P38及上游激酶MKK6对热损伤诱导Raw264.7细胞凋亡的调控作用

目的探讨P38及其上游激酶MKK6对热损伤诱导单核细胞系Raw264.7细胞凋亡的调控作用。方法应用脂质体介导的基因转染将P38的显性负效突变体P38(AF)及具有持续活性的组成性活性突变体MKK6b(E)导入Raw264.7细胞,观察P38对细胞凋亡的调控作用。结果荧光染色及Westernblot检测显示,外源基因转染进细胞,并在细胞中得到表达;蛋白激酶活性检测显示,MKK6b(E)转染细胞系P38激酶活性明显升高,而P38(AF)转染细胞系P38激酶未被激活;流式细胞术检测显示,MKK6b(E)转染可明显诱导Raw264.7细胞凋亡,并且MKK6b(E)基因转染促进热损伤诱导的Raw264.7细胞凋亡,而P38(AF)基因转染抑制热损伤诱导的Raw264.7细胞凋亡。结论MKK6-P38信号通路参与介导热损伤诱导的Raw264.7细胞凋亡。

Regulation of P38 and MKK6 kinase on heat-induced Raw264.7 cell apoptosis

Objective To elucidate the role of P38 signaling pathway on heat-induced apoptosis in monocytic cell line Raw264.7.Methods Raw cells were transfected with constitutively active mutant MKK6b(E)and dominant negative mutant P38(AF),or the empty cloning vector pcDNA3 and apoptosis was detected by flow cytometric analysis.Results The ectopic expression of P38 mutant was confirmed by immunostaining with the antibody against the Flag-epitope tag.Expression of MKK6b(E)led to a marked increase in P38 kinase activity in transfected cells and induced a 4-fold increase in the number of apoptotic cells as compared to that in cultures of control transfected cells.Meanwhile the expression of MKK6b(E)increased the apoptotic rate of Raw cells induced by heat.In contrast,the dominant-negative mutant P38(AF)inhibited Raw cells apoptosis induced by heat.Conclusion The activation of the MKK6-P38 MAP kinase signaling pathway is required for heat-induced apoptosis in Raw264.7 cells.