The Study On Growth And Chemosensitization Of Stable Hepatocellular Carcinoma Cell Line By RNA Interference To Knock Down Hepatitis B Virus X Gene

Background and Objective:There are plenty of experiments which have proved that HBx can be detected in most of the Hepatocellular carcinoma (HCC) tissue infected by hepatitis B virus. HBx is one of the principal factors of hepatocyte malignant transformation,it can increase proliferation activity of Hepatocellular carcinoma cell and promote its growth quickly.RNA interference targeting to HBx is a new approach with high performance and specificity in the gene therapy of liver cancer .Chemotherapy is still one of important methods to treat the Hepatocellular carcinoma now.Neverthless, drug resistance in chemotherapeutics is a primary cause of treatment failure in liver cancer. Related study make clear that HBx can upregurate the expression of multidrug resistance gene through special MAPK pathway . Then anti-apoptosis signal ocuppy the manage position and supress the apoptosis of Hepatocellular carcinoma cell induced by the chemotheraputics.For this reason ,we plan to adopt the RNA interference to aim directly at HBx and traditionary antinelplastic agent of liver cancer.We shoud investigate the influence of association RNA interference with traditional therapy.We should select the therapy combination of high performance and study the apoptosis mechanism.Methods:1.Identity the stable Hepatocellular carcinoma cells transfected by shRNA aimming at HBx together with independent control series:we should reanimate three kinds of Hepatocellular carcinoma cell(MHCC97-H,HK3,21543),cultivate them in the high glucose DMEM with G418 for some days,then observe the expresion of GFP in cells through inversion fluorescent microscope and initial to estimate the transfection efficiency ,take photographs at last.2.Detect the extent of HBx gene by RNA interference by semipuanitative RT-PCR : collect three kinds of cell and extract total RNA,add two different pairs of primer and carry out RT-PCR in accordance with illustration,On the basis of endogenous gene strap in the gel electrophoresis,we assessment the tempate quantity and judge the extent of HBx gene by RNA interference.3.0bserve the influence of cell growth through RNA interference:we should utilize the Cell counting kit-8 and evaluate optical density value ,then we draw the growth curve of three kinds of cell in six days.4.Detect the diversity of cell cycle by flow cytometry5.Draw the growth curve of three kinds of cell after add different density of flurouracil ,cisplatin andα-interferon and select the best combination.6.Detect the cell apoptosis by TUNEL apoptosis detection kitResult:1 .There are no difference in the shape and GFP expression intensity in two transfected cell after resuscitation.2.The result of RT-PCR demonstrate that the HBX mRNA level of 21543 cell down regulate, the HBx mRNA level of HK3 cell is not different with MHCC97-H cell.3.The growth of 21543 cell are slower than MHCC97-H cell and HK3 cell obviously. The latter two cells are not different notably.4.The cell cycle of 21543 cell showed that Hepatocellular carcinoma cell through RNA interference targeting to HBx delay to goto S stage,then proliferation activity degrade obviously.5.Three kinds of cell adding different density of flurouracil ,cisplatin grows slower than the original cell. The growth inhibiting is dependent on the density of drug and the growth inhibiting of 21543 cell is the most obvious.That of three kind of cell adding toα-interferon is not obvious.6. Flurouracil induce apoptosis in all kinds of cell . The extent of apoptosis in 21543 cell is most obvious.Conclusion: 1. RNA interference targeting to HBx can supress the growth of hepatocellular carcinoma cell.2. Hepatocellular carcinoma cell through RNA interference targeting to HBx can intensify chemo-sensitivity.3. Combination RNA interference targeting to HBx with chemotherapeutics can induced apoptosis in more hepatocellular carcinoma cell.Cell proliferation step down accordingly.