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研究抗酶(antizyme)1对人宫颈癌HeLa细胞增殖与细胞周期的影响,并分析抗酶1对细胞周期蛋白D1(cyclin D1)的表达影响.采用定点突变技术,将抗酶1的frameshift位点缺失,随后将突变基因重组至真核表达载体pEGFP-N1中,鉴定后转染HeLa细胞.通过MTT法检测细胞增殖变化,流式细胞术分析抗酶1对细胞周期的影响.RT-PCR和Western印迹检测抗酶1转染对细胞周期蛋白D1基因表达的影响.酶切结果显示,抗酶1突变基因成功克隆至pEGFP-N1中.成功转染HeLa细胞后,检测结果显示,抗酶1能够减慢HeLa细胞增殖速度,并使细胞停滞于G0/G1期,细胞周期蛋白D1基因的表达同时受到抑制.实验说明,抗酶1基因能够抑制HeLa细胞增殖,通过降低细胞周期蛋白D1的表达阻滞细胞周期.
To study the effect of antizyme 1 on the proliferation and cell cycle of human cervical cancer HeLa cells and to analyze the effect of anti-enzyme 1 on the expression of cyclin D1, a site-directed mutagenesis Point deletion, then the mutant gene was recombined into eukaryotic expression vector pEGFP-N1, identified and transfected into HeLa cells.Cell proliferation was detected by MTT assay, and the effect of anti-enzyme 1 on cell cycle was analyzed by flow cytometry.RT-PCR And western blotting were used to detect the effect of transfection of anti-enzyme 1 on the expression of cyclin D1 gene.Restriction enzyme digestion results showed that the mutant gene of anti-enzyme 1 was successfully cloned into pEGFP-N1.After transfected HeLa cells successfully, 1 can slow down the proliferation of HeLa cells and arrest the cells in G0 / G1 phase, while the expression of cyclin D1 gene is inhibited at the same time.Experiment shows that anti-enzyme 1 gene can inhibit the proliferation of HeLa cells by decreasing the expression of cyclin D1 Expression blocks cell cycle.