GPC3对肝癌细胞株MHCC97-L增殖的影响

Effect of glypican-3 on the proliferation of human hepatoma cell line MHCC97-L in vitro

目的通过构建磷脂酰肌醇蛋白聚糖3(GPC3)增强型绿色荧光蛋白真核表达载体,研究GPC3对人肝癌细胞株MHCC97-L增殖的影响。方法用基因重组及限制性内切酶技术构建并鉴定表达载体pEGFP-c3-GPC3,通过脂质体介导转染人肝癌细胞株MHCC-97L,流式细胞术和G418筛选建立稳定细胞株;采用荧光定量PCR、Western blotting分别检测MHCC-97L中GPC3 mRNA和蛋白的表达情况,并在荧光显微镜下观察细胞荧光;MTT法检测GPC3对肝癌细胞MHCC97-L体外增殖能力的影响。结果经酶切及测序鉴定,确认成功构建真核表达载体pEGFP-c3-GPC3,荧光显微镜下观察到绿色荧光表达;从mRNA和蛋白水平证实,GPC3基因在转染后的MHCC-97L中成功表达;MTT生长曲线证实MHCC97-L/GPC3较其他两株细胞增殖显著加强(P<0.001)。结论成功构建pEGFR-c3-GPC3真核表达载体;筛选出稳定表达GPC3基因的MHCC97-L/GPC3细胞株;GPC3基因表达升高可促进肝癌细胞株MHCC97-L的增殖。

Objective To construct glypican-3（GPC3）-green fluorescent protein eukaryotic expression vector pEGFP-c3-GPC3,and analyze the effect of GPC3 on the proliferation of human hepatoma cell line MHCC-97L.Methods The eukaryotic expression vector pEGFP-c3-GPC3 was constructed with recombinant DNA technique and transfected into MHCC-97L cells via Lipofectamine 2000.The cells stably expressing GPC3 were screened by flow cytometry and G418.The mRNA expression of GPC3 was detected by RT-QPCR method,and the protein expression by Western blotting and fluorescence microscope.The effect of GPC3 gene on the growth of the cells was examined by MTT assay.Results Restriction endonuclease analysis and DNA sequencing verified correct construction of the recombinant plasmid.The green fluorescence was detected in the transfected MHCC-97L cells under fluorescence microscope.RT-QPCR and Western blotting both confirmed successful expression of GPC3 in MHCC-97L cells.The growth curve showed a significant acceleration of the proliferation of the transfected MHCC97-L/GPC3 cells as compared with MHCC97-L and MHCC97-L/C3 cells（P0.001）.Conclusion We have successfully constructed the eukaryotic expression vector pEGFR-c3-GPC3,which allows stable GPC3 expression in MHCC97-L/GPC3 cells.The upregulation of GPC3 expression can stimulate the growth of hepatoma cell line MHCC97-L in vitro.