The Research On The Combination Of Human IFN-ε With Trail To Ovarian Cancer Cells In Vitro

Ovarian cancer is one of the common gynecologic tumors and the morbidity is very high. Clinical diagnosis is very difficult in early stage and 70%patient has been discoverd in the latest stage.The recidivation rate is still 60%althogh the level of therapy has been improved.The five-year- survivial is nearly 30%.But the mortality of ovarian malignant tumor is the first in female genital maligant tumors.It is necessary to find a new therapeutic approach.Gene therapy is the fouth pattern treatment after radiotherapy,chemotherapy and surgical resection.The experiment investigates the effect of interfeon-epsilon and TRAIL on the ovarian cancer cells, provides the base on gene therapy for ovarian cancer.Part 1EXTRACTION AND CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTOR OF HUMAN IFN-εGENEMany experiments has indicated interferon can inhibite tumor growth,recidivation and metastasis,hIFN-ε,discovered by Conklin in 2001,has documented the effect of antiproliferatio -n and immunomodulatory so far,and is a potent therapeutic angent.Objective:To acquire IFN-εfull length gene cDNA by the mean of RT-PCR and construct IFN-εgene eukaryotic expression vector PcDNA3.1-IFN-ε.Methods:After collecting human cervical cancer cells HeLa 3×10~6,IFN-εgene was amplified from the first ligand of cDNA by reverse transcription and then accquired underwenting the PCR reaction according to the cDNA sequence.PCR product was proved by electrophresis,then digested by rectriction endonuclease EcoRⅠand XhoⅠ.The digested product was extracted, ligated with the eukaryotic expression vector PcDNA3.1 which was digested by the same enzymes by T4 ligase over night.At last,the recombinant plasmid PcDNA3.1-IFN-εwas obtained by genetic engineering technique and identified with enzyme cleavage and DNA sequencing.Results:1 645bp cDNA was amplified by RT-PCR and proved IFN-εby electrophresis.2 The eukaryotic expression plasmid PcDNA3.1-IFN-εwas digested by EcoRⅠand XhoⅠ,and two fragments 5.4kb and 645bp were got,indicating that were the plasmid PcDNA3.1 and IFN-εgene.Then it was confirmed that the goal gene IFN-εinserted to the plasmid PcDNA3.1 by the way which the reading frame fuses by DNA sequencing.Conclusion:The eukaryotic expression Plasmid of human IFN-εgene was constructed successfully and established foundation on the research of ovarian cancer cells.Part 2 EXTRACTION AND CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTOR OF HUMAN TRAIL GENETRAIL has recently identified as a member of the TNF superfamily of cell death-inducing ligands after TNF and FasL.The effect of it on cancer therapy has seemed particularly attractive because its proapoptotic effects are lethal in transformed tumor cells but not in normal human cells.This special selectivity enables it to have very broad prospect in tumor treatment.Objective:To acquire TRAIL full length gene cDNA by the mean of RT-PCR and construct TRAIL gene eukaryotic expression vector pEGFP-N3-TRAIL.Methods:After collecting human acute promyelocyte leukemia cells HL-60 4×10~6,TRAIL gene was amplified from the cDNA by reverse transcription and then accquired underwenting the PCR reaction according to the cDNA sequence.PCR product was proved by electrophresis,then digested by rectriction endonuclease EcoRⅠand XhoⅠ.The digested product was extracted, ligated with the eukaryotic expression vector pEGFP-N3 which was digested by the same enzymes by T4 ligase over night.At last,the recombinant plasmid pEGFP-N3-TRAIL was obtained by genetic engineering technique and identified with enzyme cleavage and DNA sequencing.Results:1 865bp cDNA was amplified by RT-PCR and proved TRAIL by electrophresis.2 The eukaryotic expression plasmid pEGFP-N3-TRAIL was digested with EcoRⅠand XhoⅠ,and two fragments 4.7kb and 865bp were got,indicating that were the plasmid pEGFP-N3 and TRAIL gene.Then it was confirmed that the goal gene TRAIL inserted to the plasmid pEGFP-N3 by the way which the reading flame fuses by DNA sequencing.Conclusion:The eukaryotic expression plasmid of human TRAIL has been constructed successfu -lly and established foundation on the research of ovarian cancer cells.Part 3 EXPERIMENTAL STUDY OF TRAIL AND IFN-εIN OVARIAN CANCER CELL LINEObjective:To investigate the effects and the mechanism of pEGFP-N3-TRAIL and PcDNA3.1-IFN-εon the ovarian cancer line in vitro.Methods:Human ovarian cancer cell line SKOV3 was transfected with the plasmid pEGFP -N3-TRAIL,pEGFP-N3,PcDNA3.1-IFN-ε,PcDNA3.1,pEGFP-N3-TRAIL+PcDNA3.1-IFN -εand PBS when it was cultured in logarithm stage,examined the growth inhibitory rate of them by MTT;determined the apoptosis of them by flow cytometry(FCM)and Survivin expression was detected by RT-PCR.Results:48 hours after transfection,green fluorescent protein was detected by fluorescence micoscopy in pEGFP-N3-TRAIL group.Time-dependent cytoxicity of pEGFP-N3-TRAIL group, PcDNA3.1-IFN-εgroup and pEGFP-N3-TRAIL +PcDNA3.1-IFN-εgroup were exhibited in ovarian cancer cells.The growth inhibition rate of them was 18.00%,14.53%,36.68%after 24h; 25.51%,30.19%,56.43%after 48h;47.43%,45.05%,74.16%after 96h respectively.There were significant differences in the cytotoxicity in the same time between pEGFP-N3-TRAIL group and pEGFP-N3 group(P＜0.01);pEGFP-N3-TRAIL group and PBS group(P＜0.01); PcDNA3.1-IFN-εgroup and PcDNA3.1 group(P＜0.01);PcDNA3.1-IFN-εgroup and PBS group(P＜0.01);pEGFP-N3-TRAIL+ PcDNA3.1-IFN-εgroup and single group of them(P＜0.01);the apoptosis rate of them were 21.4%,19.1%,47.3%respectively after transfection 48h. PcDNA3.1-IFN-εgroup,pEGFP-N3-TRAIL group and the combinition of them were evidently different(P＜0.01).The difference of them and the difference of the growth inhibition rates was with one accord.Compared with pEGFP-N3 and PBS group,the Survivin expression level of pEGFP-N3-TRAIL group was decreased(P＜0.05);and PcDNA3.1-IFN-εgroup led a down-regulation of Survivin expression level compared with PcDNA3.1 and PBS group(P＜0.01);the Survivin expression level of PcDNA3.1-IFN-ε+pEGFP-N3-TRAIL group was evidently less amouts than each of them respectively(P＜0.01).Conclusion:The combination of the two treatments induced significantly higher apoptosis and cytotoxicity than exposure to TRAIL or IFN-εalone,they may offer synergistic benefits when used concurrently.The down-regulation of Survivin expression level plays a key role in TRAIL and IFN induced apoptosis of ovarian cancer cells.