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

作　　者：王冰[1] 林山[1] 王烈[1] 

机构地区：[1]福州总医院南京军区普通外科研究所

出　　处：《福州总医院学报》2010年第1期13-15,共3页Journal of Fuzhou General Hospital

基　　金：福建省青年科技人才创新项目(2005J074)

摘　　要：目的:构建GPC3基因真核表达重组质粒,研究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,应用PCR法从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(600ug/ml)筛选出抗性克隆,应用荧光显微镜及RT-PCR法对转染细胞内pEGFP-GPC3的表达进行鉴定,采用MTT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,与空质粒转染组相比有显著性意义(P<0.05),IGF2实验组与空质粒转染组相比无显著性意义(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 an eukaryotic expression recombinant plasmid na - reed pEGFP - N2 - GPC3, and study the effects of GPC3 gene on the growth promotion of growth factors in GPC3 - SK - Hep - 1 Method ： Human GPC3 full - length cDNA was obtained from pDNR- LIB- GPC3 prokaryotic amplified recombinant plasmid by PCR. The target gene GPC3 was inserted into eukaryotic expression vector pEGFP - N2. The recombinant p - lasmid was transfected into the SK - Hep - 1 human hepatoma carcinoma ceils via Lipofectin transfection. Transfected SK- Hep - [ cells were selectively screened with G418 （600μg/rrd） . The expression of GPC3 gene in transfected SK - Hep - 1 was detected by fluorescence micr - oscopy and RT PCR. Effects of GPC3 gene on the growth promotlon of growth factors in GPC3 - SK - Hep- 1 were detected by MTT. Results： The recombinant plasmid was identified to be right for ORF by restriction endonuclease analysis and DNA sequencing. The green fluorescence could be seen in transfected SK - Hep - 1 cell membrane field under fluorescen - ce microscope. GPC3 gene in transfected ceils was identified by RT - PCR. FGF2 - induced cell proliferation was proved to be significantly decreased by GPC3 regulation, whereas t - he growth effects of IGF2 was not altered by GPC3. Conclusion： DNA sequencing show - ed the sequence of the synthetic GPC3 was correct and amino acid sequence of GPC3 w - as right. The eukaryotic expression vector pEGFP - N2 - GPC3 was constructed and it expres - sed in SK - Hep - 1 cells successfully. Forced expression of GPC3 may modulate FGF2 sig -naling.

关 键 词：GPC3基因 真核表达载体 SK-Hep-1 绿色荧光蛋白 生长因子 

分 类 号：R382.5[医药卫生—医学寄生虫学]