GPC3增强型绿色荧光蛋白真核表达载体的构建及其对生长因子促细胞增殖效应的影响

Construction of GPC3 green fluorescent protein eukaryotic expression vector and its influence on growth promoting effect of growth factors in SK-Hep-1 cells

目的:通过构建GPC3增强型绿色荧光蛋白真核表达载体,研究磷脂酰肌醇蛋白聚糖-3(Glypican-3,GPC3)对成纤维细胞生长因子(fibroblast growth factor-2,FGF2)、胰岛素样生长因子(insulin-like growth factor-2,IGF2)、转化生长因子-β1(transforming growth factor-β1,TGF-β1)、骨形态发生蛋白-4(bone morphogenetic protein-4,BMP4)促细胞增殖效应的影响。方法:应用基因重组技术及限制性内切酶酶切构建并鉴定pEGFP-N2-GPC3增强型绿色荧光蛋白真核表达载体,经脂质体LipofectamineTM2000介导转染SK-Hep-1后,通过G418(600μg/ml)筛选出抗性克隆,应用逆转录-聚合酶链反应(RT-PCR)检测GPC3mRNA在真核细胞中的表达,Western印迹法检测GPC3蛋白在真核细胞中的表达,并在荧光显微镜下观察目的蛋白在真核细胞内的表达情况,采用MTT法研究GPC3对生长因子FGF2、IGF2、TGF-β1、BMP4促细胞增殖效应的影响。结果:限制性内切酶酶切分析、重组质粒测序鉴定表明为正确重组子,荧光显微镜下可见转染的真核细胞胞膜区发出强绿色荧光,RT-PCR及Western印迹法表明GPC3在真核细胞中成功表达,GPC3抑制了FGF2对SK-Hep-1细胞的增殖效应,而不抑制IGF2、TGF-β1、BMP4的促增殖作用。结论:编码的氨基酸序列与人GPC3完全一致;构建完成真核表达重组质粒pEGFP-N2-GPC3;GPC3基因在SK-Hep-1中成功表达;GPC3在FGF2信号通路中可能发挥负性调控因子的作用。

Objective：To construct a GPC3 green fluorescent protein eukaryotic expression vector pEGFP-N2-G-PC3,and analyze its effects on the growth promoting effect of growth factors （fibroblast growth factor-2, FGF2; insulin-like growth factor-2 , IGF2; transforming growth factor-β1, TGF-β1 ; and bone morphogenetic protein-4, BMP4） in human hepatoma cell line GPC3-SK-Hep-1. Methods： A eukaryotic expression vector for GPC3 genes （pEGFP-N2-GPC3） was constructed by recombinant DNA technique and was transfeeted into SK-Hep-1 cells by LipofectamineTM 2000; the cells stably expressing GPC3 were screened out by G418 （600 μg/ml）. The mRNA expression of GPC3 was detected by RT-PCR method and the protein expression of GPC3 by Western blotting and fluorescence microscope. Effects of GPC3 gene on the growth promoting effects of the above growth factors were examined by MTT. Results： The recombinant plasmid was verified to be correctly constructed by restriction endonuclease analysis and DNA sequencing. The green fluorescence was detected in the transfected SK-Hep-1 cells under fluorescence microscope. RT-PCR and Western blotting both confirmed that GPC3 was successfully expressed in SK-Hep- 1 cells. FGF2-induced cell proliferation was significantly decreased by GPC3 gene, whereas the growth promoting effects of IGF2,TGF-β1 and BMP4 were not altered by GPC3 gene. Conclusion： We have successfully obtained the synthetic GPC3 protein,which has the same amino acid sequence as that of human GPC3 protein. The eukaryotic expression vector pEGFP-N2-GPC3 has been correctly constructed and GPC3 protein has been successfully expressed in SK-Hep-1 cells. GPC3 may negatively modulate FGF2 signaling pathway.