【作者】陈压西

【导师】黄爱龙; 阮雄中

【作者基本信息】重庆医科大学，内科学，2007年，博士

【摘要】 目的细胞内的胆固醇无论是外源性摄入还是内源性合成,均通过一个依赖于胞内胆固醇水平的反馈调节系统的严密控制来保证胆固醇的内稳态平衡,即主要通过SCAP–SREBPs–LDLr/HMGcoA reductase蛋白之间的相互作用实现对细胞内脂质稳态平衡的调节。过去的研究中,我们已经证实炎性因子可以通过干扰胆固醇介导的低密度脂蛋白受体(LDLr)反馈调节来促进外周细胞,如肾系膜细胞(HMCs)、血管平滑肌细胞(VSMC)的胆固醇摄入,以及抑制ATP结合盒转运体A1(ABCA1)基因的表达来减少胆固醇外流从而导致胆固醇在外周细胞异常积聚形成泡沫细胞。本研究在我们过去研究的基础上,进一步选用HMCs和HepG2两种细胞系,(1)探讨在正常生理(非炎症)及炎症状态下外周细胞和肝细胞LDLr反馈调节的组织特异性差异;(2)观察炎症因子是否影响人HMCs和HepG2细胞中HMGCoA还原酶(HMGCoA reductase)介导的内源性胆固醇生物合成;同时探讨炎症反应导致细胞/机体对他汀类降脂药物(HMGCoA还原酶活性抑制剂)抵抗的机制。(3)进一步观察炎症因子对LDLr/HMGCoA还原酶的上游调...更多控蛋白——核转录因子SREBP-2和胆固醇敏感器SCAP的影响,探讨外周细胞HMCs和肝细胞HepG2胆固醇摄入与合成组织特异性差异的分子机制。材料和方法用酶学方法分别检测HMCs和HepG2细胞内总胆固醇(TC),游离胆固醇(FC)和胆固醇酯(CE)的水平;油红O染色观察细胞内脂质积聚情况;用14C标记的acetate(乙酸盐,胆固醇合成原料)孵育细胞,提取胆固醇,用液体闪烁计数仪测定14C含量,从而测定细胞胆固醇的合成;提取细胞总RNA,应用real-time定量PCR技术分别检测两种细胞LDLr、HMGCoA还原酶、SREBP-2和SCAP mRNA水平;用Western blotting检测LDLr、HMGCoA还原酶蛋白表达水平;进一步应用薄层层析技术测定HMGcoA还原酶活性;抗人SCAP抗体和抗高尔基抗体双重染色后,应用激光共聚焦显微镜观察两种细胞SCAP-SREBP复合物在内质网和高尔基体的定位。结果1.炎性因子IL-1β能明显促进外周细胞和肝细胞胆固醇的外源性摄入和内源性合成,从而造成胆固醇(特别是胆固醇酯)在细胞内异常积聚。外周细胞HMCs较肝细胞HepG2对炎症因子更敏感。2.正常生理(非炎症)状态下,当细胞加载不同浓度低密度脂蛋白(LDL)时,可以反馈抑制两种细胞的LDLr mRNA、蛋白水平,其中HMCs细胞LDLr的下调比HepG2更敏感(“下调易感”)。此外,Compactin(一种他汀类降脂药物,HMGCoA还原酶活性抑制剂)可以通过降低细胞内胆固醇水平,促进两种细胞LDLr mRNA和蛋白的表达,从而增强LDLr介导的外源胆固醇摄入,HepG2细胞中LDLr的上调比HMCs细胞更敏感(“上调易感”)。然而,炎症状态下,IL-1β能干扰LDL对LDLr的下调作用,促进两种细胞,尤其是HMCs细胞的LDLr mRNA和蛋白表达,使外周细胞像肝细胞一样,能大量摄取胆固醇。同时,当有炎性因子存在时,IL-1β能干扰Compactin对LDLr的上调作用,需要增加Compactin的剂量才能达到非炎症状态时同等的对LDLr上调效果。3.正常生理(非炎症)状态下,加载LDL可以反馈抑制两种细胞的HMGCoA还原酶mRNA、蛋白表达以及酶活性,这种由于胞内胆固醇水平升高而导致的反馈抑制作用,HMCs比HepG2细胞更敏感。然而,炎症状态下,炎性因子IL-1β可以打破由胞内胆固醇介导这种反馈调节,在HMCs细胞,炎性因子干扰HMGCoA还原酶反馈调节主要是通过增加HMGCoA还原酶mRNA、蛋白表达及酶活性水平来实现,而在HepG2细胞仅仅只增加HMGCoA还原酶的酶活性水平,对mRNA及蛋白表达影响不明显。Compactin可以抑制HMCs与HepG2细胞HMGCoA还原酶的酶活性、上调mRNA表达,然而当有IL-1β存在时,Compactin抑制酶活性、上调mRNA表达的作用均被明显减弱。4.进一步,我们探讨了IL-1β对LDLr/HMGCoA还原酶的上游调控蛋白——核转录因子SREBP-2和胆固醇敏感器SCAP的影响。LDL能降低SREBP-2和SCAP的mRNA水平,并抑制SCAP-SREBP复合物从内质网向高尔基体转移,这种抑制作用在HMCs更明显,而当同时有IL-1β存在时,两种细胞SREBP-2 mRNA表达均明显增加,并且SCAP-SREBP复合物从内质网向高尔基体转移明显增多,从而可以进一步促进靶基因LDLr/HMGCoA还原酶的转录。结论生理(非炎症)情况下,胞内胆固醇水平可以对两种细胞SCAP–SREBP–LDLr/HMGCoA还原酶产生负反馈调节,HMCs较HepG2细胞更敏感,使得外周细胞相对于肝细胞能在胞内胆固醇水平升高时更迅速地减少外源胆固醇摄取和内源胆固醇合成,防止了胆固醇在外周细胞的聚集。但在炎症状态下,由胆固醇介导的SCAP–SREBP–LDLr/HMGCoA还原酶的严密反馈调节被破坏,导致细胞异常摄入与合成胆固醇从而形成泡沫细胞。HMCs对炎症较HepG2细胞更敏感是外周细胞在炎症刺激下更容易形成泡沫细胞的一个原因。同时,炎症干扰了他汀类药物的作用,造成细胞对他汀类药物抵抗,其机制与炎性因子干扰了HMGCoA还原酶的转录和活性功能有关。SCAP/SREBP复合物在亚细胞上的分布定位不同是导致外周细胞和肝细胞LDLr/HMGCoA还原酶反馈调节组织特异性的一个重要因素。

【Abstract】 Objectives Cholesterol is derived from two pathways, one is the uptake of plasma via LDL receptor-mediated endocytosis, the other is from cellular synthesis. In the pathway of cholesterol synthesis, HMG-CoA reductase is the important rate-limiting enzyme. Either HMG-CoA reductase or LDLr activity is under the control via a feedback system that is dependent on intracellular cholesterol concentration. We have demonstrated that inflammatory cytokines promote foam cell formation by increasing cholesterol uptake via disrupting cholesterol-mediated LDL receptor feedback regulation, inhibiting cholesterol efflux via reducing ABCA1 gene expression in peripheral cells, such as kidney mesangial cells (HMCs) and vascular smooth muscle cells (VSMC). The aim of this study was to investigate (1) if there are differences between HMCs and human hepatic cell line HepG2 regarding regulation of cholesterol exogenous uptake via the LDL receptor under physiological and inflammatory conditions; (2) if infla...更多mmatory cytokines affect in HMGCoA reductase mediated cholesterol synthesis in HMCs and HepG2 and cause statins (competitive inhibitors of HMGCoA reductase) resistance ; (3) the molecular mechanisms by which inflammatory cytokines override the normal cholesterol-mediated suppression of the LDLr/HMGCoA reductase induced by a high concentration of LDL through examining the mRNA expression of the sterol regulatory element binding protein (SREBP-2), as well as SREBP cleavage-activating protein (SCAP) intracellular translocation between the ER and Golgi under the influence of inflammation.Material and Methods HMCs and HepG2 were used in all experiments. Intracellular total cholesterol (TC), free cholesterol (FC) and cholesterol ester (CE) were measured by enzymic assay. Oil Red O staining was used to examine cell morphology. [14C] acetic acid sodium was added to cells medium and incubation was continued for 1 hr to estimate cellular sterol synthesis. Total cellular RNA was isolated from above cells for detecting LDL receptor, HMGCoA reductase, SREBP-2 and SCAP mRNA level with real-time quantitative PCR. LDL receptor and HMGCoA reductase protein expression was measured by Western blotting. HMGcoA reductase activity was measured by Thin Layer Chromatography detecting the amount of mevalonate-3-14C that is formed from 3-14C HMGcoA. Confocal microscopy was used to investigate the translocation of SCAP-SREBP complex from the ER to Golgi after dual staining with anti-human SCAP antibody and anti-Golgin antibody.Results We demonstrated that LDL loading increased intracellular cholesterol level dose-dependently. The accumulation of intracellular cholesterol ester in HMCs is more than in HepG2 cells under IL-1βstress. Increased intracellular cholesterol reduced LDL receptor mRNA and protein expression in both HepG2 and HMCS cells, but to a greater degree in HMCs. Compactin, a HMGCoA reductase activity inhibitor, increased LDL receptor mRNA and protein expression in both cell types by decreasing intracellular cholesterol level, thereby regulating cholesterol exogenous uptake via the LDL receptor. HepG2 cell was more sensitive than HMCs regarding LDL receptor upregulation. However, IL-1βenhanced LDL receptor mRNA and protein expression, and overrode the suppression of LDL receptor induced by high concentrations of LDL cholesterol in both HepG2 and HMCs.In addition, we demonstrated that LDL loading also reduced HMGCoA reductase mRNA, protein expression and enzymatic activity in both cell types. The inhibition in HMCs was more sensitive than in HepG2 which prevent lipid accumulation in peripheral cell HMCs. However, IL-1βincreased cholesterol level in both cells in the presence of LDL by increasing HMGCoA reductase mRNA, protein expression and activity in HMCs, but only increasing activity, not mRNA and protein expression in HepG2 cells. Compactin increased HMGCoA reductase mRNA level and inhibited HMGCoA reductase activity in both cell types, but the effect of compactin was weakened in presence of IL-1β.LDL also reduced SREBP-2 and SCAP mRNA level, inhibited its translocation from the endoplasmic reticulum to the Golgi. Interestingly, there was more SCAP accumulation in the Golgi even in the presence of high concentration of LDL in HepG2 cells rather than HMCs. However, IL-1βdisrupts normal distribution of SCAP in HMCs by enhancing translocation of SCAP from the ER to the Golgi even in the presence of high concentration of LDL.Conclusion LDL receptor and HMGCoA reductase are normally regulated via a feedback system that is dependent on intracellular cholesterol level in the both cells, but to more sensitive in HMCs which prevent lipid accumulation in peripheral cell HMCs. However, IL-1βdisrupts cholesterol-mediated LDL receptor/HMGCoA reductase feedback regulation in peripheral cells, permitting intracellular accumulation of cholesterol and causing foam cell formation. HMCs is more sensitive to inflammation than HepG2 cells which could be one reason why HMCs easy to become foam cells under inflammatory stress. Inflammation also causes statin resistance by disrupting HMGCoA reductase transcriptional and activity, therefore a high concentration of statin may be required to achieve the same biological effect. SCAP location is an important factor for the sensitivity of LDL receptor and HMGCoA reductase regulation in the both cell types.
【关键词】 低密度脂蛋白受体; HMGcoA还原酶; 胆固醇; 炎症; 泡沫细胞
【Key words】 LDL receptor; HMGCoA reductase; cholesterol; inflammation; foam cell

【网络出版投稿人】重庆医科大学 【网络出版投稿时间】2007-12-29 【网络出版年期】2008/02

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