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The Effect of Recombinant Adenovirus Vector of Rat Proteoglycan Ⅱ on the Expression of Extracellular Matrix Molecules in Mesangial Cells

Author: ZhuFeng
Tutor: LiHong
School: Zhejiang University
Course: Internal Medicine
Keywords: Diabetic Nephropathy TGF- β Decorin extracellular matrix Recombinant Adenovirus
CLC: R587.2
Type: Master's thesis
Year: 2005
Downloads: 31
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Abstract

Background and ObjectiveDiabetic nephropathy (DN) is characterized by renal hypertrophy, thickening of basement membranes and accumulation of extracellular matrix (ECU) in the glomerular mesangium and the interstitium. Several extracellular matrix components, such as fibronectin, type IV collagen, tenascin and laminin, are associated with the feature of this disease.There are increasing evidences indicating that mesangial autocrine activation of transforming growth factor-β (TGF-β) seems to be crucial in the development of human and experiment diabetic glomerulosclersis. Therapeutic approaches to down-regulate TGF-β level under high glucose conditions provide one strategy to inhibit the progression of diabetic nephropathy.Proteoglycan II(Decorin, DCN) may be involved in the regulation of fundamental biologic functions, such as matrix assembly, modulatingfibronectin- and thrombospondin-mediated cell adhesions. It is more important that DCN can regulate TGF-β activity, which is a key factor in tissue fibrosis and in promoting cell proliferation . There is also evidence that the core protein of decorin can bind to TGF- β and neutralize its activity.To investigate the effect of inceased decorin synthesis on the expression of TGF-β and the ECM components fibronectin, type IV collagen, tenascin and laminin, the Recombinant Adenovirus Vector of Rat Proteoglycan II Gene will transfer to renal mesangial cells.Experiment Design and MethodsThe rat mesangial cells (RMCs) were obtained from Institute of Cell Biology, Wuhan .The culture medium was Dulbecco’s Modified Eagle’ s Medium (DMEM) supplemented with 5% fetal calf serum (FCS). About 5 X10~5 RMCs were seeded in each well of 6-well microtiter plate and were rested for 24 hours in DMEM with a D-glucoe concentration of 450mg/dl. All the cells were divided into four groups: group 1, the recombinant adenovirus vector of DCN gene (AD-DCN)were added to each cell culture, multiplicity of infection was 50. 2 hours later, after washing twice with 5%FCS-DMEM with a D-glucoe concentration of 450mg/dl, cells were incubated in the seem growth medium(2ml/well) for 24 hours to seven days. Meanwhile, cells of group 2 were treated with the recombinant adenovirus vector of LacZ gene (AD-LacZ). cells of group 3 were treated with neutralizing rabbit anti-porcine TGF-β antibody (30 μ g/ml) as the positive control. Cells of group 4 were cultured in 5%FCS-DMEM with a D-glucoe concentration of 450 mg/dl. Another group, cells were cultured in 5%FCS-DMEM with a D-glucoe concentration of 100 mg/dl all the time as the normal control. From 24 hours to 7 days , every group of cells were harvested for further RNA extraction and total RNA was isolatedand reverse- transcripted into cDNA. Equal amounts of cDNA were subjected to PCR amplification, the housekeeping gene GAPDH was co-amplified. PCR products were analyzed by 2% agarose gel electrophoresis with ethidium bromide staining. The final results were measured with ISIOOO digital image system in image density view(IDV). The mRNA levels of target genes were expressed as the radio of IDV target mRNA and IDV GAPDH mRNA. The result were expressed as means ± standard deviation and statistical analysis was carried by SPSS10.0 for Windows.Results:1. The levels of mRNA for decorin, TGF-β, collagen type IV, tenascin, fibronectin and laminin were statistically significant higher(p<0. 05) but the ratio of decorin mRNA to TGF-β , mRNA was statistically significant lower(p<0.05) in high glucose(450mg/dl) than in normal glucose conditions(100mg/dl).2. Compared with the cells in high glucose conditions, the decorin mRNA level was increased by 1. 8~2. 2-fold after the recombinant adenovirus vector of DCN gene was transferred to RMCs. The transfection of AD-DCN increased decorin synthesis and down-regulated TGF-β transcription. TGF-β mRNA level was significantly decreased (p<0. 05) from 48 hours after DCN gene was transferred. There was a statistically significant decrease of the levels of mRNA for collagen type IV, tenascin, fibronectin and laminin compa

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