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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。
Objective To construct the eukaryotic expression plasmid of green fluorescent protein (GFP) combined with gamma interferon (pEGFP-mINF-γ) and investigate the effects of IFN-γ on the growth of human ovarian cancer cell line SKOV3. Methods The recombinant plasmid was transfected into SKOV3 ovarian cancer cell line SKOV3 to obtain a stable expression of INF-γ cell line. The empty vector transfected group (skov3-pEGFP group) and blank control group (skov3 group) (RT-PCR) was used to detect the expression of the target gene. ELISA was used to determine the INF-γ secretion; MTT colorimetric assay and fluorescent microscopy The effect of INF-γ on the biological behavior of skov3 cells in vitro such as growth and apoptosis was examined by staining. Results RT-PCR and ELISA confirmed that the recombinant plasmid had been integrated into skov3 cells and stably expressed. The cell proliferation rate and cell inhibitory rate were detected by MTT assay. The cell inhibitory rate was 21.67% on day 5 in pEGFP-mINF-γ group (P <0.05). The results of Hoechst staining showed that apoptosis in skov3-mINF-γ group was more than that in skov3-pEGFP group and skov3-pEGFP group nul group, the difference was statistically significant (P <0.05), there was no significant difference between skov3-pEGFP group and skov3 group (P> 0.05). CONCLUSION: The eukaryotic expression plasmid pEGFP-mINF-γ was successfully constructed. The plasmid transfected into human ovarian cancer cell line SK-ov3 inhibited the growth and did not affect the secretion of INF-γ. The heterologous INF-γ gene may inhibit the growth of human epithelial ovarian Cancer plays an important role.