Silence Target GFP Expression In Hep G2 Cell By SiRNA

The siRNA can silence the mRNA of homologous gene sequence specially and efficiently without influencing the function of other gene, it is a new genetic therapy to genetic disease, tumor and virus infection. This thesis adopts siRNA/liposomes to transfect hepatocarcinoma cell (Hep G2) to silence the expression of target gene (GFP) in vitro test, researches the stability and cytotoxicity, the trasfection efficiency of siRNA-liposomes and the silence effect to target gene, construct a channel to silence gene, wishes to provide reference to therapy and basic study of hepatocarcinoma treatment.Objective:To silence GFP expression in Hep G2 by siRNA, research several biological indices of siRNA silencing target gene, provides information for further experimental and clinic research.Methods:(1) Through test in vitro, evaluated the stability and cytotoxicity of siRNA/liposomes;(2) Through fluorescent labeling technology with Cy3, investigated the trasfection efficiency of siRNA-liposomes;(3) Through the observation under fluorescent microscope and RT-PCR with gel electrophoresis technology, assayed the expression of mRNA of target gene, discovered the silence effect to target gene by siRNA of different type, form and dosage. Results:(1) The siRNA /liposomes maintained stability till 4 hours in culture solution without serum,degraded obviously in 5% serum after 2~4 hours;(2) There was no obvious cytotoxicity by 100nM siRNA/liposomes;(3) The transfection efficiency of siRNA-liposomes depended on time and reached 90% at 4 hour;(4) Non-special siRNA had no silent effect on target gene expression, but special siRNA/liposomes could silence target gene expression and increased with dosage(20nM,50nM,100nM).Conclusion:(1) The stablity of siRNA can meet the demand of test and there is no obvious cytotoxicity;(2) Special siRNA transfect hepatocarcinoma cell can silence target gene expression, the liposomes can improve the efficiency of transfection.(3) siRNA and RNAi provide new tool and thought for therapy of tumor and the basic study.