The Effects Of Silencing P300 And C/EBP? On The Production Of Th17 Differentiation Factor And Proliferative Lesions In Renal Tissue Of Rats With Thy-1 Nephritis And The Construction Of Cell-specific Lentiviral Vector

Part ? The effect of p300?C/EBP? gene silence on cytokine production ofpromoting Th17 cell differentiationand pathological changes of the renal tissue of rats with Thy-1 nephritisObjective:To investigate the effect of silencing renal p300 and C/EBP?gene on cytokine production of promoting Th17 cell differentiation and pathological changes of the renal tissue inrats with Thy-1 nephritis.Methods:Lentiviral vectors expressing p300 shRNA?C/EBP? shRNA or negative control shRNA were constructed and perfused into rat kidney respectively.Rat Thy-1 nephritis was induced by injection of anti-Thy-1 antibody after determining the silence of p300 and C/EBP? genes.Then the level of IL-6?TGF-?1 and IL-17 in the renal tissue at 6h and 3d after the nephritis induction were measured using Real-time PCR and Western blot.Moreover,the renal proliferative changes ofrats with Thy-1 nephritis at 7d were also observed.Results:Lentiviral vectors expressing p300 shRNA?C/EBP? shRNA or negative control shRNA were well constructed.After the gene knockdown of p300 and C/EBP?,the level of IL-6?TGF-?1 and IL-17 in the renal tissue of rat Thy-1 nephritis were notably reduced.Furthermore,the glomerular mesangial cell(GMC)proliferation and extracellular matrix accumulation as well as the urinary protein production were markedly inhibited.Conclusion:Suppression of renal p300 and C/EBP? expression could decrease the production of IL-6?TGF-?1 and IL-17 including proliferative changes in the rats with Thy-1 nephritis..Part ? Construction of lentiviral vectors carrying specific gene promoter in murine astrocyte and identification of the tissue expression and distributionObjective:To clone the sequence of specific gene promoter in murine astrocyte and to construct the specific lentiviral vector relying on this sequence.Methods:The gene promoter sequence of specific protein,namely glial fibrillary acidic protein(GFAP)in murine astrocyte was cloned from genome DNA via PCR,and then constructed into lentiviral transfer plasmid by cohesive terminal attachment to replace the original CMV promoter.These lentivirus vectors were used to infect four types of cell lines(in vitro)and murine brain(in vivo).The efficiency of astrocyte specific gene expression was determined by detecting the expression and distribution of EGFPin tissue via fluorescence microscope and RT-PCR.Results:The sequence of GFAP promoter was successfully cloned,and the lentiviral vector carrying this promoter was well constructed.Additionally,the expression of EGFP was mainly limited in the astrocytes.Conclusion:The lentiviral vector carrying astrocyte specific expressing GFAP gene promoter was well constructed and the target gene(EGFP)triggered by the promoter of the vectors was mainly expressed in murine astrocyte.