| Liver fibrosis is a necessary process in the development of liver diseases,such as hepatic cirrhosis and its complications,which is a serious threat to human health and life.Blocking the occurrence of liver fibrosis is of great significance for liver diseases.Small interfering RNA(siRNA)has been confirmed to be able to effectively regulate the expression of human related genes.It can be used as therapeutic components in the disease which caused by overexpression of a specific genes and has great potential in gene drug development.Constructing delivery system to overcome the obstacles of siRNA delivery in vivo is the most critical problem in the development of siRNA gene drugs.The topic of this paper is to construct a targeting delivery system,which is called stable nucleic acid lipid nanoparticles(SNALP).In order to develop siRNA gene drugs as the ultimate goal,we have made a very useful explorationon in the preliminary study of siRNA drugs for liver fibrosis.In selecting of liver fibrosis of siRNA,taking into account the HSP47(heat shock protein gene,mice homologous gene is gp46)expression plays a very important role in the occurrence and development of liver fibrosis,we designed 3 siRNA sequences of HSP47/gp46 for gene therapy for liver fibrosis.In this study,siRNA was transfected into NIH3T3 cells and through the real-time fluorescence quantitative PCR assay to screen the siRNA which with strongest effect of gene inhibition.The 3 sequences of gp46 gene silencing efficiency were:siRNA165.5%,siRNA264.6%,siRNA316.6%.Therefore,the siRNA1 and siRNA2 with high silencing efficiency were selected as the gene drug treatment components,and the follow-up study was carried out.In construction of siRNA lipid delivery system.Stable nucleic acid lipid nanoparticles(SNALP)were prepared by siRNA and cationic lipid DlinMC3 and auxiliary lipid such as DSPC,CHOL,PEG-DMG.In order to realize the active targeted therapy for liver fibrosis,pPB polypeptide was introduced into SNALP,which was able to identify the excessive expression of PDGF-(3.Synthesis target lipid molecules pPB-PEG-DSPE as a component of targeting-SNALP.Both siRNA,target lipid molecules pPB-PEG-DSPE and other lipid molecules include DlinMC3,DSPC,Chol,PEG-DMG together preparied a targeting stable nucleic acid lipid nanoparticles called pPB-SNALP.Characterize physical parameters such as particle size,PDI and surface charge of SNALP and pPB-SNALP,with particle size maintained at 110-130nm,PDI maintained from 0.1 to 0.3.Through Kunming mice model of the gp46 gene high expressions and liver fibrosis to verify treatment effect of pPB-SNALP and SNALP,when intravenous injection of those two animals models which SNALPs containing 0.023mg/kg siRNA.For Kunming mice model of the gp46 gene high expressions,we find conclusions as follows:1)pPB-SNALP/siRNA1 group has 37%f gene silencing efficiency.SNALP/siRNA1 group has 12%of gene silencing efficiency.2)The gene silencing efficiency of pPB-SNALP/siRNA2 was 29%,otherwise,Non-targeting SNALP groups was 13%.In summary,this project constructed targeting delivery system for liver fibrosis and test its targeting property and treatment effect both in vitro and in vivo,which laied a good foundation for targeted gene therapy of liver fibrosis. |
