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目的:构建白细胞介素8(interleukin 8,IL-8)基因真核表达载体,瞬时转染OVCAR-3卵巢癌细胞,为进一步探讨其与肿瘤发生发展关系奠定基础。方法:采用RT-PCR法自健康志愿者外周血单个核细胞扩增IL-8基因,构建pcDNA3.1(+)/IL-8重组质粒;脂质体介导将外源基因转入OVCAR-3卵巢癌细胞,半定量RT-PCR、Western印迹及ELISA法鉴定其mRNA及蛋白瞬时表达后,MTT法检测细胞转染前后生长增殖特性的改变,FCM检测细胞周期的变化。结果:重组质粒双酶切电泳结果显示,相应位置(300 bp,5 400 bp)可见2条清晰特异的DNA条带;DNA测序结果进一步显示,重组质粒中插入的基因片段全长300 bp,编码序列与GenBank报道的基因序列相符,阅读框保持不变;RT-PCR、Western印迹检测及ELISA检测结果显示,转染后OVCAR-3细胞IL-8 mRNA及蛋白表达水平均明显升高(P<0.05);MTT检测结果显示,IL-8转染细胞组转染3 d后的光密度值(D值)显著高于未转染细胞组(P<0.05);FCM分析结果显示,OVCAR-3细胞转染IL-8基因后进入S期的细胞比例显著增高,增殖指数也相应上升,与未转染及空质粒转染组细胞相比,差异显著(P<0.05)。结论:IL-8基因真核表达载体成功构建并瞬时转染OVCAR-3卵巢癌细胞;IL-8可以自分泌方式促进卵巢癌OVCAR-3细胞生长增殖。
OBJECTIVE: To construct an eukaryotic expression vector of interleukin 8 (IL-8) gene and transiently transfected ovarian cancer cell line OVCAR-3 in order to further explore its relationship with tumorigenesis. Methods: IL-8 gene was amplified from peripheral blood mononuclear cells of healthy volunteers by RT-PCR to construct recombinant plasmid pcDNA3.1 (+) / IL-8. Liposome mediated transfer of exogenous gene into OVCAR- 3 ovarian cancer cells. Semiquantitative RT-PCR, Western blotting and ELISA were used to detect the mRNA and protein expression. MTT assay was used to detect the proliferation and proliferation of cells before and after transfection. FCM was used to detect the cell cycle. Results: The results of double-enzyme digestion showed that there were two distinct DNA bands in the corresponding positions (300 bp, 5 400 bp). The DNA sequencing results further showed that the inserted gene fragment in the recombinant plasmid was 300 bp in length, encoding The results of RT-PCR, Western blot and ELISA showed that the expression of IL-8 mRNA and protein in OVCAR-3 cells were significantly increased after transfection (P < 0.05). The results of MTT assay showed that the optical density (D) value of IL-8 transfected cells was significantly higher than that of untransfected cells 3 days after transfection (P <0.05). FCM analysis showed that OVCAR-3 The percentage of cells entering the S phase after transfection of IL-8 gene was significantly increased, and the proliferation index increased correspondingly. Compared with the cells transfected with the untransfected and empty plasmids, the difference was significant (P <0.05). CONCLUSION: The eukaryotic expression vector of IL-8 gene was successfully constructed and transiently transfected into OVCAR-3 ovarian cancer cells. IL-8 can promote the growth and proliferation of ovarian cancer OVCAR-3 cells in an autocrine manner.