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目的探讨蛋白酪氨酸磷酸酶SHP-2在三氧化二砷(As2O3)诱导HEK293T细胞凋亡中的作用。方法将pIRES-GFP空载体、pIRES-GFP-SHP-2野生型和pIRES-GFP-SHP-2C459S突变体载体通过磷酸钙方法转染HEK293T细胞;在5μmol.L-1的As2O3孵育48 h后,用流式细胞术检测细胞凋亡率;Western blot方法检测Cyt-C、Caspase-3、p-ERK、ERK、p-JNK及JNK的表达变化。结果5μmol.L-1As2O3可诱导HEK293T细胞凋亡,SHP-2能抑制其作用;SHP-2C459S突变体组Cyt-C和Caspase-3表达均高于空载体组,SHP-2野生型组则低于空载体组;SHP-2野生型组p-ERK表达高于空载体组,SHP-2C459S突变体组则相反;p-JNK的表达,SHP-2C459S突变体组高于空载体组,而SHP-2野生型组低于空载体组。结论SHP-2可抑制As2O3诱导的HEK293T细胞凋亡,其机制可能与激活ERK和抑制JNK途径有关。
Objective To investigate the role of protein tyrosine phosphatase SHP-2 in the apoptosis of HEK293T cells induced by As 2 O 3. Methods HEK293T cells were transfected with empty vector pIRES-GFP, wild-type pIRES-GFP-SHP-2 and pIRES-GFP-SHP-2C459S mutants by calcium phosphate method. After incubation with 5μmol·L-1 As2O3 for 48 h, The apoptosis rate was detected by flow cytometry. The expressions of Cyt-C, Caspase-3, p-ERK, ERK, p-JNK and JNK were detected by Western blot. Results The apoptosis of HEK293T cells was induced by 5μmol·L-1As2O3, and SHP-2 inhibited the effect. The expression of Cyt-C and Caspase-3 in SHP-2C459S mutant group was higher than that in empty vector group and SHP-2 wild-type group The expression of p-ERK in SHP-2 wild-type group was higher than that in empty vector group, and SHP-2C459S mutant group was the opposite. The expression of p-JNK was higher in SHP-2C459S mutant group than in empty vector group, -2 wild-type group was lower than the empty vector group. Conclusion SHP-2 inhibits As2O3-induced apoptosis in HEK293T cells, which may be related to the activation of ERK and the inhibition of JNK pathway.