p33ING1b基因转入对SW480细胞生长增殖影响地.pdfVIP

Effect on Proliferation of SW480 Cell Line with p33ING1b Gene Transfection Abstract Objective Gene p33ING1b is a new coloned tumor suppressor gene recently, the genetic structure and expression’s abnormality of which exists in human several types of tumors. This investigation is designed to select SW480 cells as target, the expression of p33ING1b of which is low, then to observe the effect of SW480 cells and analysis the possible molecule mechanism of p33ING1b in SW480 cells after transfecting p33ING1b gene into it. Methods pcDNA3.1(+)/p33ING1b expression vector was constructed and the recombinant plasmid was transfected into SW480 by liposome transfection method as well as pcDNA3.1 (+) plasmid. After transfection, the positive cell clones of pcDNA3.1(+)/p33ING1b/SW480 and pcDNA3.1(+)/SW480 were selected by G418 and identified by RT-PCR, Western blot and S-P immunohistochemical method. In order to elucidate the effect of expression of exogenous p33ING1b gene on the colorectal cancer cell SW480, the proliferation rate in vitro of cells including SW480, pcDNA3.1(+)/p33ING1b/SW480 and pcDNA3.1(+)/SW480 were analyzed by growth curves and colony formation assay in soft agar. At same time, the apoptotic rate of cell and the cell cycle analysis were also tested by the flow cytometry. At last, using western blot analysis, we detect the expression level of the protein p53, p21WAF1, Bax and Bcl-2 in pcDNA3.1(+)/p33ING1b/SW480, pcDNA3.1(+)/SW480 and SW480, which initially indicate the molecule mechanism of inducing apoptosis by gene p33ING1b . Results The results of restriction enzyme cutting and DNA sequencing demonstra