目的 探讨Bcl-2/Bad/mPTP通路在14-3-3γ对抗脂多糖所致心肌损伤中的作用.方法 构建pFLAG-14-3-3γ重组质粒,转染至原代乳鼠心肌细胞中,然后行LPS损伤处理.处理结束后,取培养液检测LDH活性,MTT比色法检测细胞存活率,流式细胞术检测细胞凋亡,线粒体肿胀实验检测mPTP开放,Western blot检测14-3-3γ、Bad、phospho-Bad以及线粒体Bcl-2蛋白表达.结果 LPS损伤使心肌细胞LDH活性升高、细胞存活率下降、凋亡细胞增加、mPTP开放加剧,转染pFLAG-14-3-3γ重组质粒后再行LPS损伤,则LDH活性下降、细胞存活率升高、mPTP开放与细胞凋亡减少,同时phospho-Bad蛋白表达增加,线粒体Bcl-2蛋白表达增加.结论 pFLAG-14-3-3γ能够对抗脂多糖所致的心肌细胞损伤,其机制可能与磷酸化Bad,释放Bcl-2至线粒体,抑制mPTP的开放有关.%Aim To explore the role of Bcl-2／Bad／mPTP pathway in 14-3-3γprotecting against LPS-in-duced cardiomyocyte injury.Methods pFLAG-14-3-3γrecombinant lasmid was constructed and transfect-ed into primary neonatal SD rat cardiomyocytes.After treatment of LPS injury,the activity of LDH was ana-lyzed in supernatant;cell viability was detected by MTT colorimetric assay;cell apoptosis was determina-ted by flow cytometry;opening of mitochondrial perme-ability transition pore(mPTP)was detected by swelling of isolated cardiac mitochondria;the level of 14-3-3γ,Bad,phospho-Bad protein in cell and the level of Bcl-2 protein in mitochondria were analyzed bv Western blot.Results LPS damage made LDH activity increase,cell viability decrease,cell apoptosis increase,and mPTP opening aggravate.After the transfection with pFLAG-14-3-3γ,LDH activity and cell apoptosis were decreased,cell viability was increased,mPTP opening was inhibited,and level of phospho-Bad was raised,while level of Bcl-2 in mitochondria was also increased.rnConclusions pFLAG-14-3-3γcan protect against LPS-induced cardiomyocyte damage,and the mecha-nism is related to phosphorylating Bad,releasing Bcl-2rnto mitochondria,and inhibiting mPTP opening.
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