外源性细胞周期蛋白激酶抑制因子p21基因对人晶状体上皮细胞周期的影响

目的 研究细胞周期蛋白激酶抑制因子p21基因转染对人晶状体上皮细胞(1ens epithelial cells,LEC)周期调控的影响,在基因水平探讨防治后发性白内障的可能性。方法 构建含人p21基因真核表达的载体质粒pcDNA3／p21,采用基因转染技术将质粒DNA转染至永生性人LEC系HLE-B3,使用流式细胞仪观察细胞生长和周期的变化,并采用逆转录聚合酶链式反应方法检测p21mRNA的表达;分别采用免疫组化和免疫印迹(western blotting,WB)方法检测p21蛋白的表达。结果 体外构建的载体质粒pcDNA3／p21经酶切鉴定含有人p21基因全长cDNA片段。基因转染后细胞HLE—B3经传代培养,48h后出现缓慢生长且部分细胞漂浮死亡的现象,G1期细胞明显增多。与对照细胞比较,转染后细胞的p21mRNA表达明显增多;p21蛋白表达明显增强,在经筛选的阳性克隆细胞中WB方法可检测到相对分子质量为21000的阳性蛋白带。结论 外源性p21基因可在人LEC系HLE．B3中过度表达,并对细胞周期调控产生抑制作用。基因转染抑制LEC增殖可能成为防治后发性白内障的新途径。

Effects of exogenous p21 gene on cells cycle of human lens epithelial cells

OBJECTIVE: To investigate the effects of transfected exogenous p21 gene on the cells cycle of HLE-B3 cells line. The feasibility of prevention of secondary cataract by gene therapy was evaluated. METHODS: Total length of human p21 gene cDNA was cloned on the parent's plasmid pcDNA3 to construct the recombinant plasmids of pcDNA3/p21, a large amount of pcDNA3/p21 plasmid DNA was prepared by QIAGEN endofree maxi kit. After harvest of the plasmid DNA, the HLE-B3 cells line was transfected. The cell growth was observed and the cells cycle was analyzed by flow cytometry. The expression of p21 mRNA was detected by RT-PCR and the expression of p21 protein was detected by immunohistochemistry and western blot analysis. RESULTS: Forty eight hours after transfection, the growth of transfected cells became slower, some cells floated and died; the control cells and blank plasmid (blank pcDNA3) transfected cells grew normally. Flow cytometry analysis revealed that the number of cells in G(1) phase increased markedly in transfected cells. The RT-PCR showed that the product of p21 in the transfected cells (dead or alive cells) was obviously higher than that of the controls. Immunohistochemical studies showed few positive cells in the controls, and very high positive signal was detected after transfection. Western blot showed a positive band at the level of 21 000 in transfected cells, no positive band could be found in the controls. CONCLUSION: Exogenous p21 gene can transfect the HLE-B3 cells and can be expressed. The transfection can affect the cells cycle by G(1) arrest and induces cells death through the apoptosis process. This result suggests that it is possible to prevent the occurrence of secondary cataract by gene therapy.