【摘要】 目的:建立针对膜联蛋白A7(annexin A7)的shRNA(small hairpin RNA)稳定转染的小鼠肝癌细胞株Hca-F,为后续研究奠定基础。方法:构建3条针对annexin A7的shRNA(shRNA1、2、3) ,分别与pSilencer连接,构建表达载体并分别转染Hca-F细胞,在mRNA和蛋白质水平比较3条shRNA对annexin A7表达的抑制作用,筛选抑制效果最好的shRNA转染Hca-F。稳转后用含400μg/ml G418的完全培养基筛选,获得annexin A7表达稳定下调的Hca-F细胞株后传代扩增,应用Western印迹法检测Hca-F细胞annexin A7蛋白的表达,并与空载体转染组及正常对照组进行比较。结果:经DNA测序鉴定,所得到干扰序列与GenBank中序列一致;筛选出其中shRNA1对annexin A7的表达抑制效果最好。pSilencer-shRNA1转染Hca-F细胞株后,annexin A7蛋白的表达远低于空载体转染组和正常对照组(0.3186 vs 0.7987 ,0.824 3,P<0.05) ,而后二者间无统计学差异。结论:成功建立了annexin A7表达稳定下调的小鼠肝癌Hca-F细胞株,为后续研究奠定了基础。更多还原

【Abstract】 Objective:To establish a mouse hepatic cancer cell line Hca-F transfected with shRNA (small hairpin RNA) targeting annexin A7,so as to provide a basis for future study. Methods: Three shRNAs(shRNA1,2 and 3) were designed and inserted into the pSilencer vector to silence annexin A7 gene. The three pSilencer-shRNA vectors were transfected into Hca-F cells separately,and the most effective pSilencer-shRNA vector was selected based on the results of RT-PCR and Western blotting. The Hca-F cells were transfected with the most effective pSilencer-shRNA vector and the transfectants were selected by 400 μg/ml G418. The cells with annexin A7 stably knockdown were passaged and the expression of annexin A7 was confirmed by Western blotting,and the result was compared with those transfected with empty vector and normal controls.Results: The sequencing results confirmed that the sequences of the 3 shRNAs were correct,and shRNA1 was found to have the best inhibitory effect against annexin A7. Compared with normal Hca-F cells and those transfected with empty vectors,the annexin A7 protein expression was significantly down-regulated in cells transfected with pSilencer-shRNA(0.318 6 vs 0.824 3,0.798 7,P<0.05),with no significant difference found between the former 2 groups. Conclusion: We have successfully established a Hca-F cell line with annexin A7 stably down-regulated using shRNA technique,paving a way for future study.更多还原