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目的构建野生型及变异型apoptin的真核表达质粒pApoptin-EGFP、pM-apoptin-EGFP,并探讨其对人前列腺癌细胞系LNCaP、PC3的促凋亡作用。方法采用PCR的方法从变异型apoptin质粒p3×flag-m-apoptin- myc扩增出野生型apoptin片段及变异型apoptin片段m-apoptin,将其定向克隆于pEGFP-N1载体的EcoR I和BamH I酶切位点之间,构建EGFP标记的野生及变异型apoptin真核表达质粒pApoptin-EGFP及pM-apoptin-EGFP,酶切、测序鉴定,RT-PCR、荧光显微镜分析融合蛋白表达,脂质体介导瞬时转染人前列腺癌细胞系LNCaP、PC3,流式细胞仪检测凋亡及细胞周期变化。结果成功构建pApoptin-EGFP、pM-apoptin-EGFP重组质粒,并在被转染细胞检测到基因表达;瞬时转染后48h可检测到细胞凋亡,与空质粒组相比具有非常显著意义,细胞周期变化表现为G2/M期阻滞。结论apoptin对雄激素依赖及非依赖型前列腺癌均具有明显的促凋亡作用,末端突变影响其促凋亡作用,为进一步探讨apoptin应用于前列腺癌的治疗奠定基础。
Objective To construct eukaryotic expression plasmids pApoptin-EGFP and pM-apoptin-EGFP of wild type and variant apoptin, and to explore their pro-apoptotic effects on human prostate cancer cell lines LNCaP and PC3. Methods The wild-type apoptin fragment and the modified apoptin fragment m-apoptin were amplified by PCR from the apoptin plasmid p3 × flag-m-apoptin-myc and cloned into pEGFP-N1 vector EcoR I and BamH I EGFP-tagged wild and mutant apoptin eukaryotic expression plasmids pApoptin-EGFP and pM-apoptin-EGFP were constructed and identified by restriction enzyme digestion, sequencing, RT-PCR and fluorescence microscopy. Mediated transient transfection of human prostate cancer cell lines LNCaP, PC3, flow cytometry apoptosis and cell cycle changes. Results The recombinant plasmids of pApoptin-EGFP and pM-apoptin-EGFP were successfully constructed and the gene expression was detected in the transfected cells. Apoptosis was detected 48h after transient transfection, which was significantly different from that of empty plasmid group. Periodic changes showed G2 / M arrest. Conclusions apoptin can induce apoptosis in both androgen-dependent and non-prostate cancer patients. The mutation of apoptin affects the pro-apoptotic effect of apoptin, which lays the foundation for the further study of the application of apoptin in the treatment of prostate cancer.