跨膜蛋白66在血管再狭窄中的作用及机制研究

发布时间：2019-05-08 01:19

【摘要】：研究背景和目的:冠状动脉粥样硬化性心脏病(Coronary atherosclerotic heart disease,CHD)严重危害人类健康,经皮腔内冠状动脉介入治疗(Percutaneous Coronary Intervention,PCI)是治疗冠心病的重要手段,过去20多年来,PCI治疗虽然取得了长足进步,但支架内再狭窄(Restenosis,RS)的发生率仍高达10%,严重影响了PCI的临床疗效。因此,探讨RS的发生机制是指导其防治的关键。血管内皮损伤后的内膜增生是血管再狭窄的重要病理过程,血管平滑肌细胞(Vascular smooth muscle cells,VSMCs)的增殖和迁移是内膜增生的主要原因。研究表明钙库操纵性钙内流(store operated calcium entry,SOCE)在VSMCs增殖、迁移和血管内膜增生的过程中发挥着重要作用,跨膜蛋白66(transmembrane protein 66,TMEM66)是新近发现的钙库操控性钙通道(store-operated calcium channel,SOC)的重要组成部分,可以调控SOCE。我们推测TMEM66可能也是通过调控血管内皮损伤后VSMCs增殖、迁移,抑制血管内皮损伤后内膜的过度增生,从而减轻血管再狭窄。为了验证以上推测,本实验分为两部分进行。第一部分通过高脂饮食联合球囊损伤建立SD、Apo E基因敲除(ApoE-/-)两种大鼠血管损伤再狭窄模型,分析两种大鼠模型的差异。第二部分通过球囊损伤复制SD大鼠颈动脉血管再狭窄模型,观测血管中TMEM66的表达变化情况。通过慢病毒特异性转染过表达TMEM66,观测各组血管再狭窄变化情况,以明确TMEM66在血管损伤再狭窄中的作用。通过检测不同干预组VSMCs增殖、迁移,探讨TMEM66在血管损伤再狭窄中的作用机制。研究方法:1.分为SD大鼠假手术组、SD大鼠手术组、Apo E-/-大鼠假手术组、ApoE-/-大鼠手术组。两种大鼠高脂饮食喂养2周后检测血常规,甘油三酯(TG)、空腹血糖(Glu),血清总胆固醇(TC)、高密度脂蛋白(HDL)、低密度脂蛋白(LDL)、C反应蛋白(CRP)、谷草转氨酶(AST)、谷丙转氨酶(ALT)、尿素氮(BUN)、肌酐(Scr)水平。两种大鼠手术组均行左侧颈总动脉球囊损伤术,假手术组不行球囊损伤,其余过程同手术组,术后继续高脂饮食喂养2周取材。HE染色观测斑块大小,油红O染色、MASSON染色、免疫荧光染色检测斑块成分。2.SD大鼠随机分为对照组(行假手术)、手术组(行左侧颈总动脉球囊损伤)、空载病毒组(左侧颈总动脉球囊损伤后行空载病毒转染)、TMEM66过表达组(左侧颈总动脉球囊损伤后行TMEM66基因过表达转染)。实时荧光定量PCR和Western blot检测假手术组和手术组血管TMEM66表达情况。免疫组化染色观察TMEM66表达情况。HE染色观测各组血管再狭窄情况。3.VSMCs随机分为对照组、PDGF处理组(PDGF处理)、空载病毒组(空载病毒转染后,予以PDGF处理)、TMEM66过表达组(TMEM66基因过表达转染后,予以PDGF处理),EdU染色检测VSMCs增殖能力,划痕试验检测VSMCs迁移能力。结果:1.Apo E-/-大鼠组血胆固醇及炎症介质明显高于SD大鼠组;两种大鼠手术组均有明显再狭窄斑块形成;ApoE-/-大鼠血管再狭窄斑块成分以脂质浸润、纤维沉积为特点,细胞成分以巨噬细胞及VSMCs为主,SD大鼠血管再狭窄斑块成分则以VSMCs为主。2.内皮损伤后血管再狭窄,其TMEM66的表达显著下降;上调TMEM66表达可抑制内皮损伤后血管再狭窄。3.上调TMEM66表达,能够显著抑制VSMCs的增殖和迁移。结论:跨膜蛋白66可能通过调控VSMCs的增殖与迁移,抑制新生内膜的过度增生,从而减轻内皮损伤后血管再狭窄。
[Abstract]:Background and objective: coronary heart disease (CHD) is a serious threat to human health. The percutaneous transluminal coronary intervention (PCI) is an important means for the treatment of coronary heart disease. However, the incidence of restenosis (RS) in the stent was still as high as 10%, which seriously affected the clinical efficacy of PCI. Therefore, the mechanism of RS is the key to guide its prevention and control. The proliferation and migration of vascular smooth muscle cells (VSMCs) is the main cause of intimal hyperplasia. The study shows that the calcium influx (SOCE) plays an important role in the proliferation, migration and intimal hyperplasia of VSMCs, and the transmembrane protein 66 (TMEM66) is an important component of the newly discovered calcium channel (SOC). SOCE can be regulated. It is suggested that the TMEM66 may also be used to control the proliferation and migration of VSMCs after vascular endothelial injury, and to inhibit the intimal hyperplasia after vascular endothelial injury, thereby reducing the restenosis of the vessel. In order to verify the above, this experiment is divided into two parts. In the first part, SD and Apo E gene knockout (ApoE-/-) were established by high-fat diet combined with balloon injury, and the difference of the two models was analyzed. The second part is to copy SD rat carotid artery restenosis model by balloon injury, and observe the change of the expression of TMEM66 in the blood vessel. The expression of TMEM66 was specifically transfected with lentivirus, and the changes of vascular restenosis in each group were observed to determine the role of TMEM66 in the restenosis of vascular injury. The mechanism of TMEM66 in the restenosis of vascular injury was discussed by detecting the proliferation and migration of VSMCs in different intervention groups. Study method:1. It was divided into two groups: sham operation group, SD rat operation group, Apo E-/-rat pseudo-operation group, and ApoE-/-rat operation group. Blood routine, triglyceride (TG), fasting blood glucose (Glu), total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), C-reactive protein (CRP), and aspartate aminotransferase (AST) were detected after two weeks of high-fat diet. Levels of alanine aminotransferase (ALT), urea nitrogen (BUN), and muscle strength (Scr). The left common carotid artery balloon injury was performed in the operation group of both rats, the balloon injury was not allowed in the sham operation group, and the other procedure was the same as the operation group, and the high-fat diet was continued after the operation for 2 weeks. 2. SD rats were randomly divided into control group (sham operation) and operation group (left common carotid artery balloon injury). No-load virus group (no-load virus transfection after the left common carotid artery balloon injury), and the TMEM66 overexpression group (the TMEM66 gene overexpression transfection after the left common carotid artery balloon injury). The expression of TMEM66 was detected by real-time fluorescence quantitative PCR and Western blot. The expression of TMEM66 was observed by immunohistochemical staining. 3. VSMCs were randomly divided into control group, PDGF treatment group (PDGF treatment), no-load virus group (after no-load virus transfection, PDGF treatment), TMEM66 overexpression group (after the overexpression of TMEM66 gene, PDGF treatment), and EdU staining to detect the proliferation ability of VSMCs. The scratch test is used to detect the migration ability of the VSMCs. Results:1. The blood cholesterol and inflammatory mediators in Apo E-/-rats were significantly higher than that of SD rats. The cellular components were mainly macrophages and VSMCs, and the components of vascular restenosis in SD rats were mainly VSMCs. The expression of TMEM66 decreased significantly after endothelial injury, and the expression of TMEM66 could inhibit the restenosis after endothelial injury. Up-regulation of the expression of TMEM66 can significantly inhibit the proliferation and migration of VSMCs. Conclusion: The cross-membrane protein 66 may inhibit the proliferation and migration of VSMCs, inhibit the hyperproliferation of neointima, and reduce the restenosis after endothelial injury.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R541.4

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