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目的:应用构建完成的GPC3增强型绿色荧光蛋白真核表达载体,研究GPC3对SK-Hep-1肝癌细胞增殖的影响及生长因子IGF2、BMP4与GPC3抑制肝癌细胞增殖之间的关系。方法: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肝癌细胞后,通过G418(600ug/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-blot检测GPC3蛋白在真核细胞中的表达,确定GPC3已经成功转入细胞后,采用MTT法研究GPC3对SK-Hep-1肝癌细胞增殖的影响及生长因子IGF2、BMP4与GPC3抑制肝癌细胞增殖之间的关系。结果: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-blot表明GPC3在真核细胞中成功表达,基因转染组与空白对照组、空质粒转染组相比增殖明显减慢,有显著性意义(P<0.001),GPC3对生长因子IGF2、BMP4促肝癌细胞增殖无影响(P>0.05)。结论:成功筛选稳定表达GPC3蛋白的SK-Hep-1肝癌细胞系;GPC3抑制肝癌细胞增殖;GPC3与生长因子IGF2、BMP4信号通路可能无联系。
OBJECTIVE: To study the effect of GPC3 on the proliferation of SK-Hep-1 hepatocellular carcinoma cells and the relationship between the growth factors IGF2, BMP4 and GPC3 on the proliferation of hepatocellular carcinoma cells by using the constructed GPC3 enhanced green fluorescent protein eukaryotic expression vector. Methods: The eukaryotic expression vector pEGFP-N2-GPC3 enhanced green fluorescent protein was transfected into SK-Hep-1 hepatoma cells by lipofectamineTM2000. The resistant clones were screened by G418 (600ug / ml) The expression of GPC3 mRNA in eukaryotic cells was detected by polymerase chain reaction (RT-PCR), and the expression of GPC3 protein in eukaryotic cells was detected by Western-blot. After GPC3 was successfully transfected into cells, MTT was used to study the effect of GPC3 on SK Hep-1 hepatoma cells proliferation and the relationship between the growth factors IGF2, BMP4 and GPC3 on the proliferation of hepatocellular carcinoma cells. Results: The transfected eukaryotic cell membrane area under the fluorescence microscope showed a strong green fluorescence. RT-PCR and Western-blot showed that GPC3 was successfully expressed in eukaryotic cells. The transfection group and blank control group, empty plasmid transfected (P <0.001). GPC3 had no effect on the proliferation of hepatocellular carcinoma cells (IGF2 and BMP4) (P> 0.05). CONCLUSIONS: SK-Hep-1 hepatoma cell line stably expressing GPC3 protein was successfully screened; GPC3 inhibited the proliferation of hepatocellular carcinoma cell line; GPC3 might not be related to the IGF2 and BMP4 signal pathways.