P<0.001, P<0.05). Conclusion:the results demonstrated that SA could decrease size of infarciton in myocardial ischemic rats, the underlying mechanisms might be to promote angiogenesis mediated by upregulating expression of VEGF and VEGFR-2, and increasing of SDF-1 and MMP-9.
Taken all together, Salvianolic acid A is one of the effectvie material basis of the promotion of angiogenesis by SSTG the underlying mechanisms might be to promote angiogenesis mediated by upregulating expression of VEGF and VEGFR-2, and increasing of SDF-1 and MMP-9.
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