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

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1 h,LLC-PK1细胞Bcl-2/Bax(P0.05)和AIF(P0.01)基因表达以及Caspase-3(P0.01)活性从0 h开始升高(P0.01),到2 h达到最高(P0.01);HSP72基因表达和Caspase-9活性0 h即达到最高(P0.01);Cyt.c基因表达在2 h开始极显著升高(P0.01),并且此时最高,8 h以后全部因子恢复到正常水平。结果表明,42℃热应激1 h即激活LLC-PK1细胞线粒体凋亡通路,且热应激后的0~8 h线粒体凋亡通路处于活化状态,其中2 h时最为活跃。

Expression of Factors Related to Mitochondrial Apoptosis in Pig Kidney Proximal Tubular (LLC-PK1) Cells Induced by Heat Stress

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.