miRNA-20a作为CHO-K1细胞抗凋亡调控靶标的研究  

Study on miRNA-20a as targets for anti-apoptosis in CHO-K1 cells

作　　者：张兵兵[1,2] 侯汝涛[1,2] 李磊[1,2] 张莹[1,2] 潘长江[3] 

机构地区：[1]重庆大学生物工程学院,重庆400030 [2]重庆大学生物流变科学与技术教育部重点实验室,重庆400030 [3]淮阴工学院江苏省介入医疗器械研究重点实验室,江苏淮阴223003

出　　处：《中国科技论文》2015年第6期740-744,共5页China Sciencepaper

基　　金：高等学校博士学科点专项科研基金资助项目(20110191120038);中央高校基本科研业务费专项资金资助项目(CQDXWL-2012-126);江苏省介入医疗器械研究重点实验室开放基金资助项目(jr1202)

摘　　要：通过分析miRNA表达谱发现,miRNA-20a在凋亡CHO-K1细胞中的表达降低。通过构建稳定表达miRNA-20a的重组细胞株,分析该重组细胞的抗凋亡能力。分别构建携带miRNA-20a前体序列或反义序列的重组慢病毒,转染CHO-K1细胞,得到miRNA-20a功能获得或功能抑制的重组细胞。无血清过度消耗培养液应激环境下培养重组细胞,分析靶基因E2F1的表达水平,通过细胞活力和Caspase3/7活性分析评价细胞的抗凋亡能力。结果显示,miRNA-20a过表达的细胞株,E2F1的表达受到显著抑制,在无血清过度消耗的应激环境下,细胞活力显著提高,Caspase3/7活性降低。相反,miRNA-20a抑制表达的细胞株中,E2F1表达增加,而细胞活性显著降低,抗凋亡能力显著下降。上述结果提示,miRNA-20a有望作为哺乳动物宿主细胞的遗传重构靶标,构建耐受应激环境的抗凋亡细胞。The miRNA microarray analysis showed that the miR-20 aexpression was significantly downregulated in apoptotic CHO-K1 cells,which suggested that miRNA-20 awere potential target for genetic engineering cells to enhance apoptosis resistance.The genetic recombinant CHO-K1 cells with miRNA-20 astable expression or inhibiting expression were constructed using the recombinant lentivirus vectors with pre-miR-20 asequences or anti-miR-20 asequences.To evaluate anti-apoptosis ability of recombinant cells,the cells were cultured in spent medium without serum,then the expression level of target gene E2F1,cell viability and Caspase 3/7activity were analysed.The results show that the expression of E2F1 and Caspase 3/7activity are significantly inhibited,consequently the cells viability is improved.In contrast,the anti-apoptosis anbility is weakened in recombinant CHO-K1 cells with inhibited miR-20 aexpression.These results suggest that stable miR-20 aexpression is beneficial to maintain viability of cells exposed to full-stress culture and miR-20 amay become a target for developing new technologies in enhancing the resistance of mammalian cells to shear stress.

关 键 词：生物工程 微小RNA-20a 中国仓鼠卵巢细胞 抗凋亡 遗传重构 

分 类 号：Q78[生物学—分子生物学]