细胞因子和苦参碱等药物对ECM产生细胞增殖和I型胶原启动子活性的调控
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摘要
纤维化是肝、肾、肺及皮肤等多种器官组织慢性炎症病变的结果。在炎症
刺激下，原先处于静息状态或前体状态的细胞外间质（ECM）产生细胞（成纤维
细胞、HSC／MFLC等）被激活并增殖，这些处于激活状态的细胞产生过量以胶原
为主的ECM导致纤维化的发生和发展。目前的研究资料指出，在纤维化的病理
过程中有多种细胞因子参与。其中 PDGF、IGF、Insulin、TNFα等刺激 ECM产生
细胞增殖。TGF-β、IGF、Insulin等刺激以胶原蛋白为主的ECM合成，而TNF
α、IFN α、IFN γ等则抑制ECM产生细胞分泌胶原蛋白。但这其中的机制以及
这些细胞因子之间存在的复杂的相互关系均不是很清楚。
Ⅰ型胶原是纤维化时产生的主要的胶原蛋白，其基因的表达与调控是长期以
来备受关注的问题。Ⅰ型胶原α1链（COL1A1）与 α2链（COL1A2）启动子克隆
成功以来，细胞因子对Ⅰ型胶原启动子活性调控的研究取得了一定的进展，但
对细胞因子在转录水平的相互关系报道甚少，与治疗纤维化有关的药物对这些
启动子的作用也鲜见报道。
基于以上原因，本文进行了以下工作：（1）构建人COL1A1启动子pCOLH质
粒重组体，测定各重组体的活性，探讨其调控序列所存在的部位。（2）观察PDGF
-BB、IGF-1以及中药制剂苦参碱与氧化苦参碱对ECM产生细胞增值的影响，
并探讨其机理。（3）调查细胞因子和苦参碱等药物对人COL1A1启动子活性的调
控。
中文摘要
第一部分PCOLH质粒重组体的构建及活性测定
以含人COLIAI基因6．3kb～巧 的质粒为模板，PCR扩增得到六个具有
相同3’端的长短分别为0．Ikb、0．27kb、0．skb、0．gkb、1．skb、2．skb的片段
分别作为启动子，与不含启动子但含报告基因的载体pCAT3－Enhancer分别组成
重组体PCOLHO．l、PCOLHO．27、PCOLHO 5、PCOLHO．9、PCOLHI．5、PCOLHZ．5。这
些重组体经酶切鉴定正确，分别相应于人COLIAI基因上游l05～巧2b卜七68～
－42hp、－496～＋42hp、－829～－42hp、－1448～＋42hp、－2犯3～＋42hp的序列。经
测序，插入片段与 GENEBANK（accession X98705）报道的序列完全一致。用 Fugene
转染法将各重组体瞬时转染至正常人皮肤成纤维细胞，用ELISA法测定细胞报
告基因CAT表达量。以PCOLHZ．5转染细胞后CAT表达量为1，计算出其余重组
体转染细胞后的“T相对表达量。结果表明转染PCOLHO．27、PCOLHZ．5重组体
的细胞具有高CAT表达量。按活性的强弱排列为PCOLHZ．5、PCOLHO．27、
PCOLHI．5、PCOLHO．9、PCOLHO．5、PCOLHO．1，它们 的CAT表达依次为：1．0、0．97
HO．04、0．73H0．11、0．36H0．09、0．20H0．05与 0．10H0．02。
该结果提示在人COLIAI基因上游5’侧翼区－2．skb序列中存在正性和负性调
控元件。正性调控元件可能存在子－2483～1448hP、－1448～829hP、－829～
496hP、－268一105hP，而负性调控元件则可能存在于－496～268hP。采用计算
机DNAssist．0软件模拟分析《484～叶 可能存在的核蛋白因子，提示在－
101hP有NF—1识别位点、－103hP有AP－1结合位点、－123hP有SP－1结合位点，
这些转录因子识别位点可能与七68～叫 的高启动活性有关。在七483～叫
这2．skb的序列中有5个SP－1（－123，－1615，－1628，－2170，－2176）、l个NF。B（－
1571）、2个c－mvc（lll－2406）、2个AP－1（－103，－1985）核蛋白结合位点，其
中，Sp－1、Ap－l、c－myc可能与该段序列的高启动活性有关。本研究结果与Jimenez
等的报告相似，但调控序列的定位则不完全相同，这可能与不同研究中所用的
研究手段及构建的重组体所含启动子片段不完全相同有关。
本研究除了对胶原基因上游序列进行启动活性分析、了解人COLIAI基因
转录调控序列外，其意义还在于为研究相应核转录因子提供可能。另外，应用
本研究中构建的6个重组体，还能对致／抗纤维化相关因子的作用机制进行转录
水平的探讨，进一步研究相应于这些高启动活性序列的DNA结合蛋白，在激活
态胶原产生细胞中发现纤?
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 cells
Type 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.
6
Construction of pCOLH plasmids and analysis of their activity
Using 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,
刺激下，原先处于静息状态或前体状态的细胞外间质（ECM）产生细胞（成纤维
细胞、HSC／MFLC等）被激活并增殖，这些处于激活状态的细胞产生过量以胶原
为主的ECM导致纤维化的发生和发展。目前的研究资料指出，在纤维化的病理
过程中有多种细胞因子参与。其中 PDGF、IGF、Insulin、TNFα等刺激 ECM产生
细胞增殖。TGF-β、IGF、Insulin等刺激以胶原蛋白为主的ECM合成，而TNF
α、IFN α、IFN γ等则抑制ECM产生细胞分泌胶原蛋白。但这其中的机制以及
这些细胞因子之间存在的复杂的相互关系均不是很清楚。
Ⅰ型胶原是纤维化时产生的主要的胶原蛋白，其基因的表达与调控是长期以
来备受关注的问题。Ⅰ型胶原α1链（COL1A1）与 α2链（COL1A2）启动子克隆
成功以来，细胞因子对Ⅰ型胶原启动子活性调控的研究取得了一定的进展，但
对细胞因子在转录水平的相互关系报道甚少，与治疗纤维化有关的药物对这些
启动子的作用也鲜见报道。
基于以上原因，本文进行了以下工作：（1）构建人COL1A1启动子pCOLH质
粒重组体，测定各重组体的活性，探讨其调控序列所存在的部位。（2）观察PDGF
-BB、IGF-1以及中药制剂苦参碱与氧化苦参碱对ECM产生细胞增值的影响，
并探讨其机理。（3）调查细胞因子和苦参碱等药物对人COL1A1启动子活性的调
控。
中文摘要
第一部分PCOLH质粒重组体的构建及活性测定
以含人COLIAI基因6．3kb～巧 的质粒为模板，PCR扩增得到六个具有
相同3’端的长短分别为0．Ikb、0．27kb、0．skb、0．gkb、1．skb、2．skb的片段
分别作为启动子，与不含启动子但含报告基因的载体pCAT3－Enhancer分别组成
重组体PCOLHO．l、PCOLHO．27、PCOLHO 5、PCOLHO．9、PCOLHI．5、PCOLHZ．5。这
些重组体经酶切鉴定正确，分别相应于人COLIAI基因上游l05～巧2b卜七68～
－42hp、－496～＋42hp、－829～－42hp、－1448～＋42hp、－2犯3～＋42hp的序列。经
测序，插入片段与 GENEBANK（accession X98705）报道的序列完全一致。用 Fugene
转染法将各重组体瞬时转染至正常人皮肤成纤维细胞，用ELISA法测定细胞报
告基因CAT表达量。以PCOLHZ．5转染细胞后CAT表达量为1，计算出其余重组
体转染细胞后的“T相对表达量。结果表明转染PCOLHO．27、PCOLHZ．5重组体
的细胞具有高CAT表达量。按活性的强弱排列为PCOLHZ．5、PCOLHO．27、
PCOLHI．5、PCOLHO．9、PCOLHO．5、PCOLHO．1，它们 的CAT表达依次为：1．0、0．97
HO．04、0．73H0．11、0．36H0．09、0．20H0．05与 0．10H0．02。
该结果提示在人COLIAI基因上游5’侧翼区－2．skb序列中存在正性和负性调
控元件。正性调控元件可能存在子－2483～1448hP、－1448～829hP、－829～
496hP、－268一105hP，而负性调控元件则可能存在于－496～268hP。采用计算
机DNAssist．0软件模拟分析《484～叶 可能存在的核蛋白因子，提示在－
101hP有NF—1识别位点、－103hP有AP－1结合位点、－123hP有SP－1结合位点，
这些转录因子识别位点可能与七68～叫 的高启动活性有关。在七483～叫
这2．skb的序列中有5个SP－1（－123，－1615，－1628，－2170，－2176）、l个NF。B（－
1571）、2个c－mvc（lll－2406）、2个AP－1（－103，－1985）核蛋白结合位点，其
中，Sp－1、Ap－l、c－myc可能与该段序列的高启动活性有关。本研究结果与Jimenez
等的报告相似，但调控序列的定位则不完全相同，这可能与不同研究中所用的
研究手段及构建的重组体所含启动子片段不完全相同有关。
本研究除了对胶原基因上游序列进行启动活性分析、了解人COLIAI基因
转录调控序列外，其意义还在于为研究相应核转录因子提供可能。另外，应用
本研究中构建的6个重组体，还能对致／抗纤维化相关因子的作用机制进行转录
水平的探讨，进一步研究相应于这些高启动活性序列的DNA结合蛋白，在激活
态胶原产生细胞中发现纤?
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 cells
Type 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.
6
Construction of pCOLH plasmids and analysis of their activity
Using 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,
引文
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28. GoldsteinRH, Poliks CF, Pilch PF, et al Stimulation of collagen formation by insulin and insulin-like growth factor Ⅰ in cultures of human lung fibroblasts. Endocrinology, 1989;124:964-970
29．朱莉 第二十七章 血小板衍生生长因子 见：侯健，王东星，胥军民 人类细胞因子手册上海同济大学出版社，1996；316-323
30．黄沁主编 免疫药物学，第一版，上海，上海科学技术出版社，1986：168
31．陈伟忠，张俊平，许青．苦参碱对大鼠实验性肝纤维化的影响．第二军医大学学报，1996；17(5)：424—426
32．申锷．苦参碱注射液治疗黄疸型病毒性肝炎疗效观察。锦州医学院学报，1997；18(3)：41-42。
33．沈新生，王燕蓉．氧化苦参碱对CCl_4所致小鼠肝损伤修复作用的形态学研究．解剖科学进展，1997；3(3)：244—245
34．蔡雄，王国俊，瞿瑶，等。苦参素注射液治疗慢性乙型肝炎临床疗效分析．第二军医大学学报，1997；18(1)：47—49
35. Krotzsch Gomez FE, Furuzawa Carballeda J, Reyes Marguez R, et al. Cytokine expression is downregulated by collagen-polyvinylpyrrolidone in hypertrophic scars. J Invest Dermatol, 1998;111(5):828-34
36. ChungKY, Agarwal A, Uitto J. et al An Ap-1 binging sequence is essential for regulation of the human α2 (Ⅰ) collagen (COL1A2)promoter activity by transforming growth factor-β. J Biol Chem, 1996; 271(6):3272-3278
37. Hatamochi A, Mori K, Ueki H. Role of cytokines in controlling connective tissue gene expression. Arch Dermatol Res, 1994;287(1):115-21
38. Inagaki Y, Truter S, Tanaka S, et al. Overlapping pathways mediate the opposing actions of TNF alpha and TGF beta on alpha2(Ⅰ) collagen transcription. J Biol Chem, 1995;270:3353-3358
39. Kahari VM, Chen YQ, Su MW, et al Tumor necrosis factor alpha and interferon-gamma suppress the activation of human type Ⅰ collagen gene expresion by transforming growth factor-beta 1. evidence for two distinct mechanisms of inhibition at the transcriptional and posttranscriptional levels. J Clin Invest. 1990 86(5):1489-95
40. Higashi K, Kouba DJ, Song YJ.et al A proximal element within the human alpha 2(Ⅰ)collagen (COL1A2) promoter, distinct from the tumor necrosis factor-alpha response element, mediates transcriptional repression by interferon-gamma. Matrix biol 1998;16(8):447-56
41. Kouba DJ, Chung KY, Nishiyama T. et al. Nuclear factor-kappa B mediates TNF-alpha inhibitory effect on alpha 2(Ⅰ) collagen(COL1A2) gene transcripion in human dermal fibroblasts. J Immunol 1999;162(7):4226-34
42．高春芳印彤王皓等。细胞因子对小鼠α2(Ⅰ)前胶原基因启动子活性影响的研究中国免疫学杂志1999；15；296-298
43. Yuan W, Yufit T, Li L. et al Nagative modulation of alphal (Ⅰ) procollagen gene expression in human skin fibroblasts:transcriptional inhibition by inteferon-gamma. J Cell Physiol, 1999; 179(1):97-108.
44. Mallat A, Preaux AM, Blazejewski-S, et al. Interferon alfa and gamma inhibit proliferation and collagen synthesis of human Ito cells in culture. Hepatology, 1995;21(4):1003-10
45. Diaz A, Jimenez SA. Interferon-gamma regulates collagen and fibronectin gene expression by transcriptional and post-transcriptional mechanisms. Int J Biochem Cell Biol, 1997;29(1):251-60
46．钟东，姜泊，潘德寿．真核细胞总RNA的制备与分析见：姜泊，张亚历，周殿元主编 分子生物学常用实验方法 第1版，北京，人民军医出版社，1996：56-63
47．姜泊，周殿元．质粒的制备 见：姜泊，张亚历，周殿元主编 分子生物学常用实验方法 第1版，北京，人民军医出版社，1996：1-6
48. Ghahary A, shen YJ, Nedelec B, et al. Enhanced expression of mRNA for insulin-like growth factor-1 in post-burn hypertrophic scar tissue and its fibrogenic role by dermal fibroblasts. Mol Cell Biochem, 1995;148(1):25-32.
49. Scharf J-G, Knittel T, Dombrowski F, et al. Characterization of the IGF axis components in isolated rat hepatic stellate cells. Hepatology, 1998:27:1278-1284
50. Bird JL, Tyler JA. Tumor necrosis factor alpha, interferon gamma and dexamethasone regulate IGF-1-maintained collagen production in cultured human fibroblasts. J Endocrinol, 1998;147(1):167-76
51. Rose DW, Saltiel AR, Majumdar M, et al. Insulin receptor substrate 1 is required for insulin-mediated mitogenic signal transduction. Proc Natl Acad Sci USA, 1994;91:797-801.
52. Pierce GF, Mustoe-TA, Lingelbach J, et al. Transforming growth factor beta reverses the glucocorticoid-induced wound-healing deficit in rats:possible regulation in macrophages by platelet-derived growth factor. Proc Natl Acad Sci USA, 1989;86(7):2229-33
53. Tanaka H, Liang CT. Effect of platelet-derived growth factor on DNA synthesis and gene expression in bone marrow stromal cells derived from adult and old rats. J Cell Physiol, 1995;164(2):367-75.
54．王皓，高春芳，孔宪涛．抗纤复方及TGFβ对胶原基因启动子活性的影响 中国免疫学杂志 1999；15(12)：547-550．
55. Preaux A-M, Mallat A, Rosenboum J, et al. Pentoxifylline inhibits growth and collagen synthesis of cultured human hepatic myofibroblast-like cells Hepatology, 1997;26:315-322
56. Dacis BH, Vucic A. Effect of retinol on Ito cell proliferation in virto. Hepatology, 1988 8(4):788-793
57. Friedman SL. Cellular sources of collagen and regulation of collagen production in liver. Semin liver Dis, 1990;10(1):20-29
58．高春芳 王皓 孔宪涛 维生素A类对大鼠肝贮脂细胞3T3细胞增殖及前胶原
Ⅲ表达的影响新消化病杂志，1996；4(1) ：9-11
59． Krupsky M, Fine A, Berk JL et al Retinoic acid-induced inhibition of type I collagen gene expression by human lung fibroblasts Biochim Biophys Acta ,1994;1219(2) :335-41
60． Meisler NT, Parrelli J, Gendimenico GJ, et al. All-trans-retinoic acid inhibition of Pro alphal(I) collagen gene expression in fetal rat skin fibroblasts:identification of a retinoic acid response element in the Pro alphal(Ⅰ) collagen gene. J Invest Dermatol, 1997;108(4) :476-81
61． Tromp G, Kuivaniemi H, Stacey A, et al. Sreucture of a full-length cDNA clone for the prepro α1(Ⅰ)chain of human type I procollagen. Biochem J, 1988:253:919-922．
2. Roberts AB, SpornMB, Associan RK et al. Transforming growth factor type beta:rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc Natl Acad Sci USA, 1986;83,4167-4171
3. KahariVM, Heino J, Larjava H, et al. Alterations in scleroderma fibroblast surface glycoproteins assosiated with increased collagen synthesis. Acta Dermatovenereol, 1987;67:199-205
4. Uitto, J, Perejda AJ, Abergel RP et al. Altered steady state ratio of type Ⅰ/Ⅲ procollagen mRNA correlates with selectively increase type Ⅰ procollagen biosynthesis in cultureed keloid fibroblasts. Proc Natl Acad Sci USA, 1985;82:5935-5939
5. Peterson TC, IsbruckerRA. Fibroproliferation in liver disease:role of monocyte factors. Hepatology, 1992;15:191-7
6. Rockey DC, Chung JJ. Interferon γ inhibits lipocyte activation and extracellular matrix mRNA expression during experimental liver injury:implication for treatment of hepatic fibrosis. J Investig Med, 1994;42:660-70
7. Jimenez SA, Varga J, Olsen A, et al. Functional analysis of human α 1(Ⅰ) procollagen gene promoter. J Biol Chem, 1994;269(17):12684-91
8. Gao CF, Wang H, Huang C, et al. Study of activity of promoter from mouse α 2(Ⅰ) procollagen gene. Chin Med J, 1999;112(4):316-320
9．高春芳，王皓，黄超，等．小鼠α2(Ⅰ)型胶原基因启动子活性研究．第二军医大学学报，1999；20(10)：733-6
10．鄂征．基本培养技术．见：鄂征主编组织培养和分子细胞学技术．第2版．北京：北京出版社，1995：78～111
11. Anania FA, Womack L, Potter JJ, et al. Acetaldehyde enhances murine alpha2(Ⅰ) collagen promoter activity by Ca2+ -independent protein kinase Cactivation in cultured rat hepatic stellate cells. Alcol Clin Exp Res, 1999;23(2):279-84
12. Gao CF, Gressner G, Zoremba M, et al. Transforming growth factor β human fibtoblasts:regulation by cell spreading, platelet-detived growth factor and interactions with collagen fibers Matrix-Biol,1998;16(7):409-25.
expression in isolated and cultured rat hepatocytes. J Cell Physiol, 1996:167:394-405
13． Ratziu V, Lalazar A, Wong L, et al. Zf9, a Kruppel-like transcription factor up-regulated in vivo during early hepatic fibrosis. Proc Natl Acad Sci USA, 1998;95(16) :9500-5
14． Chen SJ, Artlett CM, Jimenez SA, et al. Modulation of human alphal(Ⅰ) procollagen gene activity by interaction with Spl and Sp3 transcription factors in vitro. Gene, 1998;215(1) :101-15
15． Rippe RA, Schrum LW, Stefanovic B, et al. NF-κB inhibits expression of the alphal(Ⅰ) collagen gene. DNA Cell Biol,1999:18(10) :751-61
16． Anania FA, Potter JJ, Rennie-Tankersley L, et al. Effect of acetaldehyde on nuclear protein binding to the nuclear factor I consensus sequence in the α2(Ⅰ)collagen promoter. Hepatology, 1995:21(6) :1640-8
17． Chung KY, Agarwal A, Uitto J, et al. An Ap-1 binding sequence is essential for regulation of the human α2(Ⅰ) collagen promoter activity by transforming growth factor-β. J Biol Chem, 1996:271(6) :3272-8
18． Gressner AM , Gao Chunfang. Pathology of the Liver Verh.Dtsch. Ges. Path, 1995:79,1-14．
19． Savage K, Siebert E, Swann D. The effect of platelet-derived growth factor on cell division and glycosaminoglycan synthesis by human skin and scar fibroblasts .J Invest Dermatol, 1987:89:93-99,
20． Pinzini M, Marra F, Caligiuri A, et al. Inhibition by pentoxifylline of extracellular signal-regulated kinase activation by platelet-derived growth factor in hepatic stellate cells. Br J Pharmaco, 1996:119:1117-1124．
21． Tan EM, Qin H, Kennedy SH, et al Platelet-detived growth factors-AA and BB regulate collagen and collagenase gene expression differentially in human fibroblasts. Biochem J, 1995;310(Pt 2) :585-8
22． Lepisto J, Peltonen J, Vaha-Kreula M, et al. Platelet-derived growth factor isoforms PDGF-AA,-AB and-BB exert specific effects on collagen gene expresion and mitotic activity of cultured human wound fibroblasts. Biochem-Biophys-Res-Commun, 1995:209(2) :393-9
23． Ivarsson M,Mcwhirter A, Borg TK. Type I collagen synthesis in cultured
24. Cohick WS, clemmons DR. The insulin-like growth factors. Annu Rev Physiol, 1993:55:131-153。
25. Telasky C, Tredget EE, Shen Q, et al. IFN-alpha2b suppresses the fibrogenic effects of insulin-like growth factor-1 in dermal fibroblasts. J Interferon Cytokine Res, 1998;18(8):571-7.
26. Svegliati Baroni G, Ridolfi F, Di-Sario A, et al Insulin and insulin-like growth factor-1 stimulate proliferation and type Ⅰ collagen accumulation by human hepatic stellate cells:differential effects on signal transduction pathways. Hepatology, 1999:29(6):1743-51。
27．鄂征．分子细胞学技术在研究中的应用．见：鄂征主编组织培养和分子细胞学技术．第2版．北京：北京出版社，1995：436-462
28. GoldsteinRH, Poliks CF, Pilch PF, et al Stimulation of collagen formation by insulin and insulin-like growth factor Ⅰ in cultures of human lung fibroblasts. Endocrinology, 1989;124:964-970
29．朱莉 第二十七章 血小板衍生生长因子 见：侯健，王东星，胥军民 人类细胞因子手册上海同济大学出版社，1996；316-323
30．黄沁主编 免疫药物学，第一版，上海，上海科学技术出版社，1986：168
31．陈伟忠，张俊平，许青．苦参碱对大鼠实验性肝纤维化的影响．第二军医大学学报，1996；17(5)：424—426
32．申锷．苦参碱注射液治疗黄疸型病毒性肝炎疗效观察。锦州医学院学报，1997；18(3)：41-42。
33．沈新生，王燕蓉．氧化苦参碱对CCl_4所致小鼠肝损伤修复作用的形态学研究．解剖科学进展，1997；3(3)：244—245
34．蔡雄，王国俊，瞿瑶，等。苦参素注射液治疗慢性乙型肝炎临床疗效分析．第二军医大学学报，1997；18(1)：47—49
35. Krotzsch Gomez FE, Furuzawa Carballeda J, Reyes Marguez R, et al. Cytokine expression is downregulated by collagen-polyvinylpyrrolidone in hypertrophic scars. J Invest Dermatol, 1998;111(5):828-34
36. ChungKY, Agarwal A, Uitto J. et al An Ap-1 binging sequence is essential for regulation of the human α2 (Ⅰ) collagen (COL1A2)promoter activity by transforming growth factor-β. J Biol Chem, 1996; 271(6):3272-3278
37. Hatamochi A, Mori K, Ueki H. Role of cytokines in controlling connective tissue gene expression. Arch Dermatol Res, 1994;287(1):115-21
38. Inagaki Y, Truter S, Tanaka S, et al. Overlapping pathways mediate the opposing actions of TNF alpha and TGF beta on alpha2(Ⅰ) collagen transcription. J Biol Chem, 1995;270:3353-3358
39. Kahari VM, Chen YQ, Su MW, et al Tumor necrosis factor alpha and interferon-gamma suppress the activation of human type Ⅰ collagen gene expresion by transforming growth factor-beta 1. evidence for two distinct mechanisms of inhibition at the transcriptional and posttranscriptional levels. J Clin Invest. 1990 86(5):1489-95
40. Higashi K, Kouba DJ, Song YJ.et al A proximal element within the human alpha 2(Ⅰ)collagen (COL1A2) promoter, distinct from the tumor necrosis factor-alpha response element, mediates transcriptional repression by interferon-gamma. Matrix biol 1998;16(8):447-56
41. Kouba DJ, Chung KY, Nishiyama T. et al. Nuclear factor-kappa B mediates TNF-alpha inhibitory effect on alpha 2(Ⅰ) collagen(COL1A2) gene transcripion in human dermal fibroblasts. J Immunol 1999;162(7):4226-34
42．高春芳印彤王皓等。细胞因子对小鼠α2(Ⅰ)前胶原基因启动子活性影响的研究中国免疫学杂志1999；15；296-298
43. Yuan W, Yufit T, Li L. et al Nagative modulation of alphal (Ⅰ) procollagen gene expression in human skin fibroblasts:transcriptional inhibition by inteferon-gamma. J Cell Physiol, 1999; 179(1):97-108.
44. Mallat A, Preaux AM, Blazejewski-S, et al. Interferon alfa and gamma inhibit proliferation and collagen synthesis of human Ito cells in culture. Hepatology, 1995;21(4):1003-10
45. Diaz A, Jimenez SA. Interferon-gamma regulates collagen and fibronectin gene expression by transcriptional and post-transcriptional mechanisms. Int J Biochem Cell Biol, 1997;29(1):251-60
46．钟东，姜泊，潘德寿．真核细胞总RNA的制备与分析见：姜泊，张亚历，周殿元主编 分子生物学常用实验方法 第1版，北京，人民军医出版社，1996：56-63
47．姜泊，周殿元．质粒的制备 见：姜泊，张亚历，周殿元主编 分子生物学常用实验方法 第1版，北京，人民军医出版社，1996：1-6
48. Ghahary A, shen YJ, Nedelec B, et al. Enhanced expression of mRNA for insulin-like growth factor-1 in post-burn hypertrophic scar tissue and its fibrogenic role by dermal fibroblasts. Mol Cell Biochem, 1995;148(1):25-32.
49. Scharf J-G, Knittel T, Dombrowski F, et al. Characterization of the IGF axis components in isolated rat hepatic stellate cells. Hepatology, 1998:27:1278-1284
50. Bird JL, Tyler JA. Tumor necrosis factor alpha, interferon gamma and dexamethasone regulate IGF-1-maintained collagen production in cultured human fibroblasts. J Endocrinol, 1998;147(1):167-76
51. Rose DW, Saltiel AR, Majumdar M, et al. Insulin receptor substrate 1 is required for insulin-mediated mitogenic signal transduction. Proc Natl Acad Sci USA, 1994;91:797-801.
52. Pierce GF, Mustoe-TA, Lingelbach J, et al. Transforming growth factor beta reverses the glucocorticoid-induced wound-healing deficit in rats:possible regulation in macrophages by platelet-derived growth factor. Proc Natl Acad Sci USA, 1989;86(7):2229-33
53. Tanaka H, Liang CT. Effect of platelet-derived growth factor on DNA synthesis and gene expression in bone marrow stromal cells derived from adult and old rats. J Cell Physiol, 1995;164(2):367-75.
54．王皓，高春芳，孔宪涛．抗纤复方及TGFβ对胶原基因启动子活性的影响 中国免疫学杂志 1999；15(12)：547-550．
55. Preaux A-M, Mallat A, Rosenboum J, et al. Pentoxifylline inhibits growth and collagen synthesis of cultured human hepatic myofibroblast-like cells Hepatology, 1997;26:315-322
56. Dacis BH, Vucic A. Effect of retinol on Ito cell proliferation in virto. Hepatology, 1988 8(4):788-793
57. Friedman SL. Cellular sources of collagen and regulation of collagen production in liver. Semin liver Dis, 1990;10(1):20-29
58．高春芳 王皓 孔宪涛 维生素A类对大鼠肝贮脂细胞3T3细胞增殖及前胶原
Ⅲ表达的影响新消化病杂志，1996；4(1) ：9-11
59． Krupsky M, Fine A, Berk JL et al Retinoic acid-induced inhibition of type I collagen gene expression by human lung fibroblasts Biochim Biophys Acta ,1994;1219(2) :335-41
60． Meisler NT, Parrelli J, Gendimenico GJ, et al. All-trans-retinoic acid inhibition of Pro alphal(I) collagen gene expression in fetal rat skin fibroblasts:identification of a retinoic acid response element in the Pro alphal(Ⅰ) collagen gene. J Invest Dermatol, 1997;108(4) :476-81
61． Tromp G, Kuivaniemi H, Stacey A, et al. Sreucture of a full-length cDNA clone for the prepro α1(Ⅰ)chain of human type I procollagen. Biochem J, 1988:253:919-922．