To Construct The Eukaryotic Expression Plasmid PBK-Fas And To Analyse In Vitro Anti-tumor Effects Of It's Transduction On Squamous Cell Carcinoma Of Tongue Tca8113 Cells

Objective: To Construct and Evaluate the Eukaryotic Expression Plasmid pBK-Fas.To evaluate the inhibitory effects of Fas gene transduction on squamous cell carcinoma of tongue Tca8113, To provide experimental basis for further study on Fas gene transfection on squamous cell carcinoma of tongue Tca8113 cells.Methods: Total RNA was isolated from human Lymphoid tissue .Full-length Fas fragment was amplified by reverse transcription-polymerase chain reaction(RT-PCR).Do nucleotide sequence analysis .The plasmids were prepared by double digestion;then the DNA fragment was extracted,purified and ligated in the plasmid pBK-CMV to form pBK-Fas. Plasmid including Fas gene was transfected intoTca8113 cells by lipofectamine kit, To evaluate the inhibitory effects of expression of Fas mRNA in Fas-transfected,Fas protein expression in tumor cells was detected by flow cytometry. To evaluate the inhibitory effects of Fas gene transduction on squamous cell carcinoma of tongue Tca8113 cells by cell growth curve,MTT and plating efficiency test.Results: 1. In the extraction process total RNA of Fas gene did not Occurred significant degradation,the total degradation of RNA integrity and non-degradable.It is shows that total RNA and mRNA of Fas gene is integrated.Product of RT-PCR showed a clear specific band at 1008 bp and it is show that there is the correct size consistented with the length to the estimated cDNA.2.The eukaryotic expression plasmid of pBK-Fas is Successfully constructed.Fas gene fragments in eukaryotic expression vector pBK-Fas is correct by digestion and PCR.3.The transfection in Fas gene could increase the inhibitory effects of expression of Fas mRNA in Fas-transfected and the apoptosis in cells.The expression and intensity of Fas protein was increased in no-transfected and Fas-transfected Tca8113 cells from 34.01±4.76 to 55.72±7.33(P<0.01). It is shows that the expression and intensity of Fas protein could not induced apoptosis but it reach the threshold. Fas-transfected Tca8113 cells could be degradated to the conspicuous DNA ladder by agarose gel electrophoresis for analysed Tca8113 apoptosis.4. The rowth curves of no-transfected and Fas -transfected Tca8113 cells could show the mounting time was 0.8d and the logarithmic phase time was 3d-7d,It was 2d and 4d-8d in no-transfected Tca8113 cells.The largest cells was 2.6×105 in Fas -transfected Tca8113 cells,it was more lower to the no-transfected Tca8113 cells .All this illustrated that the growth rate was more lower in Fas -transfected Tca8113 cells.5.Fas-transfected cells can stimulate apoptosis pathway by Fas antibody.Inhibitory effect of Tca8113 cells by MTT,the OD570 in Fas-transfected Tca8113 cells was Compared with in no-transfected Tca8113 cells,It was 0.186±0.007 and the Proliferation inhibition rate was 51.43%,(P<0.01).6.Colony-forming cells in the rate of Fas-transfected Tca8113 cells was 0.5%.It shows that The transfection in Fas gene could increase the inhibitory effects of cells growth.Cell growth curve and plating efficiency test revealed that Fas -transfected Tca8113 cells had longer population and lower plating efficiency compared with control cells.Conclusion: DNA(Fas)was successfully constructed and transformed into pBK-Fas,the insgenic expression of Fas gene could effectively inhibit the increase of squamous cell carcinoma of tongue Tca8113 cells.