Ap-1 Of Decoy Oligonucleotides On Renal Tubular Epithelial Cell Biology Behavior And Pathological Inflammatory Effects Of In Vitro Study

More and more experimental and clinical datas have revealed that the speed of various kinds of renal diseases progressing to the end stage of renal failure are strongly dependent on the severity of the renal tubulointerstitial injure and renal tubulointerstitial fibrosis(RIF) , don't associate with the degree of the glomerular injure and glomerulosclerosis. Tubular Epithelial Cell (TEC) is a energetic participant , not only be a passive sufferer in the course of the tubulointerstitial injure. The role of TEC in the RIF, especially the particular pathogenesis and link of which , is little understood, so the recent interests of nephrologists have focused on elucidating mechanism of the effect of TEC in the occurrenc and developing of RIF and find out a novel anti-renal fibrosis drug which target to TEC specially.The role of the activated AP-l(Activater protein-1), a nuclear transcription factor , in the renal diseases was followed with interest gradually. A little of the investigation in vivo and vitro show that AP-1 played an important role in the TEC's activation and the inflammation medium synthesis. So, we hypothesize it may be relieve the inflammation injure in renal tubulointerstitial and prevent the developing of RIF that Blocking the activated of AP-1 in TEC at gene level. As a novel strategy of gene therapy, technique of "Decoy oligonucleotides(Decoy ODN)" can black the activation of the transcription factor specially and inhibit the expression of the downstream genes, and it may be a new useful therapeutic approaches to renal diseases. In order to provid the theoretic basis and technic base for treatment of the RIF with the durg of AP-1 decoy ODN, we performed the study from following aspects by utilization AP-1 decoy ODN to block competitively the activation of AP-1 in proximal tubular epithelial cells (PTEC) and by means of the technique of gene transfection mediated by cationic lipids.The present study consists three parts:Part I : Expression of AP-1 in renal tissue of human diseased kidney and renal tubular epithelial cells.Objective: The purpose of this study was to determine the expression of AP-1 and Transforming growth factor-B1(TGF-B1) in renal tissue of human diseased kidney and the regulation of AP-1 expression in cultured tubular epithelial cells. To explored the role of AP-1 in the pathogenesis of the glomerulonephritis and renal tubulointerstitial fibrosis.Methods: Immunohistochemically was used to examine the expression of c-Jun ( subunit of AP-1) and TGF-B1 in the tissues from proliferative and non-proliferative glomerulonephritis and relationship were analyzed between the expression of c-Jun and the degree of glomreular injure or tubulointerstitial injure. Cultured LLC-PK1 cells ( Pig proximal tubular epithelial cell ) were stimulated with Tumor necrosis factor -a1(TNF-a1). Electrophoretic mobility shift assay(EMSA) were used to detect the activity of AP-1. Western blot was used to detect the protein expression of c-Jun and c-Fos(another subunit of AP-1). Gel supershift assay were used to detect the subunit of AP-1 dimer in LLC-PK1 cells.Results: (1) In the normal kidney , a constitutive expression of c-Jun was observed in the glomeruar endothelial cell, cells of the Browman's capsule and proximal tubular epithelial cells, non expression in the mesangial cells. The expression of c-Jun was up-regulation at pathologic status. The expression of c-Jun were more strong in renal chronicity progressive lesion ( glomerulosclerosis and /or renal tubulointersititial fibrosis ) than in acutereversible lesion ( proliferative lesion and /or bleeding lesion ). The expression of c-Jun were more strong in the renal tubulointerstitial portion than in the glomerulus. (2) Non relationship between the expression of c-Jun and that of TGF-B1 was observed in glomerulus. There were the positive correlation between the the expression of c-Jun and that of TGF-B1 in tubules. (3) In the normal cultured condition, a weak expression of AP-1 was observed in the nucleus of LLC-PK1 cells. EMS A showed that...