RB基因与TSC2基因介导的协同效应对血管平滑肌细胞增殖的调控机制研究

发布时间：2018-01-22 19:19

本文关键词： 血管平滑肌细胞增殖 RB基因 TSC2基因协同效应　出处：《中国人民解放军医学院》2016年博士论文　论文类型：学位论文

【摘要】：目的：血管平滑肌细胞(vascular smooth muscle cell,VSMC)的无序增殖是血管重塑后发生再狭窄的重要因素。RB/E2F通路系统和mTOR信号通路系统与细胞周期的调控和细胞的增殖有着密切的联系,RB和TSC2基因分别是两条通路的核心调控基因。本研究探索RB/E2F与mTOR信号通路系统与VSMC增殖过程的相关性；探索RB与TSC2基因双敲除后介导的协同效应对细胞增殖的抑制作用;探索活性氧自由基(ROS)是否为RB与TSC2基因双敲除介导的协同效应引起细胞增殖停止的机制。方法：1,体外培养VSMC,并施加血小板源性生长因子(PDGF-BB)干预,模拟VSMC在体内的增殖表型。2,运用RT-PCR,Wertern-Blot法检测施加PDGF-BB干预前后,RB/E2F和TSC2/mTOR通路系统靶基因表达水平的变化情况。3,慢病毒转染RB及TSC2基因,应用MTT法分别检测空载慢病毒组(为空载体对照组)、RB基因单独敲除组、TSC2基因单独敲除组、RB/TSC2基因双敲除组的细胞增殖水平。4,Brdu染色分别检测对照组、RB基因单独敲除组、TSC2基因单独敲除组、RB/TSC2基因双敲除组的DNA合成水平。5,检测对照组、RB基因单独敲除组、TSC2基因单独敲除组、RB/TSC2基因双敲除组ROS表达水平。6,施加NAC(氧自由基清除剂)干预后,应用MTT法分别检测对照组、RB基因单独敲除组、TSC2基因单独敲除组、RB/TSC2基因双敲除组的细胞增殖水平。结果：1,在施加PDGF-BB影响因子后,RB/E2F信号系统的下游靶基因CCNA2和CDK1表达水平增加了8倍,mTOR信号通路系统的下游基因s6k的蛋白表达水平有明显的升高。2,RB基因单独敲除组、TSC2基因单独敲除组的细胞增殖水平较对照组有明显提高,而RB/TSC2基因双敲除组的细胞增殖水平较对照组则出现明显的降低。3,RB基因单独敲除组、TSC2基因单独敲除组的DNA合成水平较对照组有明显提高,而RB/TSC2基因双敲除组的DNA合成水平较对照组则出现显著的降低。4, RB/TSC2基因双敲除组ROS表达水平显著高于对照组,而对照组、RB基因单独敲除组、TSC2基因单独敲除组的ROS水平相差不大。5, RB/TSC2基因双敲除组的增殖水平在施加NAC干预后显著回升,与对照组(空载慢病毒组)、RB基因单独敲除组、TSC2基因单独敲除组的增殖水平相差不大。结论：本研究发现,RB/E2F和mTOR信号通路系统参与VSMC增殖的过程之中,并具有重要的作用。RB和TSC2分别作为两条通路的核心调控基因,双敲除RB/TSC2基因介导的协同作用显著的抑制了VSMC的增殖水平。而两条通路的开放代谢产生了大量的未清除的ROS,ROS的堆积则是抑制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.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R54

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