PTEN表达载体构建及转染食管鳞癌细胞系EC9706.docVIP

PTEN表达载体构建及转染食管鳞癌细胞系EC9706 　 作者：侯桂琴，鲁照明，薛乐勋，田芳，范天黎，刘兰琦，许培荣 【摘要】 目的 筛选稳定表达野生型PTEN基因的食管鳞癌细胞株EC9706。方法 提取胎盘总RNA，设计引物，PCR扩增人野生型PTEN基因，经测序鉴定后，连接于pcDNA3.1(+)真核表达载体，构建pcDNA3.1PTEN，用脂质体介导的方法将其转染EC9706。而后加抗生素G418筛选稳定表达株，用RTPCR方法检测稳定表达株PTEN基因的mRNA水平，通过绘制生长曲线观察细胞生长情况。结果 PCR产物的电泳结果显示，在1 500bp左右有一明显亮带，测序结果与GenBank上PTEN序列完全一致；稳定表达PTEN基因的细胞株RTPCR结果显示，PTEN的mRNA水平比对照细胞株相比明显升高；生长曲线结果显示，转染后的细胞生长速度明显减慢。结论 所扩基因为野生型PTEN基因，所构建的载体为野生型PTEN基因真核表达载体，并筛选出PTEN基因稳定表达的食管鳞癌细胞株。 【关键词】 PTEN；食管鳞癌；mTOR；转染 　　Abstract：Objective To establish stable expression cell line transfected with eukaryotic expression vector of PTEN. Methods To clone the wildtype PTEN gene, total RNA was isolated from human placenta tissues. A cDNA of human PTEN was obtained by optimized RTPCR and sequenced, and then the cDNA fragment was put into the eukaryotic expression plasmid pcDNA3.1(+) to construct pcDNA3.1PTEN. Cells of esophageal squamous cell cancer cell line EC9706 were transferred with pcDNA3.1PTEN using lipofectamine. The stable expression cells were screened by G418. RTPCR and growth curve were done for identifying the screened cells. Results The cDNA fragment produced by RTPCR had 100% homology with wildtype PTEN gene sequence on GenBank by BLAST. The mRNA level of PTEN increased notably in the cell line we screened, and the growth of the cell line was very solely compared with control cell lines. Conclusion Because of cells of esophageal squamous cell cancer which stably express PTEN were successfully screened, it is concluded that a PTEN eukaryotic vector for stable expression in esophageal squamous cell cancer cell line has been constructed. 　　Key words：PTEN；Esophageal cancer；mTOR；Transfection 　　0 引言 mTOR(Mammalian target of rapamycin)是一种进化上高度保守的丝/苏氨酸蛋白酶。越来越多的证据显示， mTOR信号转导在细胞的生存、生长与增殖中起中心调控作用，其通路的异常与多种恶性肿瘤有关[1]，已成为肿瘤治疗的新靶点[2,3]，雷帕霉素是mTOR的特异性抑制剂[4]。抑癌基因PTEN (phosphatase and tensin homolog deleted from chromosome 10)是1997年发现并被克隆的第一个具有双特异性磷酸酶活性的抑癌基因,并被证实与十几种人类肿瘤的恶性增殖和转移有关[5]。据报道，PTEN的缺失或突变可以使mTOR信号通路异常