自噬在血管内皮细胞衰老中的作用及17β-雌二醇对其p53通路的干预研究
发布时间：2019-07-08 13:42
【摘要】：【研究背景与目的】人口老龄化是全世界面临的共同难题,在我国尤为严峻。衰老带来的衰老相关疾病发病率增高是社会医疗资源紧缺,社会负担过重的罪魁祸首。血管衰老是机体衰老的一大特征,而血管内皮细胞(Vascular endothelial cell,VEC)衰老是血管衰老的关键环节与始动因素,也是决定动脉粥样硬化、脑卒中、帕金森等众多衰老相关疾病转归与预后的关键因子。雌激素(Estrogen)是众所周知的心血管保护因子,17β-雌二醇(17β-estradiol,17β-E2)是其主要活性成分,也是较为明确的可延缓VEC衰老的药物之一,但目前对其具体作用机制所知甚少。自噬(Autophagy)是细胞“自我消化”现象,饥饿、氧化应激、缺氧均可改变细胞自噬活性。p53是一种肿瘤抑制蛋白,与DNA损伤后修复、细胞周期调控、凋亡、自噬密切相关,也是众多通路信号中转站,影响着多个通路正常工作。有研究表明,位于细胞核的p53可通过多种方式激活自噬,而17β-E2可通过受体作用增加核内p53的表达,再结合我们前期研究发现17β-E2可延缓VEC衰老,于是我们猜想17β-E2可能通过p53通路上调自噬发挥延缓衰老作用,但国内外仍缺乏相关系统研究。为此,我们将结合p53通路,研究自噬在VEC衰老中的作用及17β-E2的干预机制。【方法】第一部分:研究VEC衰老与自噬的关系共设4个组:空白组、过氧化氢(Hydrogen peroxide,H_2O_2)组、雷帕霉素(Rapamycin,Rapa)+H_2O_2组和3-甲基腺嘌呤(3-methyladenine,3-MA)+H_2O_2组。从细胞活力、衰老染色、内皮素-1(Endothelin,ET-1)、一氧化氮(Nitric oxide,NO)、亚细胞结构改变和磷酸化成视网膜瘤蛋白(p-Rb蛋白)表达情况检测细胞衰老程度,再检测各组细胞微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)、Beclin-1和p62表达检测细胞自噬活性;通过对比H_2O_2组与自噬阳性对照、阴性对照差异,分析VEC衰老与自噬关系。第二部分:研究17β-E2对VEC衰老及自噬活性的影响共设4个组:空白组、H_2O_2组、17β-E2组和17β-E2+H_2O_2组。从细胞活力、细胞周期、衰老染色、ET-1、NO和p-Rb蛋白表达情况检测细胞衰老程度,再检测各组细胞LC3-II、Beclin-1、p6和免疫荧光检测自噬小体来分析17β-E2延缓衰老作用和自噬的关系。第三部分:研究17β-E2对p53信号通路的影响共设3个组:空白组、17β-E2组、17β-E2+H_2O_2组。通过检测p53通路下游通路蛋白p-p53、PUMA、MDM2表达水平分析17β-E2对p53通路的作用。【结果】1.适当上调自噬可延缓H_2O_2诱导的VEC衰老Rapa+H_2O_2组LC3-II、Beclin-1蛋白表达增多,p62减少,自噬小体数量增加,与H_2O_2组相比较细胞活力升高,SA-β半糖苷酶阳染率降低,p-Rb表达减少,内皮细胞结构和ET-1、NO相对正常;而予以自噬抑制剂3-MA则得出相反结论,细胞微观结构和功能指标提示其比H_2O_2组衰老更为明显。2.17β-E2可通过上调自噬延缓H_2O_2诱导的VEC衰老与H_2O_2组相比,17β-E2+H_2O_2组LC3-II、Beclin-1表达增多,p62表达减少,自噬小体荧光数量增多,自噬活性增高,细胞活力增加SA-β半糖苷酶阳染率降低,S期细胞明显增加,NO浓度增加,ET-1减少,p-Rb蛋白表达减少;即17β-E2可延缓VEC衰老,同时增强自噬活性。3.17β-E2具有激活p53通路的作用与空白组相比,17β-E2+H_2O_2组p53、p-p53和PUMA表达明显增加,MDM2表达减少;除去H_2O_2影响,17β-E2组p53通路蛋白也明显增加,负性调节蛋白MDM2明显减少。即17β-E2可激活H_2O_2诱导VEC的p53通路。【结论】200mmol/L的H_2O_2作用24小时可成功诱导出HUVEC衰老模型;适当地上调自噬具有一定延缓VEC衰老的作用;17β-E2可能通过上调自噬延缓H_2O_2诱导的VEC衰老,该作用可能与激活p53信号通路有关。
文内图片：
图片说明：动脉粥样硬化发展的几个环节
[Abstract]:[Study Background and Objective] Population aging is a common problem facing the whole world, especially in China. The incidence of aging-related diseases caused by aging is the main cause of the shortage of social medical resources and overburdened society. The aging of vascular endothelial cells (VEC) is a key factor of the aging of the body, and the vascular endothelial cell (VEC) aging is the key factor of the outcome and prognosis of many aging-related diseases such as atherosclerosis, stroke and Parkinson's disease. Estrogen is a well-known cardiovascular protective factor, and 17%-estradiol (17-stradiol,17--E2) is one of the most important drugs for delaying the aging of the VEC, but it is very little known to its specific mechanism. p53 is a kind of tumor suppressor protein, which can be repaired after DNA damage, and can be regulated by cell cycle. [Methods] The first part: To study the relationship between the aging of VEC and autophagy in four groups: blank group, hydrogen peroxide (Hggen peroxide, H _ 2O _ 2) group, rapamycin (Rapamycin, Rapa) + H _ 2O _ 2 group and 3-methyladenine (3-methyladhenine,3-MA) + H _ 2O _ 2 group. The expression of p-p53, PUMA and MDM2 in the downstream of p53 pathway was detected by the expression of p53, PUMA and MDM2. [Results] 1. An appropriate up-regulation of autophagy could delay the expression of LC3-II, Beclin-1 protein and p62 in VEC-aging Rapa + H _ 2O _ 2 induced by H _ 2O _ 2, the number of autophagy bodies increased, the cell viability was increased compared with that of the H _ 2O _ 2 group, the positive staining rate of SA-2 half-glycosidase decreased, the expression of p-Rb was decreased, the structure of endothelial cells and ET-1 and NO were relatively normal, and the self-autophagy inhibitor 3-MA showed the opposite conclusion. The microstructural and functional index of the cells showed that it was more obvious than that of the H _ 2O _ 2 group. [Conclusion] The anti-aging model of HUVEC can be induced by H _ 2O _ 2 of 200 mmol/ L for 24 hours, and the effect of autophagy on the aging of VEC can be delayed due to the appropriate regulation of autophagy, which may be related to the activation of p53 signal pathway by up-regulating autophagy to delay the senescence of VEC induced by H _ 2O _ 2.
【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R339.38
本文编号：2511624
文内图片：
图片说明：动脉粥样硬化发展的几个环节
[Abstract]:[Study Background and Objective] Population aging is a common problem facing the whole world, especially in China. The incidence of aging-related diseases caused by aging is the main cause of the shortage of social medical resources and overburdened society. The aging of vascular endothelial cells (VEC) is a key factor of the aging of the body, and the vascular endothelial cell (VEC) aging is the key factor of the outcome and prognosis of many aging-related diseases such as atherosclerosis, stroke and Parkinson's disease. Estrogen is a well-known cardiovascular protective factor, and 17%-estradiol (17-stradiol,17--E2) is one of the most important drugs for delaying the aging of the VEC, but it is very little known to its specific mechanism. p53 is a kind of tumor suppressor protein, which can be repaired after DNA damage, and can be regulated by cell cycle. [Methods] The first part: To study the relationship between the aging of VEC and autophagy in four groups: blank group, hydrogen peroxide (Hggen peroxide, H _ 2O _ 2) group, rapamycin (Rapamycin, Rapa) + H _ 2O _ 2 group and 3-methyladenine (3-methyladhenine,3-MA) + H _ 2O _ 2 group. The expression of p-p53, PUMA and MDM2 in the downstream of p53 pathway was detected by the expression of p53, PUMA and MDM2. [Results] 1. An appropriate up-regulation of autophagy could delay the expression of LC3-II, Beclin-1 protein and p62 in VEC-aging Rapa + H _ 2O _ 2 induced by H _ 2O _ 2, the number of autophagy bodies increased, the cell viability was increased compared with that of the H _ 2O _ 2 group, the positive staining rate of SA-2 half-glycosidase decreased, the expression of p-Rb was decreased, the structure of endothelial cells and ET-1 and NO were relatively normal, and the self-autophagy inhibitor 3-MA showed the opposite conclusion. The microstructural and functional index of the cells showed that it was more obvious than that of the H _ 2O _ 2 group. [Conclusion] The anti-aging model of HUVEC can be induced by H _ 2O _ 2 of 200 mmol/ L for 24 hours, and the effect of autophagy on the aging of VEC can be delayed due to the appropriate regulation of autophagy, which may be related to the activation of p53 signal pathway by up-regulating autophagy to delay the senescence of VEC induced by H _ 2O _ 2.
【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R339.38
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