稳定表达外源性p16基因肺癌A549细胞株的建立及鉴定

ESTABLISHMENT AND IDENTIFICATION OF A549 CELL LINE WITH STABLE EXPRESSION OF EXOGENOUS P16 GENE

为构建稳定表达外源性抑癌基因 p16的肺癌 A5 49细胞株 ,用脂质体介导的基因转染方法 ,借助真核质粒表达载体 (pc DNA 3) ,将抑癌基因 p16转移入此基因缺失的人肺癌细胞株 A5 49细胞中 ,经 G418筛选 ,获得稳定表达的细胞克隆 ,用逆转录聚合酶链反应 (RT- PCR)及免疫组织化学鉴定 p16基因的表达 ,同时对克隆细胞分泌蛋白进行活性检测。结果显示转染 p16基因的 A5 49细胞中可以检测到 p16 m RNA及蛋白的表达 ,说明建立的 p16真核表达载体能在肺肿瘤细胞中分泌表达蛋白 ,表达 P16抑癌蛋白的 A5 49细胞株的建立有助于研究抑癌基因

To establish A549 cell line with stably expressing tumor suppressor gene p16,the exogenous tumor suppressor gene p16 was transfected into A549 cells with lipofectin by eukaryotic expressive vector(pcDNA3). We got clone cells after selected by G418. All the clone cells were detected by RT PCR and immunohistochemical staining on mRNA and protein levels. Meanwhile, P16 protein activity were measured by MTT. The Results showed that p16 mRNA and protein were expressed in those transfected A549 cells. It demonstrated that the p16 eukaryotic expression vector can express P16 protein in human lung cancer cells. Exogenous p16 gene may stably expressed in human lung canner cell line A549. The establishment of the cell line can help to study the functions of tumor suppression gene p16 in human lung cancer.\;