Pim-3对抗心肌细胞缺氧/复氧损伤的研究

目的研究原癌基因Pim-3对心肌细胞急性缺氧/复氧(A/R)损伤的保护作用。方法采用原代培养新生大鼠的心肌细胞A/R损伤和缺氧预适应(APC)保护模型,将已经构建好的pEGFP-N2/Pim-3质粒导入原代培养的乳鼠心肌细胞中,实验结束后测定Pim-3mRNA及蛋白表达水平(RT-PCR、Western blot法)的改变,同时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。结果RT-PCR和Western blot结果显示,Pim-3在正常组几乎不表达,A/R组表达明显升高,缺氧预适应(APC)+A/R组表达则进一步升高;转染pEGFP-N2/Pim-3质粒后24h,荧光倒置显微镜显示转染效率达30%;在A/R损伤后,Pim-3基因转染组较未转染组的心肌细胞LDH值明显降低,细胞存活率则明显升高,心肌细胞的凋亡指数明显下降。结论在细胞水平,Pim-3参与心肌APC保护作用,导入外源性的Pim-3基因能对抗急性A/R损伤。

The research of Pim-3 protective effect on cardiomyocyte anoxia-reoxygenation injury

Aim To study the protective effect of proto-oncogene Pim-3 on cardiomyocytes aganist anoxia-reoxygenation(A/R)injury.Methods The A/R and anoxia preconditioning(APC)models of primary cultured neonatal rat myocytes were established and the expression vector pEGFP-N2/Pim-3 inserted with wild type target gene Pim-3 was transfected into the cardiomyocytes.Expression of Pim-3 was detected by RT-PCR and Western blot.LDH activity,MTT and TUNEL were detected after treatment.Results Pim-3 showed little expression in the control group but did high expression in A/R injury,Furthermore,pim-3 showed a higher expression in the APC+A/R group than in the A/R group.The efficiency of transfection of exogenous genes into cardiomyocytes was as high as 30% determined by fluorescence microscope after 24 h.In the pEGFP-N2/Pim-3+A/R group,the cellular survival was significantly increased while the LDH activity and the apoptotic index of cell were markedly decreased compared with the A/R group.Conclusion Pim-3 should be involved in the role of APC protection and transfecting of exogenous Pim-3 into cardiomyocytes could protect cardiomyocytes against the A/R injury.