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目的评价丝氨酸/苏氨酸蛋白激酶(HIPK1)促进肿瘤坏死因子(TNF)-α诱导的JNK/p38激活及其对肝癌细胞凋亡的影响。方法Hepa1-6细胞,分别感染重组腺病毒rAd-Lacz-V5(107PFU/孔)和rAd-myc-HIPK1(107PFU/孔)48h,荧光显微镜检测Lacz和HIPK1的蛋白表达,TNF-α处理细胞后Western blot检测JNK/p38激活情况;HIPK1的RNA干扰检测HIPK1在JNK/p38激活中的作用;TNF-α处理病毒感染的Hepa1-6细胞24h,DAPI染色荧光显微镜下了解细胞凋亡情况。结果荧光显微镜下见Lacz和HIPK1蛋白在Hepa1-6细胞中表达效率高;HIPK1组中TNF-α介导的磷酸化型JNK/p38的表达明显高于Lacz组,相反HIPK1的RNA干扰组中TNF-α介导的磷酸化型JNK/p38的表达明显低于对照组;细胞核形态学检测HIPK1组中细胞凋亡率为(30.72±5.67)%,而Lacz组为(9.82±3.41)%,两组间差异有统计学意义(P<0.01)。结论HIPK1促进TNF-α介导的JNK/p38的激活并诱导肝癌细胞Hepa1-6凋亡。
Objective To evaluate the effect of serine / threonine protein kinase (HIPK1) on JNK / p38 activation induced by tumor necrosis factor (TNF) -α and its effect on hepatocellular carcinoma cell apoptosis. Methods Hepa1-6 cells were infected with recombinant adenovirus rAd-Lacz-V5 (107PFU / well) and rAd-myc-HIPK1 (107PFU / well) for 48h. The expression of Lacz and HIPK1 were detected by fluorescence microscope. Western blot was used to detect the activation of JNK / p38; HIPK1 was used to detect the effect of HIPK1 on JNK / p38 activation; Hepa 1-6 cells infected with TNF-α were infected with DAPI for 24 hours. Results The expression of Lacz and HIPK1 protein in Hepa1-6 cells under fluorescence microscope was high. The expression of TNF-α-mediated phosphorylated JNK / p38 in HIPK1 group was significantly higher than that in Lacz group. In contrast, in HIPK1 group, TNF- -α mediated phosphorylation of JNK / p38 was significantly lower than that of the control group. The apoptosis rate of HIPK1 group was (30.72 ± 5.67)% by nuclear morphology, while it was (9.82 ± 3.41)% by Lacz group There was significant difference between groups (P <0.01). Conclusion HIPK1 can promote TNF-α-mediated JNK / p38 activation and induce Hepa1-6 hepatocarcinoma cell apoptosis.