本文關(guān)鍵詞： 血管平滑肌細胞 增殖 RB基因 TSC2基因 協(xié)同效應　出處：《中國人民解放軍醫(yī)學院》2016年博士論文　論文類型：學位論文

【摘要】：目的：血管平滑肌細胞(vascular smooth muscle cell,VSMC)的無序增殖是血管重塑后發(fā)生再狹窄的重要因素。RB/E2F通路系統(tǒng)和mTOR信號通路系統(tǒng)與細胞周期的調(diào)控和細胞的增殖有著密切的聯(lián)系,RB和TSC2基因分別是兩條通路的核心調(diào)控基因。本研究探索RB/E2F與mTOR信號通路系統(tǒng)與VSMC增殖過程的相關(guān)性；探索RB與TSC2基因雙敲除后介導的協(xié)同效應對細胞增殖的抑制作用;探索活性氧自由基(ROS)是否為RB與TSC2基因雙敲除介導的協(xié)同效應引起細胞增殖停止的機制。方法：1,體外培養(yǎng)VSMC,并施加血小板源性生長因子(PDGF-BB)干預,模擬VSMC在體內(nèi)的增殖表型。2,運用RT-PCR,Wertern-Blot法檢測施加PDGF-BB干預前后,RB/E2F和TSC2/mTOR通路系統(tǒng)靶基因表達水平的變化情況。3,慢病毒轉(zhuǎn)染RB及TSC2基因,應用MTT法分別檢測空載慢病毒組(為空載體對照組)、RB基因單獨敲除組、TSC2基因單獨敲除組、RB/TSC2基因雙敲除組的細胞增殖水平。4,Brdu染色分別檢測對照組、RB基因單獨敲除組、TSC2基因單獨敲除組、RB/TSC2基因雙敲除組的DNA合成水平。5,檢測對照組、RB基因單獨敲除組、TSC2基因單獨敲除組、RB/TSC2基因雙敲除組ROS表達水平。6,施加NAC(氧自由基清除劑)干預后,應用MTT法分別檢測對照組、RB基因單獨敲除組、TSC2基因單獨敲除組、RB/TSC2基因雙敲除組的細胞增殖水平。結(jié)果：1,在施加PDGF-BB影響因子后,RB/E2F信號系統(tǒng)的下游靶基因CCNA2和CDK1表達水平增加了8倍,mTOR信號通路系統(tǒng)的下游基因s6k的蛋白表達水平有明顯的升高。2,RB基因單獨敲除組、TSC2基因單獨敲除組的細胞增殖水平較對照組有明顯提高,而RB/TSC2基因雙敲除組的細胞增殖水平較對照組則出現(xiàn)明顯的降低。3,RB基因單獨敲除組、TSC2基因單獨敲除組的DNA合成水平較對照組有明顯提高,而RB/TSC2基因雙敲除組的DNA合成水平較對照組則出現(xiàn)顯著的降低。4, RB/TSC2基因雙敲除組ROS表達水平顯著高于對照組,而對照組、RB基因單獨敲除組、TSC2基因單獨敲除組的ROS水平相差不大。5, RB/TSC2基因雙敲除組的增殖水平在施加NAC干預后顯著回升,與對照組(空載慢病毒組)、RB基因單獨敲除組、TSC2基因單獨敲除組的增殖水平相差不大。結(jié)論：本研究發(fā)現(xiàn),RB/E2F和mTOR信號通路系統(tǒng)參與VSMC增殖的過程之中,并具有重要的作用。RB和TSC2分別作為兩條通路的核心調(diào)控基因,雙敲除RB/TSC2基因介導的協(xié)同作用顯著的抑制了VSMC的增殖水平。而兩條通路的開放代謝產(chǎn)生了大量的未清除的ROS,ROS的堆積則是抑制VSMC增殖的主要原因。
[Abstract]:Objective: to investigate the vascular smooth muscle cell in vascular smooth muscle cells. The disorder proliferation of VSMC is an important factor of restenosis after vascular remodeling. The RBR / E2F pathway system and the mTOR signaling pathway system are closely related to the regulation of cell cycle and cell proliferation. RB and TSC2 genes are the core regulatory genes of the two pathways. This study explored the correlation between RB/E2F and mTOR signaling pathway system and the proliferation of VSMC. To explore the synergistic effect of double knockout of RB and TSC2 gene on the inhibition of cell proliferation. To explore whether Ros is the mechanism of the synergistic effect of RB and TSC2 gene double knockout on cell proliferation. Methods: 1, VSMC was cultured in vitro. Platelet derived growth factor PDGF-BBB was applied to simulate the proliferation phenotype of VSMC in vivo, and RT-PCR was used. The changes of target gene expression in RBR / E2F and TSC2/mTOR pathway system before and after PDGF-BB intervention were detected by Wertern-Blot method. The RB and TSC2 genes were transfected with lentivirus, and MTT method was used to detect the TSC2 gene knockout group in the empty vector control group (empty vector control group). The cell proliferation level of RB/TSC2 gene double knockout group was detected by Brdu staining. The level of DNA synthesis in the RB/TSC2 gene double knockout group was .5. the control group was detected by the RB gene knockout group and the TSC2 gene knockout group was detected by the single knockout group. The expression level of ROS in RB/TSC2 gene double knockout group was. 6. After the intervention of NAC (oxygen free radical scavenger), the RB gene of control group was detected by MTT method. The cell proliferation level of RBR / TSC2 double knockout group was detected in TSC2 gene knockout group. Results: 1, after applying PDGF-BB influencing factors. The expression level of CCNA2 and CDK1 in the downstream target genes of RB/E2F signal system increased 8-fold, and the protein expression level of the downstream gene s6k in the signal pathway system of mTOR increased significantly by 2.2. The proliferation level of TSC2 gene alone knockout group was significantly higher than that of the control group. The proliferation level of RB/TSC2 gene double knockout group was significantly lower than that of control group. The level of DNA synthesis in the TSC2 gene knockout group was significantly higher than that in the control group, while the DNA synthesis level in the RB/TSC2 gene double knockout group was significantly lower than that in the control group. The level of ROS expression in the double knockout group of RB/TSC2 gene was significantly higher than that in the control group, while the ROS level in the single knockout group of the RB gene alone in the control group was similar to that in the control group. The proliferation level of RB/TSC2 gene double knockout group increased significantly after NAC intervention, and was significantly higher than that of control group. Conclusion: this study found that RBP-E2F and mTOR signaling pathway system were involved in the process of VSMC proliferation. RB and TSC2 are the core regulatory genes of the two pathways, respectively. The synergism mediated by double knockout RB/TSC2 gene significantly inhibited the proliferation of VSMC, and the open metabolism of the two pathways produced a large number of ROS that were not cleared. The accumulation of ROS is the main reason to inhibit the proliferation of VSMC.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R54

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