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目的:探讨蛋白酪氨酸磷酸酶SHP-2在神经生长因子(NGF)作用下大鼠嗜铬细胞瘤PC12细胞生存及NGF撤除后细胞凋亡的过程中的作用及机制。方法:SHP-2抑制剂NSC87877作用于PC12细胞,MTT测定PC12细胞存活率;流式细胞仪检测细胞凋亡率。将pIRES-GFP空载体、pIRES-GFP-SHP-2野生型和pIRES-GFP-SHP-2C459S突变体通过脂质体方法转染PC12细胞;加入NGF作用1h和撤除NGF5h后分别用Westernblotting方法检测细胞外信号调节蛋白激酶(ERK)、磷酸化ERK(p-ERK)、c-Jun氨基末端激酶(JNK)及磷酸化JNK(p-JNK)表达变化。结果:MTT和流式细胞仪检测表明SHP-2可以促进PC12细胞生存,抑制细胞凋亡。Western blotting结果显示无SHP-2抑制剂组和转染pIRES-SHP-2野生型组p-ERK表达在加入NGF的过程中升高;撤除NGF后,各组p-ERK表达均降低,pIRES-GFP-SHP-2C459S突变体组和pIRES-GFP组p-ERK表达明显低于pIRES-GFP-SHP-2野生型组,NGF去除+SHP-2抑制剂组的表达水平明显低于NGF去除对照组;NGF去除+SHP-2抑制剂组p-JNK表达高于NGF去除对照组;pIRES-GFP-SHP-2C459S突变体组高于pIRES-GFP空载体组,pIRES-GFP-SHP-2野生型组低于pIRES-GFP空载体组。结论:SHP-2可能通过对ERK的正向激活,抑制JNK的激活,增强NGF作用下PC12细胞生存及抑制NGF撤除后所致细胞凋亡,从而在NGF的信号转导中起到一个中心环节的作用。
AIM: To investigate the role and mechanism of protein tyrosine phosphatase SHP-2 in the survival of PC12 cells and apoptosis after NGF withdrawal in rat pheochromocytoma cells treated with nerve growth factor (NGF). Methods: The SHP-2 inhibitor NSC87877 was applied to PC12 cells. The survival rate of PC12 cells was determined by MTT assay. The apoptosis rate was detected by flow cytometry. The pIRES-GFP empty vector, pIRES-GFP-SHP-2 wild-type and pIRES-GFP-SHP-2C459S mutant were transfected into PC12 cells by lipofectamine. After adding NGF for 1h and removing NGF for 5h, the cells were detected by Western blotting The expression of ERK, p-ERK, c-Jun N-terminal kinase (JNK) and phosphorylated JNK (p-JNK) Results: MTT and flow cytometry showed that SHP-2 could promote the survival of PC12 cells and inhibit apoptosis. The results of Western blotting showed that the expression of p-ERK in the non-SHP-2 inhibitor group and the wild-type pIRES-SHP-2 transfected group increased during the addition of NGF. The expression of p-ERK in each group decreased after the removal of NGF, The expression of p-ERK in GFP-SHP-2C459S mutant group and pIRES-GFP group was significantly lower than that in pIRES-GFP-SHP-2 wild-type group, and NGF + SHP-2 inhibitor group was significantly lower than that in NGF- GFP-SHP-2C459S mutant group was higher than that of pIRES-GFP empty vector group, pIRES-GFP-SHP-2 wild-type group Lower than the pIRES-GFP empty vector group. Conclusion: SHP-2 may play a central role in the signal transduction of NGF through the positive activation of ERK, inhibiting the activation of JNK, enhancing the survival of PC12 cells under NGF treatment and inhibiting the apoptosis induced by the removal of NGF Role.