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目的:探讨原癌基因Pim-3对抗心肌急性损伤的作用是否与线粒体信号转导途径介导的细胞色素C(CytC)的释放有关。方法:采用大鼠乳鼠心肌细胞,随机分为4组,对照组;缺氧复氧(A/R)组,建立A/R损伤模型;pcDNA3.1+A/R组,将pcDNA3.1转染入心肌细胞,24h后进行A/R损伤;pcDNA3.1/Pim-3+A/R组,将Pim-3重组质粒(pcDNA3.1/Pim-3)转染入心肌细胞,24h后进行A/R损伤。实验结束后,检测培养液中乳酸脱氢酶(LDH)活性,四唑盐(MTT)比色试验测定细胞存活率,TUNEL法检测细胞凋亡,蛋白印迹检测Pim-3蛋白表达水平,线粒体、胞浆CytC动态变化和caspase-3活性。结果:与对照组比较,A/R组细胞存活率明显下降,LDH值和凋亡指数明显升高,差异有统计学意义(P<0.01);与A/R组比较,pcDNA3.1/Pim-3+A/R组细胞存活率明显升高,LDH值和凋亡指数明显下降,并且CytC由线粒体向胞浆的释放明显被抑制,同时caspase-3活性也下降,差异有统计学意义(P<0.01)。结论:Pim-3对抗心肌急性损伤的机制是通过抑制CytC易位释放入胞浆激活caspase-3而致。
Objective: To investigate whether the effect of Pim-3 on acute myocardial injury is related to the release of cytochrome C (CytC) mediated by mitochondrial signal transduction pathway. Methods: Rat neonatal rat cardiomyocytes were randomly divided into four groups: control group, hypoxia-reoxygenation (A / R) group, A / R injury model and pcDNA3.1 + A / R group. The recombinant plasmid pim-3 (pcDNA3.1 / Pim-3) was transfected into cardiomyocytes and transfected into cardiomyocytes 24h after A / R injury; pcDNA3.1 / Pim-3 + Perform A / R injury. After the experiment, the activity of lactate dehydrogenase (LDH) in the culture medium was measured, the cell survival rate was measured by MTT colorimetric assay, the apoptosis was detected by TUNEL method, the expression of Pim-3 protein was detected by Western blot, Cytoplasmic CytC dynamic changes and caspase-3 activity. Results: Compared with the control group, the cell viability in A / R group was significantly decreased and the LDH value and apoptosis index were significantly increased (P <0.01). Compared with A / R group, pcDNA3.1 / Pim -3 + A / R group, the LDH value and apoptotic index decreased significantly, and the release of CytC from mitochondria to cytoplasm was significantly inhibited, and the activity of caspase-3 also decreased, with statistical significance ( P <0.01). Conclusion: The mechanism of Pim-3 against acute myocardial injury is caused by inhibiting the release of CytC translocation into the cytoplasm to activate caspase-3.