The Study Of Ultrasound And SonoVue Mediated SiRNA-survivin Promote Apoptosis Of Cell HepG2 In Vitro

Hepatoma is one of the common carcinomas in our country.The nomal traditional therapy methods include surgery,drug treatment and biotherapy and so on.But the efficiency of them are not satisfying.With the development of molecular biology,it is shown that the change of multi-oncogene and anti-oncogene are the background of genesis and growth of heptoma.Survivin is one of the genes which are especially high expressed in tumor cells and restrain the apoptosis of the cells,so it becomes one of the targets of gene therapy of carcinomas.The technology of RNA interferece is one of the gene therapy methods which transfecting the homologisation mRNA of taget gene and led to degradation and gene silencing of the target cell.This study tries to transfect siRNA survivin to hepatoma cells in vitro and promote the apoptosis of them.However,one of the important steps of the gene therapy is how to transfect the therapic genes to the target tumor cells.The traditional methods include lipidosome and virus,yet all of them have shortages such as low efficiency or low safety.Microbubble contrast agent mediated with ultrasound is one of the gene transfection methods newly emerging in the recent years wihich shows high efficiency and safety in operation.This study used microbubble contrast agnet SonoVue with ultrasound combined with lipidosome,which transfected siRNA survivin to HepG2 cells and hoped to promote the apoptosis of the cancer cells.The aim of the study is to combine the technique of RNA interfere and ultrasound,and develop a new transfection method in gene therapy.PartⅠThe study of Ultrasound and SonoVue mediated pEGFP-N1 transfection to HepG2 in vitroObjective To investigate whether ultrasound and microbubble contrast agent could enhance the transfection efficiency of pEGFP-N1 to cell HepG2.Methods The cultured cells HepG2 were divided in six groups:(1) control group,(2) ultrasound irradiated group,(3) microbubble contrast agent group,(4) contrast concentration group,which with the different dose of 5%(7.5μl),10%(15μl),20%(30μl),40%(60μl),80%(120μl) and 100%(150μl).(5) ultrasound mechanical index group,with different MI of 0.2,0.4,0.6,1.0,1.1.(6) exposing time group,with different time exposing of 1min,3min,5min,10min and 20min.The fifth group have the most efficient contrast concentration concluded in group four. The sixth group was exposed with the most efficient MI concluded in group five and have the most efficient contrast concentration concluded in group four.Each group cells were cultured 24h after transfection and then observed with fluorescence microscope and measured transfection efficiency by flow cytometry.Results The highest transfection efficiency in group four(contrast concentration group) was the cells with 40%sonoVue(18.11±1.63)%,which comparing with other group has significant difference(P＜0.01) yet the survival rate was notablely decreased with concentration dose more than 80%,and have significant difference compared with other groups(P＜0.01).In group five(ultrasound mechanical index group),the highest trasfection efficiency happens in the cells exposed with MI 1.0(27.03±2.16)%,and has significant difference with other groups(P＜0.01).The survival rate are all above 95%except the group with MI 1.1.In exposing time group,the best result happens in the cells exposed to ultrasound for 5 min(27.46±2.31)%,and has significant difference with other groups(P＜0.05).The survival rate is notablely decreased with longer exposing time.Conclusion ultrasound and microbubble contrast agent could enhance the transfection efficiency of pEGFP-N1 to cell HepG2,and the best condition is 40% sonoVue and exposing to ultrasound with MI 1.0 for 5 min.PartⅡThe study of siRNA survivin inducing cell HepG2 apoptosis in vitroObjective To screen a small interfering RNA which has active suppression of suvivin,and investigate the influence of survivin mRNA level and the apoptotic of cell HepG2.Methods Constructed the survivin and EGFP fusion gene expression vector first. According to human survivin gene order and basic principles of design,worked out 3 pieces of survivin siRNA target sites and amplifiacated siRNA express frame with two steps PCR.Then transfected psurvivin-EGFP-siRNA to cell 293T and picked out the most efficiant one.Then treated it by Taqase for 20 min and inserted siRNA to vector pMD18T by T-A clonal.The recombinant plasmid which got is the expression vector.Transfected the survivin siRNA to cell Hep2 and analysis the expression of survivin and apoptosis of the cells.Results Screened a piece of high efficiantive siRNA survivin.The results of flow cytometry shows HepG2 cells have significant apoptosis rate after transfected siRNA survivin.The ratio is 4.6%,7.8%,12.3%and 36.4%refer to the 1st,2nd,3rd,4th day after transfection.And the experiment of sensitivity to Docetaxe also showed that inhibited the expression of survivin could enhance the sensibility to chemsotherapy drugs obviously.The cells which expressed siRNA has significant difference compared to the control group and other groups when Docetaxe density≥10ng/ml(P＜0.05).Conclusions RNA interfere technique can suppress survivin expression actively and lead the HepG2 cells to apoptosis. PartⅢThe study of ultrasound and SonoVue mediated siRNA-survivin transfected to cell HepG2 and promote cell apoptosis in vitroObjective To compare the transfection rate and safety between traditional transfection method by lipidosome and ultrsound+SonoVue mediated siRNA-survivin transfect to cell HepG2.To approach the feasibility of ultrasound and SonoVue enhance gene transfection in vitro.Methods Divided the cultured HepG2 cells into four groups:control group, Sonovue+ultrasound group,lipidosome group and lipidosome+Sonovue+ultrasound group.The conditions are 40%Sonovue concentration dose,MI=1.0 and exposed under ultrasound for 5min.Then all the groups were continued culturing for 24h and analysised by Western blot and RT-PCR for the expression of survivin mRNA and apoptotic index of the cells.Results Western blot shows the expression of survivin mRNA depressed in all the groups excpet the control group and the lipidosome+SonoVue+ultrasoud group shows the best efficiency.Also has the same result by RT-PCR,there is significant difference in lipidosome+SonoVue+ultrasoud group compared with other groups(P＜0.05),yet there is also significant difference between lipidosome group and ultraound+SonoVue group(P＜0.05).The flow cytometry results shows that the apoptotic index(AI) is 23.05±0.54%in lipidosome+SonoVue+ultrasoud group. Which has significant difference compared with other groups(P＜0.05).The AI of lipidosome group is 20.55±0.38%,compared with SonoVue+ultrasound group(11.75±0.64%) has significant difference(P＜0.05).Conclusion Microbubble contrast agent SonoVue and ultrasound could enhance lipidosome mediated siRNA-survivin transfect to cell HepG2 and promote apoptosis of the cells.