Effect Of FasL CDNA Transfection On Epithelial Ovarian Carcinoma Cisplatin-Resistance

Objective:1.To detect the difference of Fas and FasL expression between the drug-resistant epithelial ovarian cancer cell SKOV3/DDP and the parent cell SKOV3.2.To construct the eukaryotic expression vector pcDNA3.1(+)/FasL containing FasL sector,re transfect it into SKOV3/DDP,and check the expression of FasL protein.3.To add the cisplatin to pre- and post-transfection drug-resistant cell respectively, observe the apoptosis of the cell.To discuss the relationship between Fas/FasL and the drug-resistance of epithelial ovarian cancer and analyse the effect of the transfection of FasL cDNA to cisplatin-resistance character.4.To assess the clinic significance of FasL accompanied with cisplatin to treat the epithelial ovarian cancer.Methods:1.Check IC50 of the drug-resistant epithelial ovarian cancer cell SKOV3/DDP to SKOV3 through MTT,and calculate resistant index.2.Extract mRNA,amplify Fas cDNA and FasL cDNA from the SKOV3/DDP and SKOV3 through RT-PCR.Compare Fas in SKOV3/DDP and SKOV3 through gel imaging system.Compare FasL cDNA in SKOV3/DDP and SKOV3 through RT Q-PCR.3.Amplify the FasL cDNA from HeLa cell through RT-PCR.Connect FasL cDNA with plasmid pcDNA3.1(+)to construct eukaryotic expression vector pcDNA3.1(+)/FasL and test the DNA sequence.4.Transfer pcDNA3.1(+)/FasL to SKOV3/DDP through liposome infection protocol.Test FasL expression before transfection and at 24,48,72 hours after transfection through western-blot.5.Observe the apoptosis of drug-resistant cell before and after transfection through TUNEL.6.Observe and compare the cisplatin-sensitivity of FasL transferred and non-transferred drug-resistant cell at 24,48 and 72 hours after adding cisplatin of different concentration through TUNEL..7.Statistics and analyze the results.Results:1.The resistant index of SKOV3/DDP is 5.54,which could be used in the test.2.Fas and FasL expression is lower in resistant cell.3.Eukaryotic expression vector pcDNA3.1(+)/FasL is constructed successfully.4.FasL protein expression is positive at 24,48 and 72 hours after transfection.5.Apoptosis is increase after transfecting FasL to resistant cell SKOV3/DDP.6.After adding cisplatin of different concentration,sensitivity is proved to be improved in transfecting resistant cell at 24,48 and 72 hours.Conclusion:1.Low expression of FasL is one of the reasons of drug-resistance of epithelial ovarian cancer.2.Treatment with cisplatin and transfection of FasL to ovarian cancer cell may be the new therapy of drug-resistant epithelial ovarian cancer.