【作者】 李燕；

【导师】 宋耀明；

【作者基本信息】 第三军医大学 ， 内科学， 2011， 硕士

【摘要】 研究背景及目的:冠心病介入术后再狭窄发病率高,成为介入手术晚期失败的主要原因。再狭窄的发生主要原因是新生内膜形成和血管重塑,其中平滑肌细胞凋亡与增殖的失衡在新生内膜形成中发挥关键作用[1]。参与平滑肌细胞凋亡的调节因子众多,调节机制也存在多条途径。主要有线粒体介导的信号通路,死亡受体介导的信号通路等。线粒体是细胞能量代谢的中心,并且在细胞凋亡过程中发挥重要的调节作用。在细胞接受凋亡刺激后,引起线粒体膜通透性增高,线粒体膜电位降低,继而释放凋亡相关蛋白,如细胞色素C(CytC)、凋亡诱导因子(AIF)等,释放的CytC与凋亡蛋白酶活化因子(Apaf-1)结合,活化半胱氨酸蛋白水解酶(caspase) 9,进而激活caspase 3途径,从而导致细胞凋亡。腺嘌呤核苷酸转位酶是定位于线粒体内膜的转运蛋白之一,属于物种进化中的保守区域,是细胞凋亡调节的关键基因[2],研究表明ANT1诱导凋亡作用与线粒体渗透性转换复合孔(mitochondrial permeability transition pore MPTP)有关[3],可能作为MPTP的一个必要成分,或是与MPTP的主要成分之一-----亲环素D( Cyclophilin D)相互作用[4],从而引起线粒体膜电位降低,触发线粒体途径的细胞凋亡。细胞实验证实ANT1可以诱导心肌细胞及肿瘤细胞的凋亡,在体实验中可以减小肿瘤瘤体体积[5,6]。因而,本研究通过构建ANT1腺病毒载体,分别用携带ANT1基因的GFP标记的腺病毒及空载体腺病毒转染体外培养的血管平滑肌细胞,检测ANT1的表达,进而对过表达ANT1的平滑肌细胞进行凋亡检测及增殖活性检测,并分别提取各组细胞的mRNA和蛋白,检测其中Bax/Bcl-2的表达。从而评价其对平滑肌细胞凋亡的诱导作用及作用机制,以此为作用靶点,可能为支架术后再狭窄等有关平滑肌细胞增殖与凋亡失衡疾病提供了新的治疗途径。实验方法:1、构建携带ANT1基因的GFP标记的重组腺病毒表达载体。采用分子克隆技术,重组穿梭质粒pShuttle-GFP-CMV-ANT1和pShuttle-GFP-CMV,与pAdxsi载体酶切后连接,采用脂质体转染法转染人胚肾HEK293细胞,包装产生ANT1腺病毒颗粒,RT-PCR鉴定为ANT1重组复制缺陷腺病毒后进行大量扩增。2、组织块法体外培养原代大鼠血管平滑肌细胞,用α-actin进行鉴定。以Ad-ANT1或Ad-GFP病毒转染VSMCs,观察不同时间的转染效率。并通过RT-PCR及Western Blotting方法检测各组细胞ANT1mRNA及蛋白的表达水平。3、以Ad-ANT1或Ad-GFP病毒转染VSMCs,分别检测细胞凋亡和细胞增殖:利用激光共聚焦观察Hochest染色凋亡细胞核,流式细胞术及TUNEL法检测细胞凋亡率,激光共聚焦观察TMRE法检测线粒体膜电位改变。细胞计数法及CCK8法检测细胞的增殖活性。4、转染后分别取各组细胞,利用RT-PCR及Western Blotting方法检测各组Bax/Bcl-2的mRNA及蛋白的表达水平。结果:1、经细胞形态学和α-actin免疫细胞化学方法鉴定,应用组织块法原代培养所得细胞即为VSMCs。可以用于后续实验。2、成功构建并扩增了Ad-ANT1和Ad-GFP(空载体对照)腺病毒,病毒滴度测定为2×1011pfu/ml。最佳感染复数MOI为80-100,分别用ANT1腺病毒载体及空载体腺病毒转染VSMCs后,转染效率高约80-90%,RT-PCR及Western Blotting显示于空载体转染组相比较,前者ANT1mRNA及蛋白的表达水平均显著增高。可以用于后续实验。3、细胞凋亡检测结果:形态学检测:激光共聚焦观察Hoechst染色后,Ad-ANT1转染组细胞染色质边聚,细胞核不规则,固缩,或碎裂等明显细胞凋亡表现。空载体组细胞核无显著性改变。TMRE法检测细胞线粒体膜电位,Ad-ANT1转染组细胞出现线粒体膜电位下降或消失,空载体转染组无显著性改变。TUNEL及流式细胞术显示Ad-ANT1转染组细胞凋亡特征明显,然后计数凋亡细胞,与空载体转染组相比较有显著性差异。4、细胞增殖活性检测结果:经细胞计数及CCK8法检测,与空载体转染组相比较,Ad-ANT1转染组细胞增殖活性显著降低。5、RT-PCR及Western Blotting检测Ad-ANT1转染组Bax的mRNA及蛋白表达水平增高,与空载体转染组相比较有显著差异。Bcl-2 mRNA及蛋白的表达水平两组间无显著差异。结论:1、成功构建了ANT1的腺病毒过表达载体,并且在体外培养的血管平滑肌细胞中有效表达。2、过表达ANT1腺病毒组能明显抑制细胞增殖活性,诱导血管平滑肌细胞出现凋亡。3、过表达ANT1腺病毒载体,诱导血管平滑肌细胞凋亡的可能机制,是通过上调Bax从而使Bax/Bcl-2升高而实现的。更多还原

【Abstract】 Background and Objective:Restenosis after percutaneous coronary intervention has been the main reason for the failure of atherosclerotic therapy. Neointimal formation and vascular reshape are the main reasons to the occurrence of restenosis. Imbalances of smooth muscle cell apoptosis and proliferation plays a key role in the formation of neointimal. Numerous factors participate in smooth muscle cell apoptosis, and there are many regulating molecular ways. They include mitochondrial mediated signaling pathways and death receptor mediated signaling pathways, etc. Mitochondria is the center of cellular energy metabolism, which plays an important role in cell apoptosis. Stimulus leads to cell apoptosis by increasing the mitochondrial membrane permeability and the reduction of mitochondrial membrane potential. Then apoptosis related proteins were released, such as CytC, apoptosis-inducing factors (AIF) etc. The union of CytC and protease activated factor (Apaf-1) activates cysteine proteolytic enzymes caspase 9 and caspase 3 ways, and eventually induced cell apoptosis.Adenine nucleotide transposition enzymes are one of the transport proteins located in the mitochondrial inner membrane. This gene maintains evolution conservative and plays a key role in regulating cell apoptosis.Many studies showed that ANT1 induced cell apoptosis is related to mitochondrial permeability transition pore (MPTP). ANT1, a necessary ingredient of MPTP interacts with Cyclophilin D, which is the major component of MPTP. The interaction reduced the mitochondrial membrane potential, triggers cell apoptosis of mitochondrial way. ANT1 can induce myocardial cells and tumor cell apoptosis in vitro; meanwhile it can reduce tumor volume tumors in vivo. Therefore, we first constructed adenine nucleotide translocase 1(ANT1)with GFP tag adenovirus vector and transfected the vascular smooth muscle cells(VSMCs) in rat. After adenovirus-mediated ANT1 (Ad-ANT1)or adenovirus-empty(Ad-GFP) transfection, the apoptosis and proliferation of VSMCs was tested, meanwhile the Bax/Bcl-2 expression were detected. Hence, the function of ANT1 in smooth muscle cell apoptosis was evaluated. Hopefully ANT1 maybe a new therapeutic target for the imbalance between vascular smooth muscle cell proliferation and apoptosis in the pathogenesis of restenosis after PCI.Methods:1、Construction of the ANT1 genes recombinant adenovirus vector with the GFP tag. Recombinant shuttle plasmid pShuttle-GFP-CMV-ANT1 and pShuttle-GFP-CMV were ligased with pAdxsi vector after enzymes digestion.The recombinant adenovirus vector was transfected into HEK293 cells by using LiporeetaminelTM 2000 and packaged for the recombinant adenovirus particles. Replication-deficient recombinant adenovirus which carries the ANT1 gene was identified by RT-PCR and amplified.2、VSMCs derived from the thoracic aortic explants of Sprague Dawley rats were primarily cultured, and confirmed by microscope andα-SM-actin immunocytochemistry. After adenovirus-mediated ANT1 (Ad-ANT1) or adenovirus-empty(Ad-GFP) transfection into VSMCs, the transfected efficiency was observed in different time by fluorescence microscope. The ANT1 mRNA and protein expression were detected by RT-PCR and Western Blotting respectively.3、After adenovirus-mediated ANT1 (Ad-ANT1) or adenovirus-empty(Ad-GFP)transfection,the apoptosis of VSMCs stained by Hoechst 33258 was observed by laser confocal microscopy.The proportion of VSMCs apoptosis marked by Annexin-V was detected by flow cytometric analysis or TUNEL assay. The viability of VSMCs was measured by CCK8 assay and cell count. The changes in mitochondrial membrane potential (△Ψm) were detected by fluorescent probe TMRE. 4、After adenovirus-mediated ANT1 (Ad-ANT1) or adenovirus-empty(Ad-GFP) transfection into VSMCs,the Bax/Bcl-2 mRNA and protein expression were detected by RT-PCR and Western Blotting respectively.Results:1、Cultured rat thoracic aortic cells were identified as VSMCs by morphology andα-SM-actin immunocytochemistry and were used in the following experiments.2、The recombinant adenovirus vector was constructed and amplification successfully, The titer of 2×l011 pfu/ml was obtained and the optimal multiplicity of infection(MOI) was 80-100.After adenovirus-mediated ANT1 (Ad-ANT1) or adenovirus-empty(Ad-GFP) transfection into VSMCs , the infection efficiency was 80% by fluorescence microscope. The expression of ANT1 mRNA and protein in VSMCs after transfection with Ad-ANT1 by RT-PCR and Western Blotting are increased significantly. They can be used in the following experiments.3、Apoptosis test Morphologic detection: After Ad-ANT1 transfection,VSMCs stained by Hoechst33258 by laser confocal microscopy showed that cell apoptosis specific features. Chromatin was dense which located mainly in the nuclear membrane. Cell nucleuses become pyknotic and apoptotic peak. There is no significant morphologic change in the Ad-GFP group. The levels of mitochondrial membrane potential (?Ψm) was decreased in Ad-ANT1 group compared with Ad-GFP group ,which was detected by fluorescent probe TMRE. Flow cytometric analysis and TUNEL assay showed a significant increase in the percentage of VSMCs apoptosis after adenovirus-mediated ANT1 (Ad-ANT1) transfection compared with cells of adenovirus-empty(Ad-GFP)group.4、Proliferation activity detection: CCK8 assay and cell count both showed the proliferation activity of Ad-ANT1 group was decreased significantly compared with Ad-GFP group.5、The Bax mRNA and protein expression were detected by RT-PCR and Western Blotting respectively. ANT1 is upregulated in VSMCs after Ad-ANT1 transfection compared Ad-GFP.No differences were observed in the mRNA and protein levels of Bcl-2 in the two groups. Conclusions:1、The recombinant adenovirus ANT1 vector was constructed successfully and can be expressed efficiently in vascular smooth muscle cells in vitro.2、Overexpression of ANT1 in VSMCs induced VSMCs apoptosis and inhibited VSMCs proliferation.3、ANT1 overexpression can induce cell apoptosis in VSMCs. The underlying mechanism may partly be attributed to up-regulate Bax expression and the rise of Bax/Bcl-2 ratio.更多还原