PART ONE:Expression Of IL-10 In Renal Biopsy Sections From Patients With Glomerulonephrits Or In HMC In Vitro

Mediators from both immnue and non-immune cells play important roles in glomerular disease processes. They are involved in induction of the immune response,initiation of acute and chronic inflammatory events, which named proinflammatory factors, and another help to limit the inflammatory effects and transit to repair or resolution. Thus attempt to reduce glomerular inflammation have focused on blocking the production and activity of proinflammatory mediators or, alternatively, on upregulating the expression of anti-inflammatory mediators. The latter have been recognized limitedly. It is another way to prevent or therapy diseases that rebuild the endogenous defense machinery through explaining the profound of anti-inflammatory factors in glomerulonephritis. IL-10 is one of the important anti-inflammatory factors, and it was first described as a cytokine synthesis inhibitor factor(CSIF). IL-10 has numerous anti-inflammatory functions including inhibition of the release of proinflammatory cytokines, chemokines, lipid mediators, and oxidants from lymphocytes, neutrophils and monocytes/macrophages. This multitude of anti-inflammatory properties suggests an important role for IL-10 in controlling immune and inflammatory responses. Since glomerulonephritis is close correlation with immunity, it is necessary for us to reveal the biological roles of IL-10 deeply in renal glomerular diseases.PART ONErExpression of IL-10 in renal biopsy sections from patients with glomerulonephritis or in HMC in vitroObjective The aim of this study was to observe the expression of interk ukin-lO(IL-lO) mRNA in human renal biopsy from patients with nephritis, and to investigate the production of IL-10 induced by proinflammatory cytokines in human mesangial cells(HMC).Methods ㎞on-radioactive in situ hybridization with DIG-labeled hIL-10 oligonucleotide probe and true-colour medical image system were used to examine the IL-lOmRNA in renal biopsy sections from 3 controls and 52 specimens from various renal glomerular diseases. (2) The production of IL-10 protein was determined by ELISA and the expression of IL-10 mRNA was measured by reverse transcription-polymerase chain reaction(RT-PCR) .Results (Din controls,IL-lOmRNA were observed in podocyte ,epithlial cells of Bowman's capsule and distal tubules, and which weakly expressed in proximal tubules. IL-lOmRNA were strongly expressed not only in above-mentioned cells, but also in mesangial cells, endothelial cells and the infiltrating inflammatory cells in renal glomerular diseases,especially in proliferative glomerulonephritis. ㏕he expression of IL-lOmRNA and protein were upregulated in a dose-dependent and time-dependent fashion in HMC stimulated by inflammatory cytokines. Both anti-IL-lp mAb and anti-TNF-cc mAb downregulated the yield of IL-10.Conclusion IL-10 act as a regulator in glomerulonephritis, and the elevation of IL-10 in glomerulonephritis should be due to renal resident cells which activated by proinflammatory cytokines.PART TWO: Effects of IL-10 on HMCObjective ㏕o observe whether IL-10 can inhibit HMC proliferation. ?To investigate the effects of IL-10 on the cell cycle proteins of P27and cyclin D,, and to clarify the mechanism of IL-10 regulating proliferation of HMC. d)To examine the effect of exogenuos/endogenous IL-10 on the proinflammatory cytokines, chemokines, and pro-fibrogentic factors expression in human mesangial cells(HMC). (4)To determine the effects of IL-10 on production of extracellular matrix(ECM) proteins such as type I collagen and fibronection. ㏕o evaluate the roles of IL-10 in the regulation of lipid mediators such as cPLA2 and COX-2 in HMC.Methods ㏕he incorporation of MTT and "H-TdR were used as the measurement of HMC proliferation, and flow cytometry were used to analyze the cell cycle or cell cycle proteins of P27and cyclin D, in HMC. (2) The semi-quantitative analysis of TNF-a, IL-la, IL-lp, MCP-1, IL-8 and TGF-P1 mRNA was detected by ribonuclease protection assay(RPA). (3) The production of type I and fibronection was m...