Effect Of Mast Cell Tryptase On Cultured Human Renal Fibroblasts

Objective: Mast cell tryptase has been implicated in regulation of skin fibroblast, lung fibroblast and hepatic stellate cell proliferation and collagen synthesis. In this study, we observed the effect of mast cell tryptase on cultured human renal interstitial fibroblasts, so as to further investigate the effect of tryptase on renal fibrosis.Methods: Normal human renal interstitial fibroblasts were isolated from nephrectomized microscopically normal renal tissue of malignant kidney tumor and cultured in vitro. The cultured fibroblasts were identified by cell shape, immunocytochemistry and electron microscope, and divided into following groups.different concentration tryptase with equal heparin(wt/wt):(1) 0ng/ml tryptase with 0ng/ml heparin(2) 10ng/ml tryptase with 10ng/ml heparin(3) 100ng/ml tryptase with 100ng/ml heparin(4) 500ng/ml tryptase with 500ng/ml heparindifferent concentration tryptase without heparin:(1) 0ng/ml tryptase(2) 10ng/ml tryptase(3) 100ng/ml tryptase(4) 500ng/ml tryptasedifferent concentration heparin without tryptase:(1) 0ng/ml heparin(2) 10ng/ml heparin(3) 100ng/ml heparin(4) 500ng/ml heparindifferent concentration tryptase with equal heparin(wt/wt) plus 200umol/L benzamidine hydrochloride hydrate (BZ):(1) 10ng/ml tryptase with 10ng/ml heparin plus 200umol/L BZ(2) 100ng/ml tryptase with 100ng/ml heparin plus 200umol/L BZ (3) 500ng/ml tryptase with 500ng/ml heparin plus 200umol/L BZ5. different concentration tryptase with equal heparin(wt/wt) plus 10ug/ml transforming growth factor-β (TGF-β) antibody(Ab):(1) 10ng/ml tryptase with 10ng/ml heparin a plus 10ug/ml TGF-βantibody(2) 100ng/ml tryptase with 100ng/ml heparin plus 10ug/ml TGF-βantibody(3) 500ng/ml tryptase with 500ng/ml heparin plus 10ug/ml TGF-βantibodyThe cells of every group, after cultured for 48 hours, were examined for the cell proliferation, the mRNA expression of collagen I, protease activated receptor-2(PAR2), monocyte chemoattractant protein-1 (MCP1), and the protein expression of alpha-smooth muscle actin(α-SMA). Proliferation of cultured renal fibroblasts was measured by means of Methylthio tetrazole(MTT) method. The expression of mRNAs by means of semi-quantitive reverse transcriptase polymerase chain reaction (RT- PCR) method. The protein expression of α-SMA by immunocytochemical method.Results: 1. Immunoctyochemistry assay showed that primary cultured renal cells had positive staining of vimentin and alpha-smooth muscle actin, but negative of keratin and CD34. The cultured cells showed fusiform shape under microscope. Under electron microscope, a lot of microfilament, Golgi complex and rough endoplasmic reticulum were observed in cultured cells. These data proved that the cultured cells were human renal fibroblasts.2. Tryptase stimulated fibroblasts proliferation in dose-depended manner at the range of 10ng/ml to 500ng/ml. The proliferation of cultured cell stimulated with 500ng/ml tryptase was 3.07-fold higher than that of control group. The proliferation of cultured cell treated with 200umol/L benzamidine hydrochloride hydrate was suppressed about 81% compared with that of tryptase stimulated cells.3. The mRNA of human PAR2, collagen I, and MCP1(respectively 324bp, 332bp, and 257bp) were amplified by RT-PCR. Theresult showed that tryptase plus heparin stimulated cultured human renal fibroblasts expressing PAR2, collagen I and MCP1 in dose-depended manner at the tryptase concentration range from 10ng/ml to500ng/ml. The cultured cells stimulated by the tryptase plus heparin had significant higher mRNA expression of the PAR2(3.14 fold), collagen I(3.71 fold) and MCP1(4.98 fold) than that of control group. The cultured cells treated with 200 umol/L benzamidine hydrochloride hydrate had less mRNA expression of the PAR2, collagen I and MCP1 for about 53～84% compared with that of tryptase stimulated cells.Conclusion:1. Human renal fibroblasts were successfully cultured in our experiment.2. Tryptase could stimulate human renal fibroblasts p...