| Fibrosis is the common consequence of different diseases characterized by chronic inflammatory damage occurred in liver, kidney, lung and skin etc. Stimulated by inflammation, quiescent or precursor ECM-producing cells (fibroblasts, HSC etc)are activated to proliferate. The activated cells produce excessive ECM with collagen as the main component , leading to fibrogenesis and further to fibrosis. Materials indicate that many cytokines are involved in the pathological process of fibrosis. PDGF, IGF, insulin and TGF a simulate proliferation of ECM- producing cells . TGF- P, IGF, insulin enhance ECM synthesis with collagen as the main component , whereas TNF a , IFN a , IFN ~' inhibit the collagen secretion by ECM-producing cellsType I collagen(COL1) ,a major constituent of extracellular matrix produced when fribrogenesis take place, has attracted much attention in its gene expression and regulation. Type I collagen is a heterometric protein composed of two a 1(I) chains and one a 2 (I) chain that are encoded by two different genes. Since the promoters of a 1(I) chains and a 2(I) chain successfully cloned, much progress has been made in regulation of COL1 promoter, but the mechanism has not been clearly defined . Especially, the complicated relations among these cytokines also remained unclear in the aspect .The effect of drugs used in fibrosis therapy on the promoter activity has been seldom reported.On the basis of the reasons mentioned above, the aims of this study are (1) to construct pCOLH plasmids of human COL1A1 promoter, to evaluate the activity of each construct and to analyze the regions of regulation sequences located;(2)to investigate the influence of PDGF-BB,IGF- 1, matrine and oxy-matrine on the growth of ECM-producing cells, and to probe its mechanisms; (3) to study the regulation of promoter activity of COL1A1 by cytokines and some drugs including t-RA, PTX, matrine and oxy-matrine.6Construction of pCOLH plasmids and analysis of their activityUsing the plasmi containing human COL1A1 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 gen pCAT3-Enhaneer (vector) respectively. The six deletion constructs obtained were named as pCOLHO. 1, pCOLHO. 27, pCOLHO. 5, pCOLHO. 9, pCOLH1. 5 and pCOLH2.5.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~---+42bp, -l448--+42bp and -2483'---+42bp respectively in the upstream to the human COL1A1 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 activity of pCOLH2. 5, the relative CAT activities of other constructs 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 (pCOLHO. 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 COL1A1 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 at -496-----268bp. Analysed by DNAssist 1. 0 software, it is indicated that the region of -2484'~+42bp contain binding sites for nuclear factors .The analysis suggest that a NF-1 binding site might be at -l0lbp and an AP-l binding site might be at -l0lbp, while at -l23bp might exist Sp-l binding site. These binding sites might be responsible for the high activity in -268----+42bp. In the 2. 5kb sequence from -2483 to +42 , there were five of Spl (-123, -1615, -1628, -2170, -2176), one of NF- K B (-1571), two of c-myc (-1118, -2406),two of AP-1(-l03, -1985) binding sites. Among them,...
|
PDF Full Text Request