Rap2b基因真核表达载体构建及其对NIH3T3细胞P38通路的影响

Construction of Eukaryotic Expression Vector of Rap2b and Its Effects on Pathway of NIH3T3 Cells

目的构建pcDNA3.1-Rap2b真核表达载体,以外源基因Rap2b转染NIH3T3细胞,以了解该基因对NIH3T3细胞AKT、ERK、JNK和P38信号转导通路的影响,为探讨该基因在人肺癌发生中的作用提供实验依据。方法构建人Rap2b真核表达载体pcDNA3.1-Rap2b并稳定转染NIH3T3细胞,利用Western blot方法对转染的细胞进行AKT、ERK、JNK和P38磷酸化蛋白及总蛋白表达水平检测。结果转染pcDNA3.1-Rap2b质粒的细胞与转染空质粒pcDNA3.1的细胞相比,其AKT、ERK、JNK磷酸化蛋白表达水平差异无统计学意义(P>0.05),而P38磷酸化蛋白表达水平明显上调(P<0.05)。结论 Rap2b基因外源性高表达可能对P38通路有活化作用,对JNK、ERK、AKT通路无活化作用。

Objective To construct pcDNA3.1-Rap2b eukaryotic expression vector and to understand the effects on AKT, ERK, JNK and P38 signaling pathway of NIH3T3 cells through transfecting Rap2b gene into NIH3T3 cells.Methods Eukaryotic expression vector pcDNA3.1-Rap2b was constructed and was stable transfected into NIH3T3 cell, and its effects on the AKT、ERK、JNK and P38 pathway of NIH3T3 cells were observed by Western blotting to measure the level of the expression of AKT、ERK、JNK and P38 Phosphorylation protein and total protein.Results The level of the expression of P38 Phosphorylation protein was higher in the pcDNA3.1-Rap2b vector than that of control（P0.05）, and the level of the expression of AKT、ERK、JNK Phosphorylation protein was not different comparing with the control（P0.05）.Conclusion Rap2b might activate P38 signaling pathway, but not activate AKT, ERK, JNK signaling pathway.