| IntroductionGlomerulosclerosis is the common pathological consequence of clinical glomerular diseases,which is caused by abnormal accumulation of extracellular matrix in glomeruli.The key role of transforming growth factor-β(TGF-β1) in the development of glomerulosclerosis has been established experimentally in vitro and in vivo. TGF-β1can stimulate the synthesis of ECM, down-regulate the protein expression of matrix metalloproteinases and up-regulate the protein level of matrix metalloproteinase inhibitors in mesangial cells (MC),.Decorin (DCN) is a leucine-rich proteoglycan secreted by glomerular mesangial cells and plays an important role in the regulation of cell growth, synthesization and composition of extracellular matrix (ECM). The over-expression of DCN can inhibit cell growth, promote the apoptosis of the mesangial cells, and down-regulate the protein levels of TGF-β1and collagen type IV in cultured mesangial cells in vitro, suggesting DCN might be a promising therapeutic target for treating mesangial proliferative glomerulonephritis and glomerulosclerosis.Polyethylenimine (PEI) is a cationic polymer which has shown potential for delivering genes in vitro and in vivo. PEI is a highly water soluble, positively charged, synthetic polymer, in which the repeat unit of PEI is two carbon atoms followed by a nitrogen atom.Under physiological conditions,approximately20%of the nitrogens are pronated. The positive charge of the PEI results in effective binding to the negatively charged plasmid DNA(pDNA),and condenses the DNA to form stable nanoparticle complexes. The major advantages of these complexes exhibit higher transfection efficiency and lower cytotoxicity.PEI is not immunogenicity and easy to be modified by others’polymers.In this study, Mixing cationic PEI with negatively charged pDCN results in the spontaneous electrostatic formation of stable nanoparticle complexes, and then delivering DCN gene to cultured mesangial cells in vitro, establishing stable transfection of DCN gene, investigating the overexpression of DCN inhibits the protein levels of TGF-β1and Col Ⅳ,which induces apoptosis and cell growth arrest in cultured mesangial cells in vitro. Then injecting the MC to glomerulus via the left renal artery of rat with ATS glomerulonephritis. We provided a promising strategy for glomerular diseases. Part I. Preparation of Polyethylenimine/decorin nanocomposites and its effect on the growth and phenotype of cultured rat MC with PEI/pDCN transfectionObjectives To mix PEI and pDNA to compose the PEI/pDNA nanoparticle complexes, delivery gene to cultured mesangial cells and investigate the effect of DCN on cell growth in cultured rat MC.Methods Mixing cationic PEI with negatively charged pDCN results in the spontaneous electrostatic formation of stable nanoparticle complexes, delivering DCN gene to cultured mesangial cells in vitro by different ratios of N atoms to P atoms and then choosing the optimum N:P ratio, Screening positive clones which stably express DCN protein. Western blot were used to analyze gene transcription and protein expression of DCN. Cell growth of MCs were assayed by flow cytometry. Expressions of Col IV and TGF-β1were analyzed using Western blot.Results The sizes of nanoparticles were measured by NicompTM380instrument and varied from146±70.91nm to151.7±72.43nm,Mixing PEI and pDNA at the ratio of N:P20results in smaller nanoparticle size and higher transfection efficiency. We chose D6clone identified by Western blot. The over-expression of DCN in MC increased protein level of DCN, arrested cells in Go/G1phase. In addition, the expression of TGF-β1and Col IV was significantly inhibited in MC/DCN.Conclusions To master the method to compose nanoparticle. Positive clone (D6) over-expressed DCN gene was established by cationic lipid-mediated transfection. In cultured rat MC, DCN could inhibit cell growth in vitro, DCN also effectively down-regulates protein expression of TGF-β1and Col IV.Part II The antagonistic effect of overpression of DCN on rat thy.l glomerulonephritis by delivering PEI/pDCN nanocomposites via renal arteryObjectives To inject D6into the rat kidney via the left renal artery and investigate the antagonistic effect of DCN in rat Thy.l glomerulonephritis Methods The rat Thy1.1glomerulonephritis model was made by tail intravenous injection with rabbit ATS serum. The positive clone was injected to rat kidneys via the left renal artery. The position of the exogenous cells in glomerulus was performed with immunohistochemistry. And then injecting D6to rat kidneys via the left renal artery. Immunohistochemistry with image analysis were performed to detect the expressions of DCN,TGF-β1,Col IV and PCNA in glomeruli.Results Successfully replicating rat Thy.l glomerulonephritis model via injecting rabbit ATS, Injecting MC/EGFP in rat left kidney via the renal artery.48h after injection,left kidneys were isolated from the rats, more65±2.13%glomerulis stained showed strong positive.the majority of the mesangial cells were located in glomerular capillary and the mesangium. Injecting D6in the same way, at3d, the protein level of DCN in injected kidneys was the highest. the expression of Col IV and PCNA markedly decreased compared to uninjected kidneys.Conclusions Successfully replicating rat Thy.l glomerulonephritis model via injecting rabbit ATS, The majority of the mesangial cells were located in glomerular capillary and the mesangium. D6injected in kidney might express DCN. the expression of Col IV and PCNA markedly decreased compared to uninjected kidneys. |
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