Abstract: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 three 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.