能稳定表达外源抑癌基因PTEN的胆管癌细胞系QBC939的建立

目的：为研究抑癌基因PTEN对胆管癌细胞生物学行为的影响，建立能稳定表达外源PTEN的胆管癌细胞系。方法：将野生型、突变型PTEN质粒和空载体质粒分别转化细菌，提取纯化后，脂质体介导法转染体外培养的胆管癌细胞系QBC939;嘌呤霉素筛选抗性克隆;Western blot 检测标签蛋白HA的表达;RT-PCR， Western blot和免疫细胞化学法分析目的基因PTEN的表达。结果：野生型、突变型PTEN和空载体转染细胞株均获得嘌呤霉素抗性;野生型和突变型PTEN转染细胞株均检测到ＨＡ的表达，而空载体转染细胞株和未转染细胞株均未检测到ＨＡ的表达;野生型及突变型PTEN转染细胞株中目的基因PTEN在mRNA和蛋白质表达水平上均较空载体转染细胞株和未转染细胞株增强。结论：成功构建了分别能稳定表达外源野生型PTEN，突变型PTEN或空载体pBP的胆管癌细胞系。

Establishment of cholangiocarcinoma QBC939 cell lines stably expressing the tumor suppressor gene PTEN

Objective To establish cholangiocarcinoma cell lines that could stably express exogenous tumor suppressor gene PTEN in order to study its effects on the biological behavior of cholangiocarcinoma cell lines. Methods Three different plasmids including a wild type PTEN, a mutant type PTEN or an empty pBP plasmid were prepared by transformation of bacterium and purification of plasmids. Then throe plasmids were separatedly transferred into cholangiocarcinoma cell line QBC939 cultured in vitro by using lipfectamine. After transfection, cells were selected by puromycin and the positive cell clones were chosen . The expression of the HA-tag protein was detected by Western blot. The expression of the tumor suppressor gene PTEN was separatedly determined by RT-PCR, Western blot and immunocytochemical staining. Results All of the cell lines transfected by wild- type PTEN, mumant- type PTEN or empty plasmid acquired resistance to puromycin ; HA-tag protein was detected by Western blot in cell lines transfected by wild type or mutant type PTEN, whereas, it did not show in cell lines un-transfected or transfected by an empty vector. RT-PCR, Western blot and immunocytochemical staining showed respectively that the tumor suppressor gene PTEN was expressed higher in cell lines transfected by wild type or mutant type PTEN than in cell lines un-transfected or transfected by an empty vector, both on mRNA lever and on protein lever. Conclusions Successful establishment of cholangiocarcinoma cells that could stably express the exogenous gene wild-type PTEN, mumant- type PTEN or empty plasmid pBP respectively was obtained.