热休克蛋白70基因转染抗心肌细胞凋亡对缺氧／复氧损伤的保护作用.pdfVIP

· 3436· 医堂盘圭 Q 生! 垄 旦筮 鲞筮 翅 Nat1 Med J China，December 25．2007．Vo1 87．No．48 ． 基础研究 ． 热休克蛋白7O基因转染抗心肌细胞凋亡对缺氧／ 复氧损伤的保护作用 刘季春 万力 何明 程晓曙 【摘要】 目的 观察比较热休克蛋白70对心肌细胞急性实验性缺氧／复氧损伤的保护作用及 其与热休克心肌细胞保护模型之间作用的异同。方法 进行大鼠乳鼠原代心肌细胞培养，分为4组： 空白组(对照)，缺氧／再灌组(A／R)，热休克蛋白组(A／R+Hs)和pCDNA热休克蛋白质粒组 (pCDNA HSP )。用脂质体对pCDNA HSP 质粒包裹，并转导入相关心肌细胞内。RT—PCR检测 HSP mRNA表达、Western印迹检测HSP 蛋白质表达。检测心肌细胞存活率(四甲基偶氮唑蓝比色 法)、培养液中乳酸脱氢酶活性、心肌细胞超微结构改变(透射电镜)及凋亡指数观察心肌细胞损伤程 度。结果 A／R+Hs组和A／R pCDNA HSP7o组细胞存活率显著提高(71．8±10．3)％、(73．4± 12．2)％；(LDH)活性明显降低(14．3±2．6)IU／L、(14．6±2．9)IU／L；细胞超微结构明显改善；A／R 组的HSP7o mRNA和蛋白质含量比A／R+HS组和A／R pCDNA HSP 组，明显偏低(1．87±0．23、 2．05±0．34倍P0．01)。二者的细胞凋亡指数也较A／R组明显降低。结论 HsP70基因高表达能 对抗缺氧／复氧对心肌细胞的损伤，其作用与抗细胞凋亡有关。 【关键词】 热休克蛋白类；心肌再灌注损伤 l~oteelion of myocardiocytes against anoxia-reoxygenerafion injury by heat shock protein 70 gene transfectlon：experiment with rats LIU Ji—chun ，WAN Li，HE ，CHENG Xiao—shu． Department ofCardiovascular Surgery，First Affiliated Hospital ofNanchang University，Nanchang 330006， China Corresponding author：CHENG Xiao—shu，Email：xiaoshumenfan@126．con 【Abstract】 Objective To study the protective role of heat shock protein(HSP)7(】expression in the myocardial ceHs undergoing anoxia／reoxygeneration．Methods Myocardiocytes of neonatM SD rats were cultured and randomly divided into 4 groups：anoxia／repeffusion (A／R)group undergoing anoxia／ reperfusion；A／R +HS group，undergoing transfection of HS and A／R，pCDNA HSP70+A／R group， undergoing transfection of liposome—coated HSP7o pCDNA and A／R，and control group． RT-PCR and Western blotting were used to detect the mRNA and protein expression of HSP7o in the cells．The cell viability was examined by MTF method．The levels of lactic dehydrogenase(LDH)，and serum creatine phophokinase isoenzyme(CPK)were detected．Transmission electronic microscopy was used to observe the ultrastru