pEGFP-mINF-γ转染人卵巢癌细胞SKOV3及其对细胞生长的抑制作用

Suppression of the reconstruct plasmid of pEGFP-mINF-γTransfected into skov3 cells

目的构建绿色荧光蛋白联合γ干扰素(pEGFP-mINF-γ)真核表达质粒,探讨γ-干扰素对卵巢癌细胞系skov3细胞生长的影响。方法将重组质粒转染入卵巢癌细胞株skov3中,获得稳定表达INF-γ细胞株,设空质粒转染组(skov3-pEGFP组)和空白对照组(skov3组);逆转录聚合酶连反应(RT-PCR)法检测目的基因的表达,双抗体夹心酶联免疫吸附实验(ELISA)方法测定INF-γ的分泌量;四甲基偶氮唑蓝(MTT)比色法和荧光显微镜下细胞染色法检测INF-γ对skov3细胞体外生长和凋亡等生物学行为的影响。结果RT-PCR和ELISA法证实重组质粒已整合到skov3细胞并获稳定表达;MTT法检测细胞的增值速度及细胞抑制率,pEGFP-mINF-γ组第5天的细胞抑制率为21.67%,明显高于skov3-pEGFP和skov3组,P<0.005;Hoechst染色检测结果显示skov3-mINF-γ组细胞凋亡,荧光显微镜下skov3-mINF-γ组凋亡细胞明显多于skov3-pEGFP组和skov3-nul组,差异有统计学意义(P<0.05),skov3-pEGFP组和skov3组间差异无统计学意义(P>0.05)。结论成功构建真核表达质粒pEGFP-mINF-γ,该质粒转染人卵巢癌细胞sk-ov3可抑制其生长,并不影响INF-γ的分泌量,异种INF-γ基因可能对人上皮性卵巢癌起重要作用。

Abjective：To establish a ovarian carcinoma cell line expressing mouseinterferon-gamma,and to analyze the role of mouse interferon-gamma on the proliferation of human ovarion carcinoma cell line（skov3）.Methods：The full-length gene of mouse intergeron-gamma（IFN-γ）was introduced into the human ovarian carcinoma cell line through retroviral vecter.The transfected cells were screened with G418,and pEGFP-mINF-γ mRNA were tested by RT-PCR and tested for expression of IFN-γ by enzyme-lined immunoadsorbent assay（ELISA）.At the same time,pEGFP-mINF-γ were transfected into skov3 as negative control.The proliferation of skov3 was tested by MTT assay,and measured the apoptotic rate by Hoechst33258.Results：The stable expression vector,pEGFP-mIFNγ,was successfully constructed and transfected into skov3 cell.It is confirm that the reconstruction plasmid of pEGFP-mIFNγ has been stably transfected into the skov3 cells by RT-PCR.And the expression properties of pEGFP-mIFNγ were investigated by ELISA that medium of the pEGFP-mIFNγ groups have been tested for high density.The result of MTT and Hoechst33258 prompt that the negative control rate and apoptotic rate was significantly higher in transfected cell group with pEGFP-mIFNγ in comparison with the skov3 group and the transfected cell with pEGFP group.The statistical significance（P0.05）.Conclusion：The stable expression plasmid of pEGFP-mIFNγ has been successfully constructed,and when the rebuild plasmid has been transfected into skov3 cells,mIFNγ expression can restrain the development of the cells.So it can summarize that different kind gene of mIFNγ may control the development and origin of the proliferation of human ovarion carcinoma cell line （skov3）.