The Study Of Apoptosis In Colorectal Cancer Cells Induced By Fas Gene Transfection

Objective:The low expression of Fas in the tumor cells is one of the major mechanisms of the apoptosis resistance and sensitivity reduction to the chemotherapy. For this reason, it is possible to induce apoptosis by the increasing the expression of Fas and enhance the sensitivity of the tumor cells to the chemotherapy. The aim of our study is to establish colorectal cancer cell strains with high level expression of Fas gene and to observe there biological characteristics in the apoptosis and the sensitivity to chemotherapy in vitro.Methods:(1) Establishing Fas-expressing colorectal cancer cell lines:Inserting the Fas eDNA into the multiple cloning site of the expressing vector pBK-CMV by molecular cloning technique, then transfecting the reconstructed plasmid into the colorectal cancer cells(Lovo cells) by lipofeetamine and choosing the positive clone containing the reconstructed plasmid by G41 8, Finally, analysing the expression level of Fas gene by Dot blot and Western blot. (2) Analysing the apoptosis of Fas-expressing colorectal cancer cells by flow cytometry and agarose gel electrophoresis. (3) Analysing the related apoptosis gene's change of Fas-transfected cells by flow cytometry (FCM). (4) Observing the sensitivity of transfected cells to chemotherapy by FCM and MTT.Results:The pBK-CMV Fas cDNA plasmid was successfully constructed. The transfected colorectal cancer cells were screened by G4 18 and 1 resistant cell strain (Lovo Fas cells) was obtained. The results suggested that Fas was expressed in both gene transfected and non-transfected cell strains, but the expression level of Fas mRNA and protein was much higher in the former. The proliferation rate was lower and the doubling time and logarithm growth period was longer than the non-transduction cells, but the discrepancy was not significant. While under the of Fas antibody, the discrepancy is significant (p