StAR对软脂酸导致的内皮细胞功能障碍的保护作用研究

发布时间：2018-04-01 21:39

本文选题：类固醇激素合成急性调节蛋白(StAR)　切入点：软脂酸(PA)　出处：《复旦大学》2012年硕士论文

【摘要】：类固醇激素合成急性调节蛋白(steroidogenic acute regulatory protein, StAR)是胆固醇的一种转运蛋白,主要参与胆固醇代谢,合成类固醇激素。本课题组前期研究表明StAR表达于肝脏、血管内皮细胞及巨噬细胞中,具有在线粒体内外膜间转运胆固醇、调节胆固醇代谢的作用。血管内皮除了维持血管壁完整外,还具有重要的稳态调节功能：首先,内皮细胞通过其生成并释放的一氧化氮(NO)、缓激肽、内皮素、前列环素调节血管张力；其次,内皮细胞通过相关血管活性物质抑制炎性细胞、血小板的聚集和粘附,抑制平滑肌的增殖和迁移等。某些内源性和外源性因子导致血管内皮功能异常,包括精神性和生理学应激、动脉粥样硬化、高血压、老龄化、炎症和糖尿病等疾病状态,使内皮从稳定状态变为失平衡状态,即内皮功能发生异常,又称内皮功能障碍。内皮功能障碍是动脉粥样硬化等心血管疾病的始发环节,是由长期暴露于心血管疾病危险因素引起的,其中最主要的危险因素是以高血脂为特征的脂质代谢紊乱,又以循环血中的高游离脂肪酸(free fatty acid, FFA)对内皮细胞的损害最为重要,也是目前该领域研究的热点之一。研究表明,FFA水平增高可损害内皮细胞一氧化氮合酶(eNOS)的活性,抑制内皮细胞依赖性的血管舒张、促进内皮细胞炎症因子的释放及诱导细胞的凋亡等。因此,针对FFA导致的内皮功能障碍的相关机制的研究,对于保护内皮细胞功能,进一步预防和治疗动脉粥样硬化具有重要意义。综上,本课题拟在原代大鼠主动脉内皮细胞中过表达StAR后,研究StAR是否对软脂酸(palmitic acid, PA)引起的内皮功能障碍具有保护作用。本研究共包括两部分内容,详细如下：第一部分：类固醇激素合成急性调节蛋白(StAR)对原代大鼠主动脉内皮细胞脂质代谢基因表达的影响我们以大鼠主动脉内皮细胞(Rat aortal endothelial cell, RAEC)为研究对象,首先用动脉环法成功培养了大鼠主动脉内皮细胞,利用倒置显微镜观察培养细胞的形态学特征；利用细胞免疫化学方法显示血管内皮细胞特异性表面抗原标志；其次利用携带StAR全长cDNA的腺病毒载体转染培养的内皮细胞,利用实时定量PCR和Wesern blot方法验证StAR在内皮细胞中过表达,同时检测转染后细胞内脂质代谢相关基因的表达变化。结果显示：动脉环法培养的大鼠主动脉内皮细胞72小时后自动脉环中爬出,具有典型血管内皮细胞特征(铺路石样、管腔样形成),CD31、vW因子等内皮细胞特异性标志表达阳性；利用腺病毒Ad-StAR,以不同的感染复数(multiplicity of infection, MOI=10,20,50,100)转染RAEC,48小时后提取细胞总RNA及蛋白,StAR的mRNA及蛋白表达随着病毒感染复数增加而递增,结果提示利用腺病毒载体在RAEC中成功过表达StAR。其次,在腺病毒转染48小时后,与转染空载体组相比,过表达StAR组FAS、ACC-1、 HMGR、LDLR、SREBP-2mRNA表达明显降低(P0.05)；最后,在腺病毒转染48小时后以氯仿／异丙醇/NP-40及氯仿/Triton X-100分别萃取细胞内胆固醇及游离脂肪酸,利用胆固醇检测试剂盒(CHO酶法)和游离脂肪酸超敏测定试剂盒检测细胞内总胆固醇和游离脂肪酸含量,结果显示：StAR转染48小时后,与转染空载体EGFP组相比,过表达StAR组细胞内总胆固醇及游离脂肪酸含量明显降低(P0.05)。第二部分：StAR对PA引起的内皮功能障碍的保护作用。我们以原代培养的大鼠主动脉内皮细胞为研究对象,利用携带StAR全长cDNA的腺病毒载体在RAEC中过表达StAR。首先,以外源性PA为刺激因素,腺病毒转染48小时后加入培养的RAEC中,于不同时间点提取细胞RNA,利用实时定量PCR检测细胞炎症因子IL-1β、TNFα、IL-6、VCAM-1的基因表达；利用ELISA试剂盒检测细胞培养上清中炎症因子含量；同时收集细胞,分别提取细胞胞浆及胞核蛋白,利用Western blot方法检测核转录因子NFκB在胞浆和胞核中的表达。结果显示：过表达StAR能抑制PA导致的炎症因子基因表达增高及释放增多,其作用机制是通过抑制NFκB的核转位,从而抑制其活化,最终抑制NFκB下游炎症因子的转录活性,进一步减少炎症因子释放。其次,在腺病毒转染RAEC48小时后加入外源性PA,在不同时间点收集细胞,提取细胞总蛋白,利用Western blot方法检测p-Akt/p-eNOS信号通路的变化,并利用NO检测试剂盒检测培养上清中NO的释放量。结果显示：PA能抑制p-Akt/p-eNOS/NO通路,减少NO的生成和释放,而过表达StAR能明显减轻PA的抑制作用,增加Akt及eNOS的磷酸化水平,增加RAEC中NO的释放,维持血管内皮舒缩功能的平衡。综上所述,本课题首先利用主动脉环贴壁法成功在体外培养了RAEC,并通过形态学及细胞表面标志物检测证实为血管内皮细胞；利用携带StAR全长cDNA的腺病毒载体可在RAEC中过表达StAR;过表达StAR可抑制RAEC中的胆固醇和脂肪酸合成关键酶的表达；在RAEC中过表达StAR可抑制PA引起的炎症反应及NO合成减少,保护内皮细胞功能,其作用是通过调节脂质代谢,降低细胞内脂肪酸的合成发挥的。本研究结果也为动脉粥样硬化、糖尿病等内皮功能障碍相关疾病的防治提供了新的实验依据和理论基础。
[Abstract]:Steroidogenic acute regulatory protein (steroidogenic acute regulatory protein, StAR) is a kind of cholesterol transporter, mainly involved in cholesterol metabolism, steroid hormone synthesis. Previous study showed that the expression of StAR in liver, vascular endothelial cells and macrophages, with transport in the mitochondrial outer membrane cholesterol, regulating the metabolism of cholesterol role.
In addition to maintain vascular endothelial integrity, but also has important functions: first, steady state regulation of endothelial cells through its generation and release of nitric oxide (NO), bradykinin, endothelin, prostacyclin in regulating vascular tension; secondly, endothelial cells by vasoactive substances inhibiting the inflammatory cells, platelet aggregation and adhesion and inhibit the proliferation and migration of smooth muscle cells. Some endogenous and exogenous factors lead to vascular endothelial dysfunction, including mental and physiological stress, atherosclerosis, hypertension, aging, inflammation and diabetes and other diseases, the endothelium from the state to the stable equilibrium state, namely endothelial function abnormal, also known as endothelial dysfunction endothelial dysfunction is the primary link. Atherosclerosis and other cardiovascular diseases, is caused by long-term exposure to risk factors of cardiovascular disease, one of the most important The risk factors of lipid metabolic disorder characterized by high blood lipids, with high free fatty acids in blood circulation (free fatty acid, FFA) on endothelial cell injury is most important, is also one of the research hotspots in this field at present. The study shows that the level of FFA increased nitric oxide synthase in endothelial cell damage (eNOS) activity, inhibition of endothelium-dependent vasodilation, promote inflammatory cytokine release and induce endothelial cell apoptosis. Therefore, study of mechanism for FFA induced endothelial dysfunction, to protect endothelial cell function, further has important significance for preventing and treating atherosclerosis. In summary, this topic over expression of StAR in primary cultured rat aortic endothelial cells after StAR of palmitic acid (palmitic acid, PA) induced endothelial dysfunction has a protective effect.
This study consists of two parts, which are as follows:
First part: the effect of steroid synthesis of acute regulatory protein (StAR) on the expression of lipid metabolism gene in primary rat aortic endothelial cells
We in rat aortic endothelial cells (Rat aortal endothelial cell, RAEC) as the research object, first with the tour de France successfully cultured arterial endothelial cells of the rat aorta, observe the morphology of the cultured cells by inverted microscope; using immunocytochemistry showed endothelial cell specific surface markers; followed by transfection of adenovirus vector carrying the StAR full-length cDNA of cultured endothelial cells, verified by real-time quantitative PCR and Wesern StAR blot expression in endothelial cells, while the expression of genes related to lipid metabolism change detection after transfection. The results showed that the tour de France culture artery rat aortic endothelial cells after 72 hours of climbing out of the aortic annulus, with the typical characteristics of vascular endothelial cells (cobblestone, tube like formation), CD31, vW factor of endothelial cell specific markers positive expression; use Adenovirus Ad-StAR with different multiplicity of infection (multiplicity of, infection, MOI=10,20,50100) RAEC 48 hours after transfection, cells were extracted total RNA and protein expression of mRNA and protein of StAR virus infection increased along with the increasing of the complex, indicated by adenovirus vector in RAEC successfully overexpressed StAR. second, within 48 hours of transfection after compared with the empty vector transfected group, overexpression of StAR group FAS, ACC-1, HMGR, LDLR, SREBP-2mRNA expression decreased significantly (P0.05); finally, in 48 hours after transfection with chloroform / isopropanol /NP-40 and /Triton X-100 respectively, chloroform extraction of intracellular cholesterol and free fatty acids, cholesterol using test kit (CHO enzyme method) and high-sensitivity determination kit for detection of intracellular total cholesterol and free fatty acid content, free fatty acids showed that StAR 48 hours after transfection, compared with the empty vector transfected group EGFP table The content of total cholesterol and free fatty acids in the cells of the StAR group decreased significantly (P0.05).
The second part: the protective effect of StAR on endothelial dysfunction caused by PA.
We in primary cultured rat aortic endothelial cells as the research object, in the RAEC over expression of StAR. using adenovirus vector carrying StAR full-length cDNA, with exogenous PA as stimuli for 48 hours after transfection to cultured RAEC cells, extracted at different time points of RNA, using real time quantitative PCR detection of inflammatory factor IL-1 beta, TNF alpha, IL-6, VCAM-1 gene expression; inflammatory cytokines content in the culture supernatant by ELISA kit to detect the cells; while collecting cells, cytoplasm and nuclear protein was extracted by using Western blot method to detect the expression of nuclear factor kappa NF B in cytoplasm and nucleus.. the results showed that overexpression of StAR can inhibit the inflammatory factor gene PA leads to increased expression and release increased, its mechanism is through inhibition of NF kappa B nuclear translocation, inhibit the activation of NF kappa B, eventually inhibit downstream inflammatory factor The transcriptional activity, further reduce the release of inflammatory factors. Secondly, the addition of exogenous PA in adenovirus RAEC48 hours after transfection, cells were collected at different time points, and total protein were extracted, detected the changes of p-Akt/p-eNOS signal pathway by Western blot method, and using the NO kit to detect the release amount of NO in the culture supernatant. The results showed PA can inhibit the p-Akt/p-eNOS/NO pathway, reduce the production and release of NO, and overexpression of StAR can significantly reduce the inhibitory effect of PA, Akt and eNOS increased the phosphorylation level of RAEC, increase in NO release, maintain vascular endothelial vasomotor function balance.
In summary, this thesis firstly uses aortic ring adherent method in vitro successfully RAEC, and the morphology and cell surface markers was confirmed by vascular endothelial cells; over expression of StAR in RAEC by adenovirus vector carrying StAR full-length cDNA; overexpression of StAR can inhibit the RAEC of cholesterol and fatty acid synthesis key the expression of the enzymes; over expression of inflammatory reaction and NO synthesis of StAR can inhibit the PA induced decrease in RAEC, protect the function of endothelial cells, which regulate lipid metabolism, reduce the use of fatty acid synthesis in cells. The research results for atherosclerosis, provides a new theoretical and experimental basis for the prevention and treatment of related diseases such as diabetes and endothelial dysfunction.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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