【作者】 霍青；

【导师】 陈晓虎；

【作者基本信息】 南京中医药大学 ， 中西医结合临床， 2010， 博士

【摘要】 本实验为山东省教育厅课题：课题编号-J05L05。目的：研究表明,原发性高血压与内皮细胞功能障碍有关。理想的抗高血压药物除降低血压外,还应具有改善高血压血管结构和功能,逆转动脉重塑,保护内皮细胞功能和保护靶器官的作用。钩藤为临床治疗高血压病的常用药物,钩藤的有效组分钩藤总生物碱有高生物活性,有较好的降压作用,其降压机制尚不明确。以往研究表明钩藤总生物碱通过抗高血压血管重塑、抑制血管平滑肌细胞增殖的途径来降血压,通过抑制内皮细胞衰老降压这一领域尚未涉及。本研究通过探索钩藤总生物碱抗高血压血管重塑、保护内皮细胞结构功能、抑制血管内皮细胞衰老的作用及其机制,试图开辟其抑制内皮细胞衰老----保护内皮细胞功能—逆转血管重塑—降低血压、保护靶器官这一防治高血压的新途径。方法：在体实验以自发性高血压大鼠(SHR)为模型,观察钩藤总生物碱对SHR尾动脉血压的影响；对大鼠胸主动脉、肠系膜动脉组织学和体视学变化的影响；对大鼠胸主动脉内皮超微结构的影响；对大鼠胸主动脉内皮β-半乳糖苷酶表达和端粒酶活性相对表达量的影响。以研究钩藤总生物碱降血压、改善血管重塑的效果及可能的机制。离体实验分别建立培养以AngⅡ诱导增殖和D-半乳糖诱导衰老的大鼠胸主动脉内皮细胞(RAEC)模型,MTT法观察钩藤总生物碱对RAEC增殖活度的影响；免疫组织化学法观察其对RAEC Ca2+浓度相对表达值的影响；流式细胞仪观察其对RAEC细胞周期的影响；扫描电镜观察其对RAEC超微结构的影响；β-半乳糖苷酶染色显示其对RAEC衰老阳性率的影响；PCR-ELISA法检测其对RAEC端粒酶活性的影响；流式细胞仪观察其对衰老RAEC凋亡的影响,以研究钩藤总生物碱抑制AngⅡ诱导的RAEC增殖、抑制D-半乳糖诱导的RAEC衰老及其促衰老RAEC凋亡的作用及可能机制。结果：在体实验表明钩藤总生物碱可以降低SHR尾动脉血压,与模型组相比有非常明显差异(P<0.01)；钩藤总生物碱组大鼠胸主动脉、肠系膜动脉中膜厚度／管腔直径的比值与模型组比较均明显降低(P<0.05)；各组胸主动脉内皮扫描电镜观察结果与模型组比较,钩藤总生物碱组内皮形态结构均有明显改善,表现在内皮细胞排列整齐,表面光滑,内皮细胞脱落明显减少,内膜损伤明显减轻；钩藤总生物碱组大鼠胸主动脉内皮β-半乳糖苷酶表达与模型组比较均明显降低(P<0.05)；钩藤总生物碱组大鼠胸主动脉内皮端粒酶活性相对表达量与模型组比较均明显降低(P<0.05)。离体实验：MTT实验表明血管紧张素Ⅱ具有明显促进RAEC增殖作用,钩藤总生物碱能抑制血管紧张素Ⅱ诱导的RAEC增殖；钩藤总生物碱组RAEC Ca2+浓度相对表达值明显降低(P<0.05)；细胞周期研究发现钩藤总生物碱组与诱导增殖组相比,G1期增高(P<0.05),S期明显降低(P<0.01),可见钩藤总生物碱可以阻断细胞由Go／Gl期向DNA合成的S期转化,从而抑制培养的RAEC衰老的作用；RAEC超微结构电镜扫描表明与D-gal诱导衰老组比较,钩藤总生物碱组的RAEC形态结构如细胞形态、大小及微绒毛数量、分布均有明显改善；β-半乳糖苷酶染色表明,D-半乳糖具有明显诱导RAEC衰老的作用,钩藤总生物碱组β-半乳糖苷酶表达的衰老细胞阳性率较D-半乳糖诱导衰老组非常明显降低(P<0.01)；钩藤总生物碱组端粒酶活性检测与模型组比较均明显降低(P<0.05)；钩藤总生物碱可以显著促进衰老内皮细胞的凋亡(P<0.05)。结论：研究表明钩藤总生物碱可以显著降低SHR尾动脉血压,有明显的降血压的作用；具有明显的改善、逆转高血压血管重塑的效应；具有明显改善血管内皮结构和功能,保护血管内皮的功能；具有明显抑制血管内皮衰老的作用。钩藤总生物碱可抑制AngⅡ的促RAEC增殖效应,对D-半乳糖介导的RAEC衰老具有明显的抑制作用,可明显改善RAEC结构和功能,降低衰老细胞阳性率和端粒酶活性,有显著的抑制RAEC衰老的作用,有明显的促进衰老的RAEC凋亡的作用。更多还原

【Abstract】 This experiment had come from shandong provincial projects:topic Numbers--J05L05.Objective:The recent studies indicate that, primary hypertension pertains to endothelial function obstacle. The ideal antihypertensive medication besides lower blood pressure, still should improve the structure and function of the vascular hypertension, reverse arterial remodeling, and make the function of normal, protect endothelial function and protection of target organs. The Uncaria rhynchOphylla is commonly used medicine for clinical treatment of hypertension. The effective components of the Uncaria rhynchOphylla total alkaloids have high biological activity, having good step-down, resisting myocardial reconstruction and antiarrhythmic effect, but the hypotensive mechanism is unclear. Previous research shows that, total alkaloids lower blood pressure through antihypertensive vascular remodeling, inhibit antihypertensive vascular smooth muscle cell proliferation. The field Inhibiting endothelial cell ageing step-down has not involved. Therefore, this research explored the function and mechanism of the Uncaria rhynchOphylla that antihypertensive vascular remodeling, inhibit endothelial cell aging. And try to open up its by inhibiting endothelial cell senescence-to protect endothelial cell function-reverse the vascular remodeling-lower blood pressure-protect the target organs, an effective way to control the field of hypertension.Methods:In vivo experiments, we took spontaneously hypertensive rats (SHR) as a model. Then we observed the influence of Uncaria alkaloids on the blood pressure of SHR tail artery, on the rat thoracic aorta, mesenteric artery histological and stereological changes, on the rat aorta endothelial ultrastructure, on the Rat aortic endothelialβ-galactosidase expression and telomerase activity expression of the relative. On this basis, we study the effect and possible mechanism of Uncaria rhynchOphylla total alkaloids step-down, improve the effect of vascular remodeling. In vitro experiments, we build RAEC model AngⅡinduced by the proliferation and D-galacitiol induced agingl respectively. We observed the effects of total alkaloid on RAEC proliferation activity by MTT method, on RAEC Ca2+concentration the value of the relative expression by immunohistochemistry, on RAEC group cell cycle By flow cytometry, on RAEC ultrastructure by scanning electron microscopy,on positive rate of aging RAEC byβ-galactosidase staining, on the expression of telomerase activity in RAEC by PCR-ELISA assay and on the apoptosis of aging RAEC. To study the effect and the possible mechanism of Uncaria rhynchOphylla total alkaloids that AngⅡRAEC induced by inhibiting proliferation, restrain D-galacitiol RAEC induced aging and promote aging RAEC apoptosis.Results:The vivo experiments show that RTA can reduce the total alkaloid SHR tail arterial blood pressure and has very obvious difference compared with the model group(P<0.01). Comparing with the model group, the RTA group, mesenteric artery and the thoracic aorta film thickness/lumen diameter ratio reduced significantly(P<0.05). The results of thoracic aortic endothelial scanning electron microscope shows that the RTA group s endothelial form structure were obviously improvedCompared comparing with the model group. It performances that endothelial cells aligned with a smooth surface, the cell shedding reduced obviously and lining damage ligtening obviously. The thoracic aortic endothelial rats beta galacitiol nucleoside enzyme expression of the RTA group reduced significantly compareing with the model group (P <0.05). AT the same time, the thoracic aortic endothelial rats relative express telomerase activity of the RTA group reduced significantly compareing too with the model group (P<0.05). The vitro experiments indicate that:The MTT experiments showed thatⅡangiotensin significantly promote RAEC proliferation, the Total alkaloid can inhibit the proliferation of angiotensinⅡ-induced RAEC, the RAEC Ca2+concentration of the RTA group significantly reduce relative to express (P<0.05), It was found that G1 period became high (P<0.05) and S period became lower (P<0.01) in the RTA group, and the RTA blocked the transform from G0/G1 period to synthesis of DNA period, so that theRAEC proliferation was inhibited. RAEC ultrastructure scanning electrometry microscopy showed that the RAEC morphological structure of the RTA group improved obviously, such as the cell morphology and size and microvilli quantity and distribution. Beta galacitiol nucleoside enzymes stain shows that D-galacitiol has obvious effect of RAEC induced aging, the rate of senescent cells of the RTA group reduced obviously (P<0.01). Detection of telomerase activity were significantly lower compareing with the model group (P<0.05). The RTA can improve senescence endothelial cell apoptosis significantly(P<0.05).Conclusion:This medicine opened a new field for traditional Chinese medicine to control of hypertension,though the effective way that inhibitting endothelial cells-protecting endothelial function-reversing vascular remodeling-reducing blood pressure-protecting target organs. Research shows that RTA can significantly reduce the total alkaloid SHR tail arterial blood pressure.It has an significant function to lower blood pressure. The RTA has the obvious improvement, vascular remodeling of reverse hypertension. It can improve sendothelial function and structure and protect endothelial function and inhibit endothelial function of aging significantly. The RTA can inhibit Ang II RAEC proliferation-promoting effect. For D-galacitiol mediated RAEC aging has obvious inhibition. The RTA can obviously improve the structure and function of the RAEC. The RTA can reduce positive senescent cells and the telomerase activity. The RTA can inhibit the RAEC aging significantly. The RTA can improve senescence endothelial cell apoptosis significantly.更多还原