ANXA11表达稳定下调小鼠肝癌Hca-F细胞株的构建  

Construction of mouse hepatoma Hca-F cell with stable down-regulation of ANXA11

作　　者：王嘉盛[1] 郭春梅[1] 刘淑清[2] 孙明忠[1] 唐建武[3] 

机构地区：[1]大连医科大学生物技术系,辽宁大连116044 [2]大连医科大学生物化学教研室,辽宁大连116044 [3]大连医科大学病理教研室,辽宁大连116044

出　　处：《中华肿瘤防治杂志》2014年第2期86-90,共5页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(81050010;81171957;81272186)

摘　　要：目的:构建膜联蛋白A11(annexin A11,ANXA11)稳定下调的小鼠肝癌高淋巴转移力细胞株Hca-F。方法:合成2条针对ANXA11的发夹RNA(short-hairpin RNA,shRNA)序列shRNA1和shRNA2,连接到pGPU6/GFP/Neo质粒中,并转染到Hca-F细胞,获得了稳定转染的pGPU6/GFP/Neo-ANXA11-Hca-F细胞株。通过有限稀释法筛选单克隆细胞,并利用RT-PCR和蛋白质印迹法验证ANXA11的表达变化。结果:成功构建了pGPU6/GFP/Neo-ANXA11重组表达质粒,并筛选出单克隆细胞;RT-PCR及蛋白质印迹法检测结果表明,与空白对照组比较,重组质粒pGPU6/GFP/Neo-A11-Hca-F-1和2在mRNA水平,对ANXA11干扰率分别为(27.3±5.9)%和(58.3±12.4)%,P=0.001;在蛋白质水平,对ANXA11的下调率分别为(57.4±0.9)%和(87.1±6.1)%,P<0.001;shRNA-2对ANXA11的表达抑制效果更为显著,P<0.001。结论:成功建立了ANXA11表达稳定下调的小鼠肝癌Hca-F细胞株,为进一步研究ANXA11在淋巴转移中的作用奠定前期基础。OBJECTIVE：To construct and obtain Annexin All （ANXAll） stable down-regulated mouse hepatoma cells from Hca-F,a cell line with high lymphatic metastatic potential. METHODS： Two artificial synthesized shRNA se- quences targeting ANXAll were ligated to pGPU6/GFP/Neo plasmid and transfected into Hca-F cell, then the mono- clonal pGPU6/ GFP/Neo-ANXAll-Hca-F cell with the stable knockdown of ANXAll was obtained by limited dilution, and the expression of ANXAll at mRNA and protein levels were validated by RT-PCR and Western blot, respectively. RESULTS： The recombinant expression vector of pGPU6/GFP/Neo-ANXAll and monoclonal pGPU6/C-FP/ Neo-ANXAll-Hca-F cell lines were successfully constructed and screened. RT-PCR results indicated that the mRNA lev els of ANXAll in Hca-F cells pGPU6/GFP/Neo-All-Hca-F-1 and -2 transfected with shRNA-1 and -2 specifically de- signed for ANXA11 were down-regulated by （27.3±5.9）% and （58.3±12.4）% （P=0.001） compared with normal Hca-F cell. Western blot assay indicated that ANXAll was down-regulated by （57.4±0.9）% and （87.1±6.1）% （P〈 0. 001） at protein level. The shRNA-2 showed better inhibitory effect against ANXAll expression （P〈0. 001 ）. CON- CLUSION：Mouse hepatoma Hca-F cells with stably down-regulation of ANXAll are constructed, which provides a solid base for further study of ANXAll in hepatoma lymphatic metastasis.

关 键 词：ANXA 肿瘤淋巴转移 HCA-F RNA干扰 

分 类 号：R735.7[医药卫生—肿瘤]