热应激诱导猪肾小管上皮(LLC-PK1)细胞线粒体凋亡相关因子的时效表达

摘要

拟探讨热应激诱导LLC-PK1细胞线粒体凋亡相关因子的表达和活性情况,以及激活线粒体凋亡通路的时间.采用qRT-PCR方法和Caspase活性检测试剂盒分别检测42℃热应激处理后0,2,4,8,16 h以及37℃培养的LLC-PK1细胞中HSP72、Bcl-2、Bax、AIF和Cyt.c的基因表达以及Caspase-9和Caspase-3的活性.结果显示,42℃热应激1h,LLC-PK1细胞Bcl-2/Bax(P＜0.05)和AIF(P＜0.01)基因表达以及Caspase-3(P＜0.01)活性从0h开始升高(P＜0.01),到2h达到最高(P ＜0.01);HSP72基因表达和Caspase-9活性0h即达到最高(P ＜0.01);Cyt.c基因表达在2h开始极显著升高(P＜0.01),并且此时最高,8h以后全部因子恢复到正常水平.结果表明,42℃热应激1h即激活LLC-PK1细胞线粒体凋亡通路,且热应激后的0～8h线粒体凋亡通路处于活化状态,其中2h时最为活跃.%To investigate the expression and the activity of factors related to mitochondrial apoptosis in LLCPK1 cells induced by heat stress,and the time of activation of mitochondrial apoptotic pathway,we assessed the HSP72,Bcl-2,Bax,AIF,Cyt.c expressions in LLC-PK1 cells by qRT-PCR and Caspase-9,Caspase-3 activity by caspase activity assay kit at 0,2,4,8,16 h after 42 ℃ heat stress treatment for 1 h.The above factors were also measured in cells cultured at 37 ℃ as control.Results demonstrated that Bcl-2/Bax and AIF gene expressions and Caspase-3 activity significantly increased from 0 h,and reached the highest point at 2 h after heat stress;HSP72 gene expression and Caspase-9 activity reached the highest point at 0 h;Cyt.c gene expression significantly increased and reached the highest point at 2 h,and all the measured factors returned to normal level at 8 h after heat stress.The above results reveal that mitochondrial apoptotic pathway in LLC-PK1 cells is in a state of activation in 0-8 h after 42 ℃ heat stress for 1 h and is the most active at 2 h.