The Protective Effect Of Pim-2on H9c2Injury Induced By Anoxia-reoxygenation

Objective:To study the protective effect of Pim-2under A/R, the A／R model of H9c2cellswas established, and the expression of Pim-2was detected by Western blotting indifferent anoxia-reoxygenation time points. Then the Pim-2expression vector wastransfected into H9c2cells in order to study the protective effect of Pim-2under A/R.Methods:H9c2cells were provided from cell bank of Chinese academy of sciencesShanghai branch, incubated in a mixture of DMEM with10%FBS at37°C in5%CO2in a humidified atmosphere, harvested just as reaching approximate90%confluency for experiment.1、H9c2cells A/R model was established by anoxia-reoxygenation device.After H9c2cells were exposed to anoxia for0h、2h、4h、6h、8h、10h、12h、24h,the expression of Pim-2was detected by Western blotting. Then H9c2cells wereexposed to reoxygenation for0h,2h,4h,8h,10h, the expression of Pim-2was detectedby Western blotting.2、The Pim-2expression vector was transfected into H9c2cells. Theexperiment was divided into6groups:(1)control group;(2)A/Rgroup:using the bestanoxia-reoxygenation time points which was detected in step1;(3) PCMV-Pim-2-Flag+A/Rgroup;(4) PCMV-Pim-2-Flag group;(5) PCMV-Flag+A/Rgroup;(6)PCMV-Flaggroup. The rate of apoptosis was detected by Annexin-v FITC/PI double-dyed flowcytometry.The expression of Pim-2was detected by RT-PCR and Western blotting. Results:1. The percentage of H9c2cells apoptosis in different group.Compared with the control group, the rate of the H9c2apoptosis in A/R groupwas significantly increased(5.103±0.136%vs22.81±0.425%, P＜0.05);Comparedwith the A/R group and PMCV-flag+A/R group, the rate of H9c2apoptosis ofPCMV-Pim-2-Flag+A/R was decreased(22.81±0.425%vs14.55±0.021%, P＜0.05;23.267±0.41%vs14.55±0.021%, P＜0.05).Compared with the control group,the rateof H9c2apoptosis of PCMV-Pim-2-Flag and PCMV-Flag showed little change（5.1035±0.136%vs5.25±0.595, P＞0.05;5.103±0.136%vs5.45±0.15%, P＞0.05）.2.The expression of Pim-2mRNA.The expression of Pim-2mRNA was significantly increased in A/R group bycomparison with that of the control group(0.399±0.110vs0.441±0.014, P＜0.05).The expression of Pim-2mRNA was further elevated in PCMV-Pim-2-Flag+A/Rgroup，by comparison with that of A/R and PCMV-Pim-2-Flag group (0.441±0.014vs0.694±0.018, P＜0.05;0.541±0.015vs0.694±0.018, P＜0.05).However, in controland PCMV-Flag group, there showed little change (0.399±0.110vs0.389±0.010, P＞0.05).3.The expression of Pim-2protein.The expression of Pim-2at the different time points of A/R were higher thanthose of the control group, peaking at12/2hour(1.672±0.065vs1.514±0.067, P＜0.05).After the Pim-2expression vector transfected, the expression of Pim-2proteinincreased significantly in A/R group by comparison with that of the controlgroup(1.514±0.067vs1.636±0.029, P＜0.05). The expression of Pim-2protein wasfurther elevated in PCMV-Pim-2-Flag+A/R group, by comparison that of A/R andPCMV-Pim-2-Flag group(1.636±0.029vs1.930±0.065, P＜0.05;1.784±0.062vs1.930±0.065, P＜0.05). However, in control and PCMV-Flag group, there showedlittle change (1.514±0.067vs1.517±0.052, P＞0.05). Conclusions:Our study show that the Pim-2gene is expressed in H9c2cells in low level. A/Rinduced higher Pim-2expression, peaking at12/2hour. Furthermore, the transfectionof Pim-2into H9c2cells may play a protective role against A/R.