本文關(guān)鍵詞： 姜黃素 內(nèi)皮細(xì)胞衰老 SIRT1 過氧化氫　出處：《武漢大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：材料：內(nèi)皮細(xì)胞衰老、內(nèi)皮功能障礙與動(dòng)脈粥樣硬化等多種心血管疾病的形成有著密切的聯(lián)系。姜黃素提取于姜科、天南星科的植物根莖中,是一種多酚類化合物,既往研究認(rèn)為姜黃素能夠發(fā)揮抗氧化應(yīng)激和抗炎作用,可能在疾病治療中具有潛在價(jià)值。然而,氧化應(yīng)激、DNA損傷等刺激因素是促使細(xì)胞衰老的常見病因。因此,姜黃素可能通過其抗氧化作用等,對(duì)內(nèi)皮細(xì)胞衰老產(chǎn)生抑制作用,從而在動(dòng)脈粥樣硬化等疾病中發(fā)揮保護(hù)效果。本研究通過過氧化氫(H2O2)誘導(dǎo)人臍靜脈內(nèi)皮細(xì)胞(HUVECs)衰老的發(fā)生,旨在探討姜黃素在細(xì)胞衰老過程中的作用及其可能機(jī)制。方法：1. HUVECs采用含5%胎牛血清、1%內(nèi)皮細(xì)胞生長(zhǎng)因子、100U/ml青霉素、100μg/ml鏈霉素的完全內(nèi)皮細(xì)胞培養(yǎng)基進(jìn)行細(xì)胞培養(yǎng),并置于37℃含5%CO2、95%O2的無菌培養(yǎng)箱中培養(yǎng)。采用CCK-8試劑盒對(duì)不同濃度的姜黃素(0、5、10、25、50和100μM)預(yù)處理的培養(yǎng)細(xì)胞進(jìn)行細(xì)胞增殖檢測(cè)；另外,使用100 μM H2O2誘導(dǎo)HUVECs衰老的發(fā)生,同時(shí)應(yīng)用不同濃度的姜黃素(10、25μM)作為細(xì)胞培養(yǎng)的干預(yù)條件。細(xì)胞衰老的鑒定通過衰老相關(guān)的β半乳糖苷酶(SA-βG)染色,并使用流式細(xì)胞儀測(cè)定細(xì)胞周期。2.采用SIRT1 siRNA轉(zhuǎn)染HUVECs,使細(xì)胞內(nèi)SIRT1沉默表達(dá)。仍使用100μM H2O2誘導(dǎo)HUVECs衰老,同時(shí)以10μM姜黃素及構(gòu)建的SIRT1 siRNA轉(zhuǎn)染的HUVECs作為干預(yù)條件細(xì)胞培養(yǎng)一定時(shí)間后,采用Western blotting法測(cè)定SIRT1、p21、p-eNOS (Ser1177)和eNOS等蛋白表達(dá)水平,同時(shí)測(cè)定細(xì)胞培養(yǎng)基中一氧化氮(NO)水平。結(jié)果：1.與陰性對(duì)照組相比,不同濃度(0、5、10、25、50和100μM)的姜黃素對(duì)細(xì)胞增殖能力影響不同,高劑量(如50,100μM)預(yù)處理時(shí)可以在一定程度上降低細(xì)胞的增殖能力(均p0.05),而低劑量(如10,25μM)預(yù)處理對(duì)細(xì)胞增殖能力無顯著影響。不僅如此,與陰性對(duì)照組相比,經(jīng)100μM H2O2刺激后,HUVECs中SA-βG陽(yáng)性細(xì)胞比例顯著增加,流式細(xì)胞儀測(cè)定細(xì)胞周期明顯受到阻滯(均p0.05)。2.與H2O2對(duì)照組相比,姜黃素(10,25μM)可以顯著減少HUVECs中SA-βG陽(yáng)性細(xì)胞比例及細(xì)胞生長(zhǎng)停滯狀態(tài)(均p0.05)；相對(duì)于陰性對(duì)照組,H2O2刺激可提高HUVECs中p21的表達(dá),降低HUVECs中eNOS的磷酸化和培養(yǎng)基中NO的水平,還抑制HUVECs中SIRT1的表達(dá)(均p0.05)；相對(duì)于H2O2對(duì)照組,姜黃素(10μM)預(yù)處理可以顯著抑制HUVECs中p21表達(dá),提高HUVECs中eNOS的磷酸化以及細(xì)胞培養(yǎng)基中NO的水平,并增加SIRT1的表達(dá)(均p0.05)。然而,HUVECs經(jīng)SIRT1 siRNA處理后,姜黃素預(yù)處理對(duì)H2O2誘導(dǎo)的細(xì)胞衰老的保護(hù)作用消除。結(jié)論：1.H2O2能夠促進(jìn)HUVECs衰老及其功能失調(diào)。2.細(xì)胞水平研究證實(shí)姜黃素可以通過上調(diào)SIRT1表達(dá),減緩H2O2誘導(dǎo)的HUVECs衰老。3. HUVECs經(jīng)SIRT1 siRNA轉(zhuǎn)染后,姜黃素預(yù)處理對(duì)H2O2誘導(dǎo)的細(xì)胞衰老的保護(hù)作用消除。
[Abstract]:Materials: endothelial cell senescence, endothelial dysfunction and the formation of various cardiovascular diseases, such as atherosclerosis, are closely related. Curcumin is extracted from the rhizome of the family Curcumae, which is a polyphenolic compound. Previous studies have suggested that curcumin may have potential value in the treatment of diseases by exerting antioxidant and anti-inflammatory effects. However, oxidative stress and DNA damage are common causes of cell senescence. Curcumin may inhibit the aging of endothelial cells through its antioxidation effect and thus play a protective role in diseases such as atherosclerosis. In this study, human umbilical vein endothelial cells (HUVECs) were induced to senescence by hydrogen peroxide (H _ 2O _ 2). To investigate the role of curcumin in cell senescence and its possible mechanism, HUVECs was cultured in 100 渭 g / ml streptomycin (100 渭 g / ml) of 100 渭 g / ml streptomycin (100 渭 g / ml streptomycin) culture medium containing 5% fetal bovine serum and 1% endothelial cell growth factor. The cultured cells were cultured in a sterile incubator containing 5CO2 and 95O2 at 37 鈩，

本文編號(hào)：1549681