Research On The Relationship Between Smac、XIAP And Ovarian Cancer Drug Resistance And Apoptosis

Objective:1. To observe the differential expression of Smac and XIAP in ovarian car-cinoma cell lines of SKOV3and SKOV3/DDP.2. An eukaryotic expressing vector pcDNA3.1(+)/EGFP+Smac was constructed and trasfected into epithelial ovarian carcinoma SKOV3/DDP cell line.3. Observe the effect of transfer exogenous Smac gene on the expression of Sm-ac and XIAP in epithelial ovarian carcmoma drug-resistance cell,to discusses the relationship of Smac and the drug resistance of ovarian carcinoma and apoptosis mechanisms.Methods:1. Check IC50of the drug-resistant epithelial ovarian cancer cell SKOV3/DDP to SKOV3through MTT,and calculate resistant index.2. To observe the expression of Smac and XIAP in ovarian carcinoma cell lines of SKOV3and SKOV3/DDP by RT-PCR and western blot.3. Two-step RT-PCR was performed to get mature Smac cDNA. After the single digestion to Smae cDNA and the mammalian expression vector pcDNA3.1(+)/EGFP with KpnI, two purified target fragments were ligated by T4DNAligase. Then the ligation products were transformed intoTransl-T1Phage Resistant competent cells. Recombinant vector was amplified and examined by sequencing.4. Transfer pcDNA3.1(+)/EGFP+Smac to SKOV3/DDP through liposome infe-ction protocol. Test Smac and XIAP expression after transfection through Western blot. And test cell growth inhibition rate after the invitro cellular growth activitise were estimated by MTT. Observe the apoptosis of drug-resistant cell after transfection of Smac.Results:1. The resistant index of SKOV3/DDP is4.56693. which could be used in the test.2. Smac and XIAP were detected in ovarian cell lines SKOV3and SKOV3/ DDP, the relative content of Smac/DIABLO mRNA in SKOV3was higher than that in SKOV3/DDP,XIAP was on the contrary.3. Eukaryotic express vector pcDNA3.1(+)/EGFP+Smac was constructed succe-ssfully and examined by sequencing.4. Smac protein expression is positive after transfection through Western blot and the Expression of XIAP not change significantly.5. Apoptosis is increase after transfecting Smac to resistant cell SKOV3/DDP48h and72h by MTT.Conclusions:Smac protein expression is lower in ovariall cell lines and XIAP is hig-her.Overexpression of Smac was definitely related to the rise of ovarian cancer but he expression of XIAP was not change significantly. Suggesting Smac was definitely related to promoting apoptosis in ovarian cancer, but the mechanism of apoptosis is not to promote XIAP degradation. Smac combined with XIAP and then inhibited the combination of XIAP and caspase,activated the caspase and thus play a pro-apoptot-ic effect.