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目的探讨JWA蛋白PKC磷酸化位点对TPA诱导的MCF-7细胞分化的影响。方法利用连续PCR引入点突变技术构建JWA编码区PKC磷酸化位点1、2以及两位点同时突变的三种pEGFP-N1载体,脂质体稳定转染获得相应的三种MCF-7细胞株,油红O染色观察细胞分化状态。结果20μmol/L TPA处理MCF-7-N1(稳定转染pEGFP-N1载体的MCF-7细胞)、MCF-7-JWA(稳定转染pEGFP-N1-JWA载体的MCF-7细胞)、MCF-7-JWA-1、MCF-7-JWA-2、MCF-7-JWA-1+2细胞2d后,油红O染色阳性细胞百分比分别为48%、67%、69%、67%、70%。与MCF-7-N1相比,TPA诱导MCF-7- JWA、MCF-7-JWA-1、MCF-7-JWA-2、MCF-7-JWA-1+2细胞分化率(油红O染色阳性细胞百分比)显著升高(P<0.05),但MCF-7-JWA、MCF-7-JWA-1、MCF-7-JWA-2、MCF-7-JWA-1+2细胞分化率,差异无统计学意义(P>0.05)。结论稳定转染JWA能增强TPA诱导的MCF-7细胞分化;JWA编码区PKC磷酸化位点突变并不影响TPA诱导MCF-7细胞分化,可能存在其他的分子机制。
Objective To investigate the effect of PKA phosphorylation site of JWA protein on the differentiation of MCF-7 cells induced by TPA. METHODS: Three pEGFP-N1 vectors with PKC phosphorylation sites 1, 2 and two sites in JWA coding region were constructed by using continuous PCR technique. Stable transfection of liposomes resulted in three MCF-7 cell lines , Oil red O staining observed cell differentiation status. Results MCF-7-N1 (MCF-7 cells stably transfected with pEGFP-N1 vector), MCF-7-JWA (MCF-7 cells stably transfected with pEGFP-N1-JWA vector), MCF- The percentages of oily red O positive cells were 48%, 67%, 69%, 67% and 70% after 2 days of 7-JWA-1, MCF-7-JWA-2 and MCF-7-JWA- . Compared with MCF-7-N1, the differentiation rate of MCF-7-JWA, MCF-7-JWA-1, MCF-7-JWA-2 and MCF-7-JWA- The positive rate of MCF-7-JWA-1, MCF-7-JWA-2 and MCF-7-JWA-1 + 2 were significantly increased No statistical significance (P> 0.05). Conclusion Stable transfection of JWA can enhance the differentiation of MCF-7 cells induced by TPA. The mutation of PKC phosphorylation site in JWA coding region does not affect the differentiation of MCF-7 cells induced by TPA. Other molecular mechanisms may exist.