【摘要】：研究背景和目的:冠狀動脈粥樣硬化性心臟病(Coronary atherosclerotic heart disease,CHD)嚴重危害人類健康,經皮腔內冠狀動脈介入治療(Percutaneous Coronary Intervention,PCI)是治療冠心病的重要手段,過去20多年來,PCI治療雖然取得了長足進步,但支架內再狹窄(Restenosis,RS)的發(fā)生率仍高達10%,嚴重影響了PCI的臨床療效。因此,探討RS的發(fā)生機制是指導其防治的關鍵。血管內皮損傷后的內膜增生是血管再狹窄的重要病理過程,血管平滑肌細胞(Vascular smooth muscle cells,VSMCs)的增殖和遷移是內膜增生的主要原因。研究表明鈣庫操縱性鈣內流(store operated calcium entry,SOCE)在VSMCs增殖、遷移和血管內膜增生的過程中發(fā)揮著重要作用,跨膜蛋白66(transmembrane protein 66,TMEM66)是新近發(fā)現的鈣庫操控性鈣通道(store-operated calcium channel,SOC)的重要組成部分,可以調控SOCE。我們推測TMEM66可能也是通過調控血管內皮損傷后VSMCs增殖、遷移,抑制血管內皮損傷后內膜的過度增生,從而減輕血管再狹窄。為了驗證以上推測,本實驗分為兩部分進行。第一部分通過高脂飲食聯合球囊損傷建立SD、Apo E基因敲除(ApoE-/-)兩種大鼠血管損傷再狹窄模型,分析兩種大鼠模型的差異。第二部分通過球囊損傷復制SD大鼠頸動脈血管再狹窄模型,觀測血管中TMEM66的表達變化情況。通過慢病毒特異性轉染過表達TMEM66,觀測各組血管再狹窄變化情況,以明確TMEM66在血管損傷再狹窄中的作用。通過檢測不同干預組VSMCs增殖、遷移,探討TMEM66在血管損傷再狹窄中的作用機制。研究方法:1.分為SD大鼠假手術組、SD大鼠手術組、Apo E-/-大鼠假手術組、ApoE-/-大鼠手術組。兩種大鼠高脂飲食喂養(yǎng)2周后檢測血常規(guī),甘油三酯(TG)、空腹血糖(Glu),血清總膽固醇(TC)、高密度脂蛋白(HDL)、低密度脂蛋白(LDL)、C反應蛋白(CRP)、谷草轉氨酶(AST)、谷丙轉氨酶(ALT)、尿素氮(BUN)、肌酐(Scr)水平。兩種大鼠手術組均行左側頸總動脈球囊損傷術,假手術組不行球囊損傷,其余過程同手術組,術后繼續(xù)高脂飲食喂養(yǎng)2周取材。HE染色觀測斑塊大小,油紅O染色、MASSON染色、免疫熒光染色檢測斑塊成分。2.SD大鼠隨機分為對照組(行假手術)、手術組(行左側頸總動脈球囊損傷)、空載病毒組(左側頸總動脈球囊損傷后行空載病毒轉染)、TMEM66過表達組(左側頸總動脈球囊損傷后行TMEM66基因過表達轉染)。實時熒光定量PCR和Western blot檢測假手術組和手術組血管TMEM66表達情況。免疫組化染色觀察TMEM66表達情況。HE染色觀測各組血管再狹窄情況。3.VSMCs隨機分為對照組、PDGF處理組(PDGF處理)、空載病毒組(空載病毒轉染后,予以PDGF處理)、TMEM66過表達組(TMEM66基因過表達轉染后,予以PDGF處理),EdU染色檢測VSMCs增殖能力,劃痕試驗檢測VSMCs遷移能力。結果:1.Apo E-/-大鼠組血膽固醇及炎癥介質明顯高于SD大鼠組;兩種大鼠手術組均有明顯再狹窄斑塊形成;ApoE-/-大鼠血管再狹窄斑塊成分以脂質浸潤、纖維沉積為特點,細胞成分以巨噬細胞及VSMCs為主,SD大鼠血管再狹窄斑塊成分則以VSMCs為主。2.內皮損傷后血管再狹窄,其TMEM66的表達顯著下降;上調TMEM66表達可抑制內皮損傷后血管再狹窄。3.上調TMEM66表達,能夠顯著抑制VSMCs的增殖和遷移。結論:跨膜蛋白66可能通過調控VSMCs的增殖與遷移,抑制新生內膜的過度增生,從而減輕內皮損傷后血管再狹窄。
[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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R541.4

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