本文選題：類固醇激素合成急性調(diào)節(jié)蛋白(StAR)　切入點：軟脂酸(PA)　出處：《復旦大學》2012年碩士論文

【摘要】：類固醇激素合成急性調(diào)節(jié)蛋白(steroidogenic acute regulatory protein, StAR)是膽固醇的一種轉(zhuǎn)運蛋白,主要參與膽固醇代謝,合成類固醇激素。本課題組前期研究表明StAR表達于肝臟、血管內(nèi)皮細胞及巨噬細胞中,具有在線粒體內(nèi)外膜間轉(zhuǎn)運膽固醇、調(diào)節(jié)膽固醇代謝的作用。 血管內(nèi)皮除了維持血管壁完整外,還具有重要的穩(wěn)態(tài)調(diào)節(jié)功能：首先,內(nèi)皮細胞通過其生成并釋放的一氧化氮(NO)、緩激肽、內(nèi)皮素、前列環(huán)素調(diào)節(jié)血管張力；其次,內(nèi)皮細胞通過相關(guān)血管活性物質(zhì)抑制炎性細胞、血小板的聚集和粘附,抑制平滑肌的增殖和遷移等。某些內(nèi)源性和外源性因子導致血管內(nèi)皮功能異常,包括精神性和生理學應激、動脈粥樣硬化、高血壓、老齡化、炎癥和糖尿病等疾病狀態(tài),使內(nèi)皮從穩(wěn)定狀態(tài)變?yōu)槭胶鉅顟B(tài),即內(nèi)皮功能發(fā)生異常,又稱內(nèi)皮功能障礙。內(nèi)皮功能障礙是動脈粥樣硬化等心血管疾病的始發(fā)環(huán)節(jié),是由長期暴露于心血管疾病危險因素引起的,其中最主要的危險因素是以高血脂為特征的脂質(zhì)代謝紊亂,又以循環(huán)血中的高游離脂肪酸(free fatty acid, FFA)對內(nèi)皮細胞的損害最為重要,也是目前該領(lǐng)域研究的熱點之一。研究表明,FFA水平增高可損害內(nèi)皮細胞一氧化氮合酶(eNOS)的活性,抑制內(nèi)皮細胞依賴性的血管舒張、促進內(nèi)皮細胞炎癥因子的釋放及誘導細胞的凋亡等。因此,針對FFA導致的內(nèi)皮功能障礙的相關(guān)機制的研究,對于保護內(nèi)皮細胞功能,進一步預防和治療動脈粥樣硬化具有重要意義。綜上,本課題擬在原代大鼠主動脈內(nèi)皮細胞中過表達StAR后,研究StAR是否對軟脂酸(palmitic acid, PA)引起的內(nèi)皮功能障礙具有保護作用。 本研究共包括兩部分內(nèi)容,詳細如下： 第一部分：類固醇激素合成急性調(diào)節(jié)蛋白(StAR)對原代大鼠主動脈內(nèi)皮細胞脂質(zhì)代謝基因表達的影響 我們以大鼠主動脈內(nèi)皮細胞(Rat aortal endothelial cell, RAEC)為研究對象,首先用動脈環(huán)法成功培養(yǎng)了大鼠主動脈內(nèi)皮細胞,利用倒置顯微鏡觀察培養(yǎng)細胞的形態(tài)學特征；利用細胞免疫化學方法顯示血管內(nèi)皮細胞特異性表面抗原標志；其次利用攜帶StAR全長cDNA的腺病毒載體轉(zhuǎn)染培養(yǎng)的內(nèi)皮細胞,利用實時定量PCR和Wesern blot方法驗證StAR在內(nèi)皮細胞中過表達,同時檢測轉(zhuǎn)染后細胞內(nèi)脂質(zhì)代謝相關(guān)基因的表達變化。結(jié)果顯示：動脈環(huán)法培養(yǎng)的大鼠主動脈內(nèi)皮細胞72小時后自動脈環(huán)中爬出,具有典型血管內(nèi)皮細胞特征(鋪路石樣、管腔樣形成),CD31、vW因子等內(nèi)皮細胞特異性標志表達陽性；利用腺病毒Ad-StAR,以不同的感染復數(shù)(multiplicity of infection, MOI=10,20,50,100)轉(zhuǎn)染RAEC,48小時后提取細胞總RNA及蛋白,StAR的mRNA及蛋白表達隨著病毒感染復數(shù)增加而遞增,結(jié)果提示利用腺病毒載體在RAEC中成功過表達StAR。其次,在腺病毒轉(zhuǎn)染48小時后,與轉(zhuǎn)染空載體組相比,過表達StAR組FAS、ACC-1、 HMGR、LDLR、SREBP-2mRNA表達明顯降低(P0.05)；最后,在腺病毒轉(zhuǎn)染48小時后以氯仿／異丙醇/NP-40及氯仿/Triton X-100分別萃取細胞內(nèi)膽固醇及游離脂肪酸,利用膽固醇檢測試劑盒(CHO酶法)和游離脂肪酸超敏測定試劑盒檢測細胞內(nèi)總膽固醇和游離脂肪酸含量,結(jié)果顯示：StAR轉(zhuǎn)染48小時后,與轉(zhuǎn)染空載體EGFP組相比,過表達StAR組細胞內(nèi)總膽固醇及游離脂肪酸含量明顯降低(P0.05)。 第二部分：StAR對PA引起的內(nèi)皮功能障礙的保護作用。 我們以原代培養(yǎng)的大鼠主動脈內(nèi)皮細胞為研究對象,利用攜帶StAR全長cDNA的腺病毒載體在RAEC中過表達StAR。首先,以外源性PA為刺激因素,腺病毒轉(zhuǎn)染48小時后加入培養(yǎng)的RAEC中,于不同時間點提取細胞RNA,利用實時定量PCR檢測細胞炎癥因子IL-1β、TNFα、IL-6、VCAM-1的基因表達；利用ELISA試劑盒檢測細胞培養(yǎng)上清中炎癥因子含量；同時收集細胞,分別提取細胞胞漿及胞核蛋白,利用Western blot方法檢測核轉(zhuǎn)錄因子NFκB在胞漿和胞核中的表達。結(jié)果顯示：過表達StAR能抑制PA導致的炎癥因子基因表達增高及釋放增多,其作用機制是通過抑制NFκB的核轉(zhuǎn)位,從而抑制其活化,最終抑制NFκB下游炎癥因子的轉(zhuǎn)錄活性,進一步減少炎癥因子釋放。其次,在腺病毒轉(zhuǎn)染RAEC48小時后加入外源性PA,在不同時間點收集細胞,提取細胞總蛋白,利用Western blot方法檢測p-Akt/p-eNOS信號通路的變化,并利用NO檢測試劑盒檢測培養(yǎng)上清中NO的釋放量。結(jié)果顯示：PA能抑制p-Akt/p-eNOS/NO通路,減少NO的生成和釋放,而過表達StAR能明顯減輕PA的抑制作用,增加Akt及eNOS的磷酸化水平,增加RAEC中NO的釋放,維持血管內(nèi)皮舒縮功能的平衡。 綜上所述,本課題首先利用主動脈環(huán)貼壁法成功在體外培養(yǎng)了RAEC,并通過形態(tài)學及細胞表面標志物檢測證實為血管內(nèi)皮細胞；利用攜帶StAR全長cDNA的腺病毒載體可在RAEC中過表達StAR;過表達StAR可抑制RAEC中的膽固醇和脂肪酸合成關(guān)鍵酶的表達；在RAEC中過表達StAR可抑制PA引起的炎癥反應及NO合成減少,保護內(nèi)皮細胞功能,其作用是通過調(diào)節(jié)脂質(zhì)代謝,降低細胞內(nèi)脂肪酸的合成發(fā)揮的。本研究結(jié)果也為動脈粥樣硬化、糖尿病等內(nèi)皮功能障礙相關(guān)疾病的防治提供了新的實驗依據(jù)和理論基礎(chǔ)。
[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.

【學位授予單位】：復旦大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R363

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