Construction And Expression Of Lentivirus Vectorparticle LV-SMAC-RNAi In The Lens Epithelial Cells

Cataract has been the main cause of blind eye.It is predicted that by2020the number of cataract surgery each year will up to2to2.5million. This will no doubt become a serious social and economic problems. Therefore, exploring the etiology and pathogenesis of cataract, delaying and preventing the occurrence of cataract has become medical workers’ most important problems. In recent years,there has evidence that lens epithelial cell apoptosis may be the root cause of cataract,except the congenital cataract.Therefore, to understand the relationship between the fomation of cataract and apoptosis provide us a new way to treat cataract.Smac is called second mitochondria-derived activator of caspase, Smac is identified as a mitochondrial protein that is released from mitochondria into the cytosol in response to apoptotic stimuli. Study showed that Smac played a regulatory role in apoptosis of lens epithelial cells.If we koncked down the Smac gene in the lens epithelial cells,it may decrease the morbidity of cataract.RNA interference (RNAi) is a form of posttranscriptional gene silencing mediated by short double-stranded RNA, known as small interfering RNA (siRNA). These siRNA are capable of binding to a specific mRNA sequence and causing its degradation. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger. RNAi has been rapidly developed in a variety of organisms as a tool for gene silence. In this study,we use RNAi to knock down the Smac gene,to explore the relationship between cataract and apoptosis.ObjectiveTo investigate the expression of Smac in the lens epithelial cell after konck down the Smac gene by lentivirus vector particle with GFP reporter,it accomplishes a groundwork in understanding the regulatory effects of Smac in apoptosis of lens epithelial cells and the molecular pathological mechanism of cataract.Materials and Methods1. HLE-B3Cultured:Human lens epithelial cell line HLE-B3were cultured with DMEM(including20%FBS), in5%CO2,37℃saturated atmosphere.2. To construct lentivirus vector particle to produce lentivirus.3. Immortalized human lens epithelial cell (HLEC-B3) was divided into3groups,about16hours after infection,we changed the liquid waste, after72hours,we detect the expression of GFP(Green Fluorescent Protein, GFP) in HLE-B3by fluorescent inverted microscope.4. Total RNA was extracted from lens epithelial cells and96hours after infection with shRNA expression vectors we make Real-time PCR for three times. Data Was analyzed using GAPDH as the housekeeping gene control.Result1.HLE-B3cells were transfered successfully by Smac-RNAi,GFP can expressed in HLEs.The fluorescence expressed in cell is uniform and stable.Epithelial cells were imaged via fluorescence microscopy after72hours transduction with siRNA constructs and we found that in each case approximately80%of cells were transduced.2.Real-time PCR indicates that the expression of Smac significantly decreased in the knock down group as compared to negetive control groups. The knock down efficiency is about66.5%(p<0.05).ConclusionWe Contructed Lentiviral Vector Particle successfully and decreased the expression of Smac.It is essentially to investigate the regulatory effects of Smac in apoptosis of lens epithelial cells and the molecular pathological mechanism of cataract.