骨形态发生蛋白-7基因转染对新生大鼠心肌细胞缺氧-复氧时凋亡的影响

Effects of transfection with bone morphogenetic protein-7 on apoptosis in neonate rate cardiomyocytes induced by hypoxia-reoxygenation

目的探讨骨形态发生蛋白-7（BMP-7）基因转染对新生大鼠心肌细胞缺氧-复氧时凋亡的影响。方法体外培养的新生大鼠心肌细胞分为3组：正常对照组（C组）、缺氧-复氧组（HR组）培养的心肌细胞置于95%N2-5%CO2混合气体饱和的缺氧罐中缺氧2h后,放回孵育箱中继续培养4h;BMP-7基因转染组（BT组）将含pcDNA3.1-BMP-7基因的质粒在转染试剂作用下转染心肌细胞后72h再行缺氧2h,复氧4h。每组重复8次。计数心肌细胞搏动次数,采用全自动生化分析仪测定乳酸脱氢酶（LDH）活性,倒置显微镜下观察心肌细胞形态。采用流式细胞仪检测心肌细胞凋亡情况,免疫细胞化学方法测定Bcl-2、Bax蛋白的表达。结果与C组相比,HR组和BT组心肌细胞LDH活性、细胞凋亡率增加,Bax蛋白表达上调,Bcl-2蛋白表达下调（P〈0.05）;与HR组相比,BT组心肌细胞LDH活性、细胞凋亡率降低,Bax蛋白表达下调,Bcl-2蛋白表达上调（P〈0.05）。结论BMP-7基因转染可减轻新生大鼠心肌细胞缺氧-复氧损伤,其机制可能与调控Bcl-2、Bax蛋白的表达,抑制心肌细胞凋亡有关。

Objective To investigate the effects of transfection with bone morphogenetic protein-7 （BMP-7） on apoptosis in rat cardiomyocytes induced by hypoxia-reoxygenation （H/R）.Methods Cardiomyocytes were isolated from 3 day-old Wistar rats of both sexes and cultured in DMEM liquid culture medium for 3 days and then randomly divided into 3 groups：groupⅠnormal control （C）;groupⅡH/R and groupⅢBMP-7 gene transfection （BT）.In H/R group the cardiomyocytes were subjected to 2 h hypoxia and 4 h reoxygenation.In groupⅢthe cardiomyocytes were transfected with plasmid containing pcDNA3.1-BMP-7 gene and then subjected to 2 h hypoxia and 4 h reoxygenation.The lactate dehydrogenase （LDH） activity,the frequency of cardiomyocyte contraction and morphologic changes were determined.The apoptosis in cardiomyocytes were determined by flow cytometry and Bcl-2 and Bax protein expression in cardiomyocytes was measured by immuno-cytochemistry.Results H/R significantly increased LDH activity and apoptosis rate,up-regulated Bax protein expression and downregulated Bcl-2 protein expression in H/R and BT groups （ⅡandⅢ） as compared with control group. Transfection with BMP-7 gene significantly attenuated the above-mentioned changes induced by H/R.Conclusion Transfection with BMP-7 gene can significantly protect cardiomyocytes against H/R injury by inhibiting the Bax protein expression and increasing Bcl-2 protein expression leading to suppression of cardiomyocyte apoptosis.