抗酶1基因转染对HeLa细胞增殖及细胞周期的抑制作用

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.

Inhibitory Effect of Antizyme 1 Transfection on Cell Proliferation and Cell Cycle in HeLa Cells

To investigate the effect of antizyme 1 on the cell proliferation and cell cyclein connection with cyclin D1 expression in HeLa cells, a frame-shift mutant antizyme 1 was created by site-directed mutagenesis, then cloned into a pEGFP-N1 vector. After transfection into HeLa cells,the cell proliferation was determined by MTT assay and the cell cycle was characterized by flow cytometry. The level of cyclin D1 in response to antizyme 1 was determined by RT-PCR and Western blotting. The results showed that the mutant antizyme 1 inhibited cell proliferation and the cell cycle was arrested at G_０/G₁ phase. Meanwhile, the expression of cyclin D1 was down-regulated. The data suggested that the reduced expression of cyclin D1 might be important for the anti-proliferation effect of antizymelin HeLa cells.