重组质粒pEGFP-N2-GPC3的构建及GPC3对生长因子FGF2、IGF2促进细胞增殖的影响

Construction of recombinant plasmid pEGFP-N2-GPC3 and effects of GPC3 gene on the growth promotion of growth factors in GPC3-SK-Hep-1

目的构建GPC3基因真核表达重组质粒，观察GPC3对生长因子FGF2、IGF2促进肝癌细胞增殖的影响。方法自行设计引物从GPC3原核扩增重组质粒pDNR．LIB．GPC3中获得编码GPC3的基因片段。应用基因重组技术，将GPC3基因片段克隆到真核表达载体pEGFP-N2中，然后经脂质体介导转染SK-Hep-1，并通过G418（600mg／L）筛选出抗性克隆，应用荧光显微镜及逆转录．聚合酶链反应（RT-PCR）法对转染细胞内pEGFP-GPC3的表达进行鉴定，采用噻唑蓝（MTT）比色法研究GPC3对生长因子FGF2、IGF2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SK-Hep-1胞膜区发出强绿色荧光，RT-PCR表明GPC3在SK-Hep-1中成功表达，生长因子实验中，GPC3明显抑制FGF2促细胞增殖效应，与空质粒转染组比较差异有统计学意义（P〈0．05），IGF2实验组与空质粒转染组比较差异无统计学意义（P〉0．05）。结论测序表明，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 eukaryotic expression recombinant plasmid named pEGFP- N2-GPC3, and study the effects of GPC3 gene on the growth promotion of growth factors in GPC3-SK-Hep- 1. Methods Human GPC3 full-length cDNA was obtained from pDNR-LIB-GPC3 prokaryotic amplified recombinant plasmid by PCR. The target gene GPC3 was inserted into the eukaryotic expression vector pEGFP-N2. The recombinant plasmid was transfected into the SK-Hep-1 human hepatoma carcinoma cells via Lipofectin transfection. Transfected SK-Hep-1 cells were selectively screened with G418 （600 mg/L）. The expression of GPC3 gene in transfected SK-Hep-1 was detected by fluorescence microscopy and RTPCR. Effects of GPC3 gene on the growth promotion of growth factors in GPC3-SK-Hep-1 were assayed by MTT. Results The recombinant plasmid was identified to be right for ORF by restriction endonuclease a- nalysis and DNA sequencing. The green fluorescence could be seen in transfected SK-Hep-1 cell membrane field under fluorescence microscope. GPC3 gene in transfected ceils was detected by RT-PCR. FGF2- induced cell proliferation was proved to be significantly decreased by GPC3 regulation, whereas the growth effects of IGF2 was not altered by GPC3. Conclusion DNA sequencing showed the sequence of the synthetic GPC3 was correct and aminoacid sequence of GPC3 was right. The eukaryotic expression vector pEGFP-N2-GPC3 was constructed and expressed in SK-Hep-I cells successfully. Forced expression of GPC3 may modulate FGF2 signaling.