Inhibitory Effects Of PTEN Gene Mediated Combine With L-OHP On Human Cholangiocarcinoma Cell Line

Objectives To investigate the inhibitory effects of retrovirus intervention to supprosessgene PTEN in combination with L-OHP on human cholangio-carcinoma cell line , to mouse out the way of gene therapy on human biliary duct carcinoma.Methods 1. Transfection : First take pBP-PTEN and plasmid pBP transfected in DH5ct, and to clone and amplify their DNA. Then pick-up and sublime DNA of plasmid, determine by EcoR-I digestionandelectrophoresis Containing PTEN gene which was transfected into human QBC939 cell under mediation of lipofec|amine, and positive cell clones were selected and amplified.we named the group of pBp-QBC, pBp-PTEN-QBC. 2. Cell growth curve and MTT: we observe cells of QBC939 and pBP-PTEN-QBC growth, protracted the pBP-PTEN-QBC and QBC939 cell growth curve for 7 days. 3. Immunohistochemistry: Expression of PTEN gene was detected by S-P immunohistochemistry. 4. Test of cell invasion: We used millipore filter cell invasion platr , supperlayer contain DMEM medium and cell numbers of 1× 106/ml with pBP-QBC group and pBP-PTEN group, two groups cells swing in with of L-OHP ( 0.5mg/L). The down layer contain tendency factor medium, under microscope take count of cell numbers on lowerlayer. 5. Electronmicroscope: The cell cycle, ultra-structure and cell shape. 6. Flow cytometry: the cell apoptosis and cell cycline Gl to S period change were detected by flow cytometry. 7 Tumor growth in nudemice: Nudemice were inoculated with 1× 10Vml of QBC939 cells and PTEN-QBC cells. The tumor appeared in QBC group, we injected 0.2ml L-OHP(0.5mg/l)in celiac with each one of QBC group and PTEN-QBCgroup.and anther groups was injected sodium chloride. Them were respectively named QBC group PTEN group QBC+L-OHPgruop and PTEN+L-OHPgroup. we take out sampler tumor from QBCgroup andQBC + L-OHPgroup nudemice and measured size by electronmicroscope scanning.Results 1. PTEN was expressed in the QBC939 transfected with PTEN gene by immunohistochemistry showing that QBC939 group is 23.37 ± 11.07, pBP- QBC group is 28.82 ± 10.05, PTEN-QBC group is 52.02 ± 14.38, (p<0.05); 2. Cell growth curve showing pBP-PTEN-QBC cell gowths is slower than anther groups cell (p>0.05); 3. Electronmicroscope showing that the ultra-structure has been changed in cell science PTEN gene when transfected, and the cells growth becoming mature;4. The resulted cells invasion rate into PTEN-QBC+L-OHP group is 18%, QBC+L-OHP group is 40%, PTEN-QBC is 33% and QBC939 group is 56%. 5. In transfected PTEN gene cells, not only the progression of cell cycle was arrested Gl to S phase, but also the rate of apoptosis hoist by flow cytometry even shown; 6.In nudemice was inoculated PTEN-QBC cells have been not grown,but the QBC939 group tumour diameter 7.63±3.808 (mm), QBC+L-OHP group tumour diameter 3.833±1.650(mm), in comparison group (QBC) tumor growth is obvious. (p<0.05).Conclusions QBC939 cholangiocarcinoma cell could be arrested at Gl/S phase and the growth of cells inoculated in nudemice could be significantly suppressed by exogenous PTEN gene, by combining with L-OHP the effect of suppression will get strengthened.