论文部分内容阅读
血清和糖皮质激素调节蛋白激酶(SGK)家族参与调节生长因子和激素的信号转导.为了研究SGK家族成员SGK2α在细胞中的功能,构建了真核表达质粒pEGFP-N1-SGK2α并瞬时转染HEK293细胞,通过激光共聚焦显微镜观察发现融合蛋白SGK2α-GFP主要定位于细胞浆,免疫共沉淀实验发现SGK2α与糖原合成激酶3β(GSK3β)存在相互作用.利用PCDNA6-V5-HisB-SGK2α质粒转染肝癌BEL7402细胞,建立稳定表达SGK2α蛋白的细胞系,通过细胞增殖实验发现,SGK2α的过表达使BEL7402细胞生长速度减慢、细胞倍增时间延长.裸鼠成瘤实验发现,与对照组细胞相比表达SGK2α的BEL7402细胞在裸鼠中的成瘤能力明显降低.免疫印迹实验证实,SGK2α的过表达不影响GSK3β的表达,但却使β-catenin和Cyclin D1的表达下调.提示影响Wnt/β-catenin信号通路关键分子的表达可能是外源性SGK2α蛋白过表达抑制BEL7402细胞增殖的分子机制.
Serum and glucocorticoid-regulated protein kinase (SGK) families are involved in the regulation of growth factor and hormone signaling.In order to study the function of SGK2a in cells, a eukaryotic expression plasmid pEGFP-N1-SGK2α was constructed and transiently transfected HEK293 cells by confocal laser scanning microscopy found that the fusion protein SGK2α-GFP mainly located in the cytoplasm, co-immunoprecipitation found that SGK2α and glycogen synthesis kinase 3β (GSK3β) interaction exists using PCDNA6-V5-HisB-SGK2α plasmid The hepatoma BEL7402 cells were stained to establish a cell line stably expressing SGK2α protein.The results of cell proliferation showed that the over-expression of SGK2α slowed down the growth of BEL7402 cells and prolonged the cell doubling time.Compared with the control group, The tumorigenic ability of BEL7402 cells expressing SGK2α was significantly reduced in nude mice.The results of Western blotting showed that the over-expression of SGK2α did not affect the expression of GSK3β, but down-regulated the expression of β-catenin and Cyclin D1, The expression of key molecules of catenin signaling pathway may be the molecular mechanism of exogenous SGK2α protein overexpression inhibiting the proliferation of BEL7402 cells.