| Type I collagen is a major constituent of extracellular matrix produced when fibrogenesis and its expression is controlled by transcription and post-transcriptional. Most studies before is concentrated in the post-transcriptional, but the evidence is not sufficient for making the mechanism of hyper-activation of type I collagen expression during fribrogenesis and sclerogenesis in the molecule level other than protein level clear. So this study tries to find the mechanisms and intervention role of cytokines in the fibrogenesis form the terminal of the signal transducers and activators of transcription by genetic recombination technique, gene transfection technique, report gene technique.1.Construction of pCOLH plasmids and analysis of their activity Using the plasmi containing human a 1 (I) collagen gene-5.3kb---+42bp as the template, six fragments with the same 3' end were obtained by PCR method. The six promoter fragments of 0.1kb, 0.27kb, 0.5kb, 0.9kb, 1.5kb and 2.5kb were ligated to the chloramphenicol acetyltransferase (CAT) reporter gene---pCAT3--Enhancer respectively. The six deletion constructs botained were named as pCOLHO. 1, pCOLH 0.5, pCOLH 0.9, pCOLH 1.5 and pCOLH 2.5. They were analyzed to be corrected by digested with restriction enzymes. They were correspondent to the sequences of -105～+42bp, -268～+42bp, -496～+42bp, -829～442bp,-1 448-+42bp and -24 83-I-42bp respectively in the upstream to the human a 1 (I) collagen gene. These constructs were transiently transfected into human normal skin fibroblasts. The CAT activity of the reporter gene was assessed by ELISA method. Compared with the activity8cooperated with other cytokines had effect of regulation or not still need to investigate.There is few research on the regulation of type I collagen transcript by AngII and AT1 receptor antagonist. This study showed that AngII promoted the activity of pCOLH1 .5 and pCOLH2.5. In the further study on Losartan, we find that Losartan not only inhibited the expression of recombinant CAT report gene, but also antagonize activation of pCOLH2.5 by TGF P -1. In conclusion, the mechanism that Losartan inhibited the synthesis of type I collagen and decreased the accumulation of glomerulus is that it may 1) directly or by way of antagonizing the AT1 receptor inhibit proliferation of mensangial cell; 2) directly inhibit transcript activity of ci 1 (I) collagen gene promoter; 3) prevent increase of activity promoter transcript induced by TGF P -1.This study investigated the regulation of human a 1 (I) collagen promoter using a method of transient transfection and result showed that verapamil could not only inhibit the activity of pCOLH1 .5 and pCOLH2.5, but also antagonized activation of a 1 (I) collagen promoter induced by TGF P -1. So verapamil could decrease the mRNA level of collagen and the synthesis of collagen by directly regulation of type I collagen gene promoter.Several studies show that there is glucocorticoid regulatory elements (GRE) in the type I collagen promoter of human, mouse and rat. This experiment showed that dexamethasone didn't only inhibit the activity of CAT of rat mensangial cell transfected by pCOLH1 .5 and pCOLH2.5, but also inhibit the activity of CAT induced increase by TGF P -1. So dexamethasone inhibit the synthesis of type I collagen not only by decreasing proliferation of mensangial cell, but also by negatively regulation of collagen gene promoter in the level of transcript.13of pCOLH2.5, the relative CAT activities of other construts were calculated. The CAT values were 1.0 (pCOLH2.5),0.97 ± 0.04 (pCOLHO.27), 0.73±0. 11 (pCOLH1 .5), 0. 36±0. 09 (pCOLHO.9), 0.20±0.05 (pCOLH 0.5) and 0.10±0.02 (pCOLHO.1).The results suggest that there might be positive and negative regulatory elements in the S'flank region of -2.5kb upstream to a 1 (I) collagen gene. The positive regulatory element might be located at-2483---1448bp, -1448---829bp, -829---496bp, -268--- lOSbp, whereas the negative regulatory elements might be located...
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