Suppress Proliferation Of NIH3T3Cells By Stable Expression Of Perlecan ShRNA Lentiviral Particles

1、Objectiveto investigate the role and mechanism of perlecan on f ibroblast synthesis. And explore the impact of suppression of proliferation of NIH3T3cells by stable expression of Perlecan shRNA Lentiviral Particles.2、Methodin this study, Fibroblasts cells were cultured, using LV-GFP with MOI=10,30,50to transfect cells, then observe the GFP expression in a fluorescence microscope during one week, to estimate the proper MOI and the time of GFP expression. Detecting the transfection efficiency of LV-GFP with the proper MOI by fluorescence-activated cell sorting. The stable transfected cell lines were developed by puromycin screening for more than2weeks. Randomly divided the third generation HFF in good condition into three group:objective infected gene, empty lentivirus vector, and contrastive group. Semi quantify RT-PCR, Western blot assay and MTT assay were used to detected the expression of perlecan mRNA and protein in the three groups, the level of cell proliferation.3、Result1) Fluorescence microscopic observation:a) The GFP expressed better as the MOI increased. The group with MOI=30lready showed satisfactory GFP expression.b) After four days of transfection。GFP expression became significant. It showed no reducti on at one week.2) The expression of perlecan gene was examined by RT-PCRandWestern blotting.It was showed that the expression of perlecan mRNA and protein were significantly reduced in objective infected gene group compared with empty lentivirus vector and contrastive group. Cultivate in the DMEM/F12culture solution, the shRNA transfected cells showed a reduced proliferation rate while the contrastive group and empty lentivirus vector group grew rapidly.3)The proliferation ability of NIH3T3cells was examined by MTT. Cultivate in the DMEM/F12culture solution, the shRNA transfected cells showed a reduced proliferation rate while the contrastive group and empty lentivirus vector group grew rapidly.4、ConclusionAn appropriate condition for lentiviral vector to transfect has been learned. For the cell in this experiment, MOI=30is enough the relatively. The target geneS expression can be well detected afer four days.growth of NIH3T3cells couldbe inhib i teds ignifi cant lybyPerlecanshRNAL entiviralParticles transfect ion.