【作者】 黄健安；

【导师】 张敏州；

【作者基本信息】 广州中医药大学 ， 中西医结合临床， 2010， 硕士

【摘要】 研究背景内皮祖细胞是一类能增殖并分化为血管内皮的前体细胞,内皮祖细胞在维持血管内皮的功能及血管新生中起重要作用。益气活血中药通冠胶囊对血管内皮细胞具有保护作用,能促进冠心病患者介入术后心功能的恢复,抑制介入术后再狭窄的发生。通冠胶囊的生物学作用与内皮祖细胞介导的再内皮化和改善缺血组织血供的生物学功能相似。同时,在通冠胶囊组方中,部分药物具有显著改善内皮祖细胞的增殖、粘附、迁移等生物学功能。研究目的探讨益气活血中药通冠胶囊对内皮祖细胞的生物学功能的作用及其机制。研究方法1临床资料回顾分析,选择符合入选条件的60例冠心病患者,其中冠心病基础用药30例和治疗组(冠心病基础用药+通冠胶囊)30例,回顾分析两组患者一般情况、冠脉造影和手术方式；对比分析术前、术后1月左室射血分数、室壁运动指数和心功能分级；术后1月,观察两组患者主要心血管事件发生率；对比分析术前和术后1月两组患者血清VEGF水平。2实验部分,给予SD大鼠通冠胶囊水煎剂灌胃,生理盐水灌胃做对照组,9天后提取大鼠外周血单个核细胞,用EGM-2完全培养基诱导其分化,通过观察细胞生长的形态学特征和激光共聚焦显微镜观察其具有内吞acLDL和结合UEA-1的内皮能力,鉴定为内皮祖细胞。对比两组的集落数量、迁移能力、粘附能力和血清VEGF水平。分离并培养健康志愿者循环内皮祖细胞,使用激光共聚焦显微镜鉴定其具有内皮功能,流式细胞仪检测其细胞表面抗原CD34+与VEGFR+2+表达。通过血清药理学研究方法,制备通冠胶囊含药血清,同时给予大鼠生理盐水灌胃,作为空白对照组。细胞经同步化后,随机分为四组：生理盐水组、通冠胶囊含药血清组、辛伐他汀组和通冠胶囊含药血清与PI3K/Akt抑制剂(Wortmannin+LY294002)组,干预24小时后,检测细胞迁移能力、粘附能力、增殖能力和细胞增殖周期比例。研究结果1临床资料回顾分析,两组患者术前基本资料、手术方式、左室射血分数以及室壁运动指数、心功能分级和血清VEGF水平无明显差异。术后1月,两组左室射血分数、室壁运动积分均较术前改善(均P＜0.05),治疗组较对照组可进一步改善左室射血分数及室壁运动积分(P＜0.05)。对照组有1例因重症左心功能不全,于术后4周死亡；两组患者在观察时间内均无靶血管血运重建。对照组新发的心功能衰竭发生率明显高于治疗组(P=0.045)；两组主要心血管事件发生率比较,治疗组为6.7%,对照组为33.3%,治疗组明显低于对照组(P=0.024)。组术前比较,差异无统计学意义(P＞0.05)。两组术后30天较术前血清VEGF水平均减低,差异有显著性意义(P均＜0.05)。但是术后30天治疗组血清VEGF水平均明显高于安慰剂对照组,差异有显著性意义(P均＜0.01)。2大鼠内皮祖细胞的生长及形态学观察和鉴定：接种第5天,贴壁的梭状细胞开始从集落的边缘长出,形成典型的集落现象。第7天后形成了长梭状细胞。通过激光共聚焦显微镜观察,鉴定其为内皮祖细胞。3大鼠内皮祖细胞集落计数、迁移、粘附能力及血清VEGF水平结果：通冠胶囊组与生理盐水组相比,细胞集落数量和迁移能力明显增强,具有显著性差异(均P＜0.01),两组的粘附能力和血清VEGF水平无明显差异(均P＞0.05)。4人内皮祖细胞的鉴定结果：培养第5天,贴壁的梭状细胞开始从集落的边缘长出,形成典型的集落现象。第7天后形成了长梭状细胞。通过激光共聚焦显微镜观察,鉴定其为内皮祖细胞。经流式细胞仪检测,贴壁细胞同时表达CD34+和VEGFR-2+的为15.4±1.8%。5各分组人内皮祖细胞的迁移、粘附、增殖能力和增殖周期比例结果：1umol／L辛伐他汀细胞的增殖能力和增殖周期比例,与其它辛伐他汀浓度组比具有显著差异(P＜0.01)。各组与生理盐水组相比,细胞的迁移、粘附、增殖能力和细胞增殖周期期比例均显著显著增高(P＜0.01)。通冠胶囊组与辛伐他汀组相比,粘附能力、迁移和增殖周期比例明显降低(P＜0.01),但增殖能力明显提高(P＜0.05)。通冠胶囊与PI3K通道蛋白阻滞剂组相比,细胞迁移、粘附、增殖、细胞增殖周期比例均显著增高(P＜0.01)。结论1.通冠胶囊能够明显改善冠心病PCI术后患者左室射血分数、室壁运动积分和心功能分级,并上调PCI术后患者血清VEGF的水平。2.通冠胶囊能够显著动员大鼠骨髓内皮祖细胞至外周血循环,并增加其迁移能力。通冠胶囊含药血清能显著提高体外培养人内皮祖细胞的迁移、粘附能力、增殖能力和增殖周期比例,但作用效果不及辛伐他汀。PI3K/AKT通道蛋白阻滞剂明显减弱通冠胶囊的生物学功能。生理条件下,通冠胶囊改善内皮祖细胞的生物学功能机制可能其机制与PI3K/AKT通道的活化相关,但不完全依赖PI3K通道,同时通冠胶囊动员内皮祖细胞的机制可能不依赖于提高血清VEGF水平。更多还原

【Abstract】 BackgroundEndothelial Progenitor cells can proliferate and differentiate into vascular endothelial cells (EPC). EPC derived from bone marrow play an important role in vascular functions and angiogenesis after Ischemia. Tongguan Capsule, a protective effect on vascular endothelial cells, not only promotes the recovery of cardiac function of the patients after percutaneous coronary intervention, but also significantly inhibits the restenosis in stent. The effect of Tongguan Capsule is so similar to EPC biology, which mediated the re-endothelialization and improved the blood supply of ischemic tissue. Some Herbs, in Tongguan capsule prescription, have significantly improved the proliferation, migration, adhesion capacity of EPC.Research PurposesTo explore the effects and Mechanisms of Tongguan Capsul mobilizing bone marrow—derived endothelial progenitor cells.Methodology1 Retrospective analysis of clinical data:60 cases of eligible patients with coronary heart disease, who were treated with percutaneous coronary intervention (PCI), Of which 30 cases of patients were treated with conventional western medicine (the control group) and other 30 cases were treated with Tongguan Capsule and conventional western medicine(the treatment group). The clinical characteristics, coronary angiography and surgical methods were Analysis. Comparative Analysis the left ventricular ejection fraction (LVEF), ventricular wall motion parameters and cardiac function classification of NYHA before the operation and 30 days after the operation. The Major adverse cardiac events (MACE) were Analysis in 30 days after PCI. The level of the serum VEGF before the operation, and 30 days after the operation were Analysis.2 The peripheral blood mononuclear cells were extracted from SD rat, which were given Tongguan Capsule for 9 days. Mononuclear cells were cultivated with endothelial growth medium-2. After being cultured for 7d, the attached cells were identified as EPC by uptaking DiI-acLDL and binding FITC-lectin-1. We measured the number of colony-forming units of EPC. Cell Migration ability was examined by migration assay. The adhesion activity was determined by cell counts. The level of the serum VEGF was measure by ELISA. Isolate and culture the circulating EPC from healthy volunteers. The attached cells were characterized by uptaking Dil-acLDL and binding FITC-lectin-1. And also the attached cells were determined expressing CD34+ and VEGFR-2+ by flow cytometry. Preparation Tongguan Capsule serum by serum pharmacology method. Saline were given to the SD rat as the control. Cultured EPC were incubated with Tongguan Capsule serum and Tongguan Capsule serum with PI3K/Akt inhibitors (wortmannin and LY294002), simvastatin as a positive control. The cell migration ability was examined by migration assay, the adhesion activity was determined by cell counts The proliferation was determined by MTT assay and cell proliferation cycle analysis assay.Research Results1 Clinical Retrospective analysis:Before the operation the clinical characteristics, coronary angiography, surgical methods, LVEF, ventricular wall motion parameters and cardiac function classification of NYHA were no difference in two groups.30 days after the operation, the LVEF, ventricular wall motion parameters of two groups are improved (P<0.05), Meanwhile, compared with the control group, the LVEF and ventricular wall motion scores of the treatment group were further improved. Duing to the severe left ventricular dysfunction, one case in control group was died at 4 weeks after the operation.30 days after the operation, there were on difference in nonfatal myocardial infarction, target vessel revascularization between two groups. The incidence of new heart failure of the treatment group was statistically lower than the control group (P=0.045), thus, the incidence of MACE in the treatment group was statistically lower than the control group (P=0.024). The level of VEGF were decreased significantly at 30 days in two groups. The level of VEGF at 30 days after the operation in the treatment group had significant differences compared with the control group(P<0.01).2 At the fifth day, rat mononuclear cells changed from round to spindle-shaped and grow into a colony. At the seventh day, some of the long spindle-shaped cell showing the distribution of cord-like or tubular. Cells were Identified as EPC that it could uptaking Dil-ac-LDL and binding FITC-lectin-1.3 Result of rat EPC colony-forming units, migration assay, adhesion assay and serum VEGF level assay:compared with the saline group, the numbers of colony-forming units and migration in Tongguan capsule group were significant (P<0.01). The number of adhesion and VEGF level in Tongguan capsule group, were no differences (P>0.05)4 At the fifth day, human mononuclear cells cells changed from round to spindle-shaped and grow into a colony. Cells were Identified as EPC that it could uptaking Dil-ac-LDL and binding FITC-lectin-1. The attached cells positive in expressing both CD34+and VEGFR-2+ were about 15.4±1.8%.5 Result of human EPC migration assay, adhesion assay, proliferation assay and cell proliferation cycle analysis assay:simvastatin as a positive control, compared with other Concentration, 1umol/L simvastatin has a were significant effect on EPC proliferation and proliferation cycle. Compared with the saline group, the numbers of EPC migration, adhesion, proliferation and cell proliferation cycle in Tongguan capsule serum group, lumol/L simvastatin group, and PI3K/Akt inhibitors group were significant (P<0.01). Compared with lumol/L simvastatin group, Tongguan capsule serum group have a lower effect on EPC adhesion and cell proliferation cycle (P<0.01), but have a higher effect on proliferation(P <0.01). Compared with PI3K/Akt inhibitors group, Tongguan capsule serum group have a higher effect on the numbers of EPC migration, adhesion, proliferation and cell proliferation cycle (P<0.01).Conclusion1 Tongguan capsule can significantly improve left ventricular ejection fraction, ventricular wall motion parameters, cardiac function classification of NYHA and serum VEGF levels in patients with coronary heart disease after percutaneous coronary intervention.2 Tongguan capsule can significantly mobilized rat bone marrow EPC to peripheral blood and enhance the biological function of migration. In vitro, Tongguan capsule serum significantly increased in human EPC migration, adhesion and proliferation, but effects were not so as simvastatin. PI3K/AKT inhibitors significantly reduced the biological functions of Tongguan capsule. The mechanisms on the effect of Tongguan Capsule mobilizing bone marrow—derived EPC may not be by increasing the serum VEGF levels and do not rely entirely on the activation of PI3K/AKT signal.更多还原