| Abnormal wound healing results in hypertrophic scars or keloids. Although their clinic symptoms are obvious, their mechanisms are unknown. It was reported that pathologic scars originate from unbalance of metabolism in ECM especial type I collagen, and that cytokines play an important role in development of fibrosis. However, many are not clear about the transcriptional mechanism of type I collagen gene, activation of target genes in transcription by cytokines, and interaction of DNA-binding proteins related to fibrosis with their cis-acting elements in pathologic scars.This study is to analyze activity of promoter from human a 1 ( I ) procollagen gene, to investigate the interaction of cytokines with target genes in transcriptional level, and to analyze the DNA-binding proteins in pathologic scars.oPart one:Activity analysis of promoter from human a 1(I)procollagen geneThis part arms to investigate the fragments with high promoter activity in human a 1(I) procollagen gene . Six chimeric genes were constructed in which various lengths of sequences of promoter from the human a 1(I)5procollagen gene were fused to chloramphenicol acetyltransferase (CAT) reporter gene.They were named as phCOLO.1, phCOLO.27, phCOLO.5, phCOLO.9, phCOL 1.5, phCOL2.5, which were correspondent to the sequences of -105-'+42bp, -268---+42bp, 496-+42bp, -829~+42bp,-1448---H-42bp and -2483~+42bp respectively in the upstream of promoter from the human COL1A1 gene. These recombinant plasmids were transfected transiently to normal human skin fibroblast with liposomal transfection method. The putative promoters activities were observed and compared by means of CAT measurement in transfected cells. Results: The higher CAT expression was observed in recombinant plasmids phCOLO.27 and phCOL2.5, the lower CAT expression was observed in phCOLO. 1 and phCOLO.5. This showed Fragments from -2483 to +42bp,-268 to +42bp have higher promoter activities in human a 1 ( I ) procollagen gene, fragments from-105 to +42bp, -4g~to +42bp have lower promoter activities The results also suggested that there might be positive and negative regulatory elements in the S'flank region of -2.5kb promoter of COL1AL gene. The positive regulatory elements might be located at -2483---1448bp, -1448----829bp, -829---496bp,-26&-- 1 OSbp, whereas the negative regulatory elements might be located at-49&--268bp. Meantime they are ideal target sequences for consensus DNA-binding protein research in fibrosis.oPart two: Dexamethasone and bFGF modulate theproliferation of human skin fibroblasts and the activity ofpromoter from human al(1) procollagen gene.1. Effects of dexamethasone and bFGF on the proliferation of human skin fibroblasts and hypertrophic scar fibroblastsThis study aims to investigate the effects of dexamethasone and bFGF on the proliferation of human skin fibroblasts and hypertrophic scar fibroblasts. Fibroblasts from human skin and hypertrophic scar were primary cultured and subcultured. The effects of dexamethasone and bFGF on the fibroblasts were determined by BrdU incorporation into DNA of fibroblasts. After 24h of treatment on the fibroblasts with 1 Os- 1 W4mol/L dexamethasone in DMEM containing 2% and 10% FCS, BrdU incorporation into DNA was determined. It showed that dexamethasone with 1W9'-104mo1/L had no effects (P>0.05)on both of the fibroblasts, and also suggested that inhibition of collagen synthesis by dexamethasone resulted from metabolism procession of collagen. After 24h of treatment on the fibroblasts with 0.25ng/ml~- 64ngIml bFGF in DMEM containing 2% or 10% FCS, BrdU incorporation into DNA was determined, which had difference in both of the fibroblasts(P<0.05). It showed that bFGF with 0.25ngIml- 64ngIml had effects on the proliferation of both fibroblasts, and also suggested that inhibition of collagen synthesis by bFGF resulted from inhibition of the flbroblasts proliferation partly.72.Effects of dexamethasone , bFGF and TGF beta-i on the promoter a...
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