稳定表达stathmin Ser25磷酸化位点突变的肝癌细胞株的建立  

CONSTRUCTION OF STATHMIN SER25 PHOSPHORYLATION SITE-DIRECTED MUTAGENESIS HCC CELLS

作　　者：甘淋[1] 陶忠桦[1] 刘晓燕[1] 刘银坤[2] 

机构地区：[1]泸州医学院医学基础研究中心,四川泸州646000 [2]复旦大学中山医院肝癌研究所

出　　处：《泸州医学院学报》2011年第5期487-490,共4页Journal of Luzhou Medical College

基　　金：国家863高科技发展计划项目(2006AA02A308)

摘　　要：目的:建立稳定表达stathmin Ser25磷酸化位点点突变(stathmin S25A)的肝癌细胞株。方法:采用PCR定点突变的方法构建stathmin S25A的重组质粒,并用酶切和测序技术鉴定突变结果;采用脂质体转染的方法,筛选建立stathmin野生型(wt)和突变型(S25A)的肝癌细胞HCCLM6,用Western blotting进行鉴定。结果:定点突变构建stathmin S25A的重组质粒,测序证实stathmin 25位丝氨酸突变为丙氨酸。将stathmin wt和S25A转染HCCLM6,筛选建立稳定表达的肝癌细胞株,Westernblotting检测发现stathmin pS25在stathmin S25A细胞中的表达较stathmin wt和对照组细胞明显下降(P<0.05)。结论:建立了稳定表达stathmin S25A的肝癌细胞株,为研究stathmin位点磷酸化在肝癌发病中的分子机制提供实验模型。Objective： To construct stathmin Ser25 phosphorylation site-directed mutagenesis（stathmin S25A） hepatocellular carcinoma（HCC） cells.Methods：Site-directed mutagenesis based on PCR,was used to construct stathmin S25A plasmid,and verified by restriction enzyme cleavage and sequencing technology.Lipidosome transfection method was used to establish,stathmin wt and S25A HCCLM6 cells and identified by Western blotting.Results： Sequencing report of stathmin S25A plasmid showed that stathmin serine25 had mutated into alanine.The stable cells transfected with stathmin wt and S25A plasmid were constructed.And the expression of stathmin pS25 in stathmin S25A cells was depressed compared with stathmin wt and HCCLM6 control cells（P〈0.05）.Conclusion： The stathmin S25A HCCLM6 cells were constructed,which offered experimental model for further investigating the molecular mechanism of stathmin phosphorylation in hepatocarcinogenesis.

关 键 词：点突变 磷酸化 肝癌 STATHMIN 

分 类 号：Q78[生物学—分子生物学]