【摘要】：【研究背景與目的】人口老齡化是全世界面臨的共同難題,在我國尤為嚴(yán)峻。衰老帶來的衰老相關(guān)疾病發(fā)病率增高是社會(huì)醫(yī)療資源緊缺,社會(huì)負(fù)擔(dān)過重的罪魁禍?zhǔn)住Ｑ芩ダ鲜菣C(jī)體衰老的一大特征,而血管內(nèi)皮細(xì)胞(Vascular endothelial cell,VEC)衰老是血管衰老的關(guān)鍵環(huán)節(jié)與始動(dòng)因素,也是決定動(dòng)脈粥樣硬化、腦卒中、帕金森等眾多衰老相關(guān)疾病轉(zhuǎn)歸與預(yù)后的關(guān)鍵因子。雌激素(Estrogen)是眾所周知的心血管保護(hù)因子,17β-雌二醇(17β-estradiol,17β-E2)是其主要活性成分,也是較為明確的可延緩VEC衰老的藥物之一,但目前對(duì)其具體作用機(jī)制所知甚少。自噬(Autophagy)是細(xì)胞“自我消化”現(xiàn)象,饑餓、氧化應(yīng)激、缺氧均可改變細(xì)胞自噬活性。p53是一種腫瘤抑制蛋白,與DNA損傷后修復(fù)、細(xì)胞周期調(diào)控、凋亡、自噬密切相關(guān),也是眾多通路信號(hào)中轉(zhuǎn)站,影響著多個(gè)通路正常工作。有研究表明,位于細(xì)胞核的p53可通過多種方式激活自噬,而17β-E2可通過受體作用增加核內(nèi)p53的表達(dá),再結(jié)合我們前期研究發(fā)現(xiàn)17β-E2可延緩VEC衰老,于是我們猜想17β-E2可能通過p53通路上調(diào)自噬發(fā)揮延緩衰老作用,但國內(nèi)外仍缺乏相關(guān)系統(tǒng)研究。為此,我們將結(jié)合p53通路,研究自噬在VEC衰老中的作用及17β-E2的干預(yù)機(jī)制�！痉椒ā康谝徊糠�:研究VEC衰老與自噬的關(guān)系共設(shè)4個(gè)組:空白組、過氧化氫(Hydrogen peroxide,H_2O_2)組、雷帕霉素(Rapamycin,Rapa)+H_2O_2組和3-甲基腺嘌呤(3-methyladenine,3-MA)+H_2O_2組。從細(xì)胞活力、衰老染色、內(nèi)皮素-1(Endothelin,ET-1)、一氧化氮(Nitric oxide,NO)、亞細(xì)胞結(jié)構(gòu)改變和磷酸化成視網(wǎng)膜瘤蛋白(p-Rb蛋白)表達(dá)情況檢測(cè)細(xì)胞衰老程度,再檢測(cè)各組細(xì)胞微管相關(guān)蛋白1輕鏈3(microtubule-associated protein 1 light chain 3,LC3)、Beclin-1和p62表達(dá)檢測(cè)細(xì)胞自噬活性;通過對(duì)比H_2O_2組與自噬陽性對(duì)照、陰性對(duì)照差異,分析VEC衰老與自噬關(guān)系。第二部分:研究17β-E2對(duì)VEC衰老及自噬活性的影響共設(shè)4個(gè)組:空白組、H_2O_2組、17β-E2組和17β-E2+H_2O_2組。從細(xì)胞活力、細(xì)胞周期、衰老染色、ET-1、NO和p-Rb蛋白表達(dá)情況檢測(cè)細(xì)胞衰老程度,再檢測(cè)各組細(xì)胞LC3-II、Beclin-1、p6和免疫熒光檢測(cè)自噬小體來分析17β-E2延緩衰老作用和自噬的關(guān)系。第三部分:研究17β-E2對(duì)p53信號(hào)通路的影響共設(shè)3個(gè)組:空白組、17β-E2組、17β-E2+H_2O_2組。通過檢測(cè)p53通路下游通路蛋白p-p53、PUMA、MDM2表達(dá)水平分析17β-E2對(duì)p53通路的作用�！窘Y(jié)果】1.適當(dāng)上調(diào)自噬可延緩H_2O_2誘導(dǎo)的VEC衰老Rapa+H_2O_2組LC3-II、Beclin-1蛋白表達(dá)增多,p62減少,自噬小體數(shù)量增加,與H_2O_2組相比較細(xì)胞活力升高,SA-β半糖苷酶陽染率降低,p-Rb表達(dá)減少,內(nèi)皮細(xì)胞結(jié)構(gòu)和ET-1、NO相對(duì)正常;而予以自噬抑制劑3-MA則得出相反結(jié)論,細(xì)胞微觀結(jié)構(gòu)和功能指標(biāo)提示其比H_2O_2組衰老更為明顯。2.17β-E2可通過上調(diào)自噬延緩H_2O_2誘導(dǎo)的VEC衰老與H_2O_2組相比,17β-E2+H_2O_2組LC3-II、Beclin-1表達(dá)增多,p62表達(dá)減少,自噬小體熒光數(shù)量增多,自噬活性增高,細(xì)胞活力增加SA-β半糖苷酶陽染率降低,S期細(xì)胞明顯增加,NO濃度增加,ET-1減少,p-Rb蛋白表達(dá)減少;即17β-E2可延緩VEC衰老,同時(shí)增強(qiáng)自噬活性。3.17β-E2具有激活p53通路的作用與空白組相比,17β-E2+H_2O_2組p53、p-p53和PUMA表達(dá)明顯增加,MDM2表達(dá)減少;除去H_2O_2影響,17β-E2組p53通路蛋白也明顯增加,負(fù)性調(diào)節(jié)蛋白MDM2明顯減少。即17β-E2可激活H_2O_2誘導(dǎo)VEC的p53通路。【結(jié)論】200mmol/L的H_2O_2作用24小時(shí)可成功誘導(dǎo)出HUVEC衰老模型;適當(dāng)?shù)厣险{(diào)自噬具有一定延緩VEC衰老的作用;17β-E2可能通過上調(diào)自噬延緩H_2O_2誘導(dǎo)的VEC衰老,該作用可能與激活p53信號(hào)通路有關(guān)。
文內(nèi)圖片：
圖片說明：動(dòng)脈粥樣硬化發(fā)展的幾個(gè)環(huán)節(jié)
[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.
【學(xué)位授予單位】：廣東藥科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R339.38

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