含启动子序列的单链寡核苷酸拮抗胰岛素对人α1（Ⅰ）型胶原启动子的激活
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摘要
目的:1.探讨含启动子序列的单链寡脱氧核糖核苷酸(ssPTODN)抑制胰岛素对人 α1(I)型胶原(COL1A1)基因启动子的激活。2.进一步确定胰岛素在人成 纤维细胞COL1A1 基因上的反应元件
方法:1. 将含有氯霉素乙酰基转移酶(CAT) 基因与人COL1A1 基因-2483～ +42bp 片段的重组质粒(pCOLH12.5)稳定转染至人胚肺成纤维细胞 WI-38 中, 建立稳定转染的WI-38 细胞株,于该细胞株中加入不同剂量 的胰岛素,测CAT 值;2.合成全硫代的不同序列ssPTODN,其中 ssPTODN1 的序列为COL1A1 基因-129 至-105 间的25 个碱基。瞬时转染 不同剂量的ssPTODN1 于克隆的细胞株中,静息后,分别加入2.5μmol/ml 的胰岛素,测CAT 值。
结果:1.成功建立了整合有pCOLH12.5 的WI-38 细胞株PN1。2.胰岛素浓度在 0、0.25、2.5、10μmol/ml 时的CAT 值(pg/ml)分别是920±94、1021 ±98、1595±101、1711±117。2.5μmol/ml 及10μmol/ml 的胰岛素显著 增加了COL1A1 基因启动子活性(P<0.01),二者的作用无显著性差异 (P>0.05)。3.胰岛素剂量为2.5μmol/ml,ssPTODN1 浓度分别为0、 40nM、80nM 、120nM、160nM时,细胞株PN1的各组校正后CAT值(pg/ml) 相应为:1593±87、1535±49、1370±54、1147±93、899±85;单独胰 岛素作用组的CAT 校正值为1601±23(pg/ml);ssPTODN1 浓度为120nM 和160nM 时,受胰岛素刺激所增加的CAT 值得到显著抑制(P<0.01), 但160nM 剂量时所抑制的水平低于启动子的基础CAT 值。
结论: 1. ssPTODN1 能够抑制胰岛素对人成纤维细胞COL1A1 基因启动子的激 活。2.胰岛素在人成纤维细胞COL1A1 基因上的反应元件被定位于该 基因启动子近端-129 至-105 区域内。
Objective:(1)To investigate the effect of sense single-strandphosphorothioate oligodeoxynucleotide(ssPTODN) conciding with promotergenes on promoter activity of human COL1A1 gene induced by insulin.(2)Tolocate the response element of insulin on human COL1A1 gene moreprecisely.
Methods: (1) The construct of pCOLH_12.5 containing –2483 to +42 bp ofthe promoter and CAT gene as a reporter was stably transfected into WI-38cells.The cell clone integrated with pCOLH_12.5 were treated with insulinof various dosages.(2) Four different ssPTODNs were synthesized. Variousdosage’s ssPTODN1 coinciding with position -129 to -105 of COL1A1 genewere transitently transfected into the cell clone, consequently treatedwith insulin of 2.5μmol/ml.
Results: (1) the WI-38 cell clone named PN1 containing pCOLH12.5 wasSuccessfully constructed. (2) At 0、0.25、2.5、10μmol/ml insulin,the CATvalues(pg/ml)were 920±94、1021±98、1595±101(P<0.01) and 1711±117a(P<0.01) respectively. But it showed no significance between 2.5 and10μmol/ml insulin (P>0.05).(3) At 0、40、80 、120、160nM ssPTODN1, theCAT values(pg/ml)were 1593±87、1535±49、1370±54、1147±93(P<0.01)and 899±85(P<0.01) respectively in the presence of 2.5μmol/ml insulin.At 120nM ssPTODN1, insulin-induced increase in CAT value was abrogated
to basal level.Conclusions: (1)The ssPTODN coinciding with position -129 to -105 ofCOL1A1 gene can inhibit the promoter activity of human α1(I) collagen geneinduced by insulin.(2)The location of insulin response element on humanCOL1A1 gene is suggested between -129 and -105 in the promoter.
方法:1. 将含有氯霉素乙酰基转移酶(CAT) 基因与人COL1A1 基因-2483～ +42bp 片段的重组质粒(pCOLH12.5)稳定转染至人胚肺成纤维细胞 WI-38 中, 建立稳定转染的WI-38 细胞株,于该细胞株中加入不同剂量 的胰岛素,测CAT 值;2.合成全硫代的不同序列ssPTODN,其中 ssPTODN1 的序列为COL1A1 基因-129 至-105 间的25 个碱基。瞬时转染 不同剂量的ssPTODN1 于克隆的细胞株中,静息后,分别加入2.5μmol/ml 的胰岛素,测CAT 值。
结果:1.成功建立了整合有pCOLH12.5 的WI-38 细胞株PN1。2.胰岛素浓度在 0、0.25、2.5、10μmol/ml 时的CAT 值(pg/ml)分别是920±94、1021 ±98、1595±101、1711±117。2.5μmol/ml 及10μmol/ml 的胰岛素显著 增加了COL1A1 基因启动子活性(P<0.01),二者的作用无显著性差异 (P>0.05)。3.胰岛素剂量为2.5μmol/ml,ssPTODN1 浓度分别为0、 40nM、80nM 、120nM、160nM时,细胞株PN1的各组校正后CAT值(pg/ml) 相应为:1593±87、1535±49、1370±54、1147±93、899±85;单独胰 岛素作用组的CAT 校正值为1601±23(pg/ml);ssPTODN1 浓度为120nM 和160nM 时,受胰岛素刺激所增加的CAT 值得到显著抑制(P<0.01), 但160nM 剂量时所抑制的水平低于启动子的基础CAT 值。
结论: 1. ssPTODN1 能够抑制胰岛素对人成纤维细胞COL1A1 基因启动子的激 活。2.胰岛素在人成纤维细胞COL1A1 基因上的反应元件被定位于该 基因启动子近端-129 至-105 区域内。
Objective:(1)To investigate the effect of sense single-strandphosphorothioate oligodeoxynucleotide(ssPTODN) conciding with promotergenes on promoter activity of human COL1A1 gene induced by insulin.(2)Tolocate the response element of insulin on human COL1A1 gene moreprecisely.
Methods: (1) The construct of pCOLH_12.5 containing –2483 to +42 bp ofthe promoter and CAT gene as a reporter was stably transfected into WI-38cells.The cell clone integrated with pCOLH_12.5 were treated with insulinof various dosages.(2) Four different ssPTODNs were synthesized. Variousdosage’s ssPTODN1 coinciding with position -129 to -105 of COL1A1 genewere transitently transfected into the cell clone, consequently treatedwith insulin of 2.5μmol/ml.
Results: (1) the WI-38 cell clone named PN1 containing pCOLH12.5 wasSuccessfully constructed. (2) At 0、0.25、2.5、10μmol/ml insulin,the CATvalues(pg/ml)were 920±94、1021±98、1595±101(P<0.01) and 1711±117a(P<0.01) respectively. But it showed no significance between 2.5 and10μmol/ml insulin (P>0.05).(3) At 0、40、80 、120、160nM ssPTODN1, theCAT values(pg/ml)were 1593±87、1535±49、1370±54、1147±93(P<0.01)and 899±85(P<0.01) respectively in the presence of 2.5μmol/ml insulin.At 120nM ssPTODN1, insulin-induced increase in CAT value was abrogated
to basal level.Conclusions: (1)The ssPTODN coinciding with position -129 to -105 ofCOL1A1 gene can inhibit the promoter activity of human α1(I) collagen geneinduced by insulin.(2)The location of insulin response element on humanCOL1A1 gene is suggested between -129 and -105 in the promoter.
引文
1 Schuppan D,Herbst H,milanni,S. Matrix synthesis and molecular net-works in epatic fibrosis.Extracellular matrix,chemistry, biology with emphasis on the liver. New York:Marcel dekker, 1993,201.
2 Giuliano Ramadori,Thomas Knittel,Bernhard Saile.Fibrosis and Altered Matrix Synthesis. Digestion,1998,59(4):372-375.
3 Myllyharju J,Kivirikko KI. Collagens and collagen-related diseases.Ann Med, 2001,33(1):7-21.
4 伍严安. I 型胶原基因启动子研究进展. 国外医学分子生物学分册,2001,23 (6):328-331.
5 Rossert J,Terraz C,Dupont S. Regulation of type ? collagen genes expression. Nephrol Dial Transplant, 2000,15[Suppl 6]:66-68.
6 Peterkofsky B, GosiewskaA, Singh K, et al. Species differences in cis-elements of the proalpha1(I) procollagen promoter and their binding proteins.J Cell Biochem,1999, 73(3):408-422.
7 Vergeer WP, Sogo JM, Pretorius PJ, et al. Interaction ofAp1,Ap2, and Sp1 with the regulatory regions of the human pro-alpha1(I) collagen gene. Arch Biochem Biophys, 2000,377(1):69-79.
8 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):1743-1751.
9 Bénédicte Fournier, Annie Gravel, David C. et al. Strength and Regulation of the Different Promoters for Chromosomal β-Lactamases of Klebsiella oxytoca. Antimicrob Agents Chemother,1999 April,43(4): 850–855.
10 Cicchillitti L,Jimenez SA,Sala A, et al. B-Myb acts as a repressor of human COL1A1 collagen gene expression by interacting with SP1 and CBF factors in scleroderma fibroblasts. Biochem J,2004,378(Pt 2):609-616.
11 JorgeAlemany,Teresa Borras,Flora de Pablo.Transcriptional stimulation of the б1-crystallin gene by insulin-like growth factorⅠand insulin requires DNAcis elements in chiken.Cell Biology,1990, 87:3353-3357.
12 Morishita R,Higaki J,Tomita N, et al.Application of transcription factor “decoy”strategy as a means of gene therapy and study of gene expression in cardiovascular diseases.Circ Res, 1998,82:1023-1028.
13 伍严安,高春芳,王皓,等.胰岛素样生长因子1 和胰岛素对人α1(Ⅰ)前 胶原启动子活性的影响.中国免疫学杂志,2001,17(11):601-603.
14 pCAT3 Reporter Vectors. TECHNICAL MANUAL,Promega,4/01:6.
15 PCl-neo Mammalian Expression Vector. ECHNICAL MANUAL,Promega, No.215:5.
16 J Sambrook E .Molecular Cloning a laboratory manual 3rd, Cold Spring Harbor Laboratory Press 1998.
17 J.萨姆布鲁克D.W.拉塞尔著黄培堂等译. 分子克隆试验指南(第三 版)北京:科学出版社,2003:1272.
18 曹亚. 实用分子生物学操作指南北京:人民卫生出版社,2003:185-193.
19 J.萨姆布鲁克D.W.拉塞尔著黄培堂等译. 分子克隆试验指南(第三 版)北京:科学出版社,2003:1322-1413.
20 高春芳,陆伦根. 纤维化疾病的基础和临床上海:上海科学技术出版社, 2004:222-225
21 Tokudome T, Horio T, Yoshihara F, et al.Direct effects of high glucose and insulin on protein synthesis in cultured cardiac myocytes and DNA and collagen synthesis in cardiac fibroblasts. Metabolism, 2004 Jun,53(6):710-715.
22 Madibally SV, Solomon V, Mitchell RN, et al.Influence of insulin therapy on burn wound healing in rats. J Surg Res, 2003, 109(2):92-100.
23 Ruiz-TorresA, Melon J, Munoz FJ.Insulin stimulates collagen synthesis in vascular smooth muscle cells from elderly patients. Gerontology, 1998, 44(3):144-148.
24 Marandi S, De Keyser N, SaliezA, et al. Insulin signal transduction in rat small intestine: role of MAP kinases in expression of mucosal hydrolases.Am J Physiol
Gastrointest Liver Physiol, 2001,Feb;280(2):G229-240.
25 Anderson PW, Zhang XY, Tian J, et al.Insulin and angiotensin II are additive in stimulating TGF-beta 1 and matrix mRNAs in mesangial cells. Kidney Int, 1996,Sep;50(3):745-753.
26 Krupsky M, FineA, Kuang PP, et al.Regulation of type I collagen production by insulin and transforming growth factor-beta in human lung fibroblasts.Connect Tissue Res, 1996,34(1):53-62.
27 Skorman SE.Use of injectable collagen to treat chronic diabetic foot ulcers. J Foot Surg,1987 Nov-Dec;26(6):511-515.
28 Kronenwett R,Stridl U,Kirsch M, et al. Oligodeoxyribonucleotide uptake in primary human hematopoetic cells in enhanced by cationic lipids and depends on the hematopoietic cell rubact.J Blood,1998,91,852.
29 Cutroneo KR, Chiu JF.Asense phosphorothioate oligodeoxynucleotide containing the transforming growth factor beta regulatory element acts as a novel local nonsteroidal antifibrotic drug. Wound Repair Regen,2000,8(5):399-404.
30 Kenneth R. Cutroneo1 and Kenneth M, et al. How do Glucocorticoids Compare to Oligo Decoys as Inhibitors of Collagen Synthesis and Potential Toxicity of These Therapeutics? Journal of Cellular Biochemistry,2004 , 92:6–15.
31 Meisler NT,Chiu JF,Cutroneo KR. Promoter competitors as novel antifibrotics that inhibit transforming growth factor-beta induction of collagen and noncollagen protein synthesis in fibroblasts. J Cell Biochem, 1999,75(2):196-205.
32 Yamagiwa H, Yamada Y, Bolander ME,et al.Oligonucleotid decoy mimicking alphaA-crystallin-binding protein 1 binding site on mouse Col2a1 enhancer stimulates transcription from the adjacent Col2a1 promoter in chondrogenic ATDC5 cell. Mol Biotechnol, 2004, Sep;28(1):1-8.
33 高春芳,王皓,徐玲玲,等.人皮肤成纤维细胞中α1( Ⅰ) 前胶原基因转录调 控研究. 中国生物化学与分子生物学报, 2002,18(3):293～298.
34 Inagaki Y, Truter S. Ramirez F. Transforming Growth factorβstimulates α2 ( Ⅰ) collagen gene expression through a cis-acting element that contains an SP-1 binding site. J Biol Chem ,1994 ,269 :14828～14834.
2 Giuliano Ramadori,Thomas Knittel,Bernhard Saile.Fibrosis and Altered Matrix Synthesis. Digestion,1998,59(4):372-375.
3 Myllyharju J,Kivirikko KI. Collagens and collagen-related diseases.Ann Med, 2001,33(1):7-21.
4 伍严安. I 型胶原基因启动子研究进展. 国外医学分子生物学分册,2001,23 (6):328-331.
5 Rossert J,Terraz C,Dupont S. Regulation of type ? collagen genes expression. Nephrol Dial Transplant, 2000,15[Suppl 6]:66-68.
6 Peterkofsky B, GosiewskaA, Singh K, et al. Species differences in cis-elements of the proalpha1(I) procollagen promoter and their binding proteins.J Cell Biochem,1999, 73(3):408-422.
7 Vergeer WP, Sogo JM, Pretorius PJ, et al. Interaction ofAp1,Ap2, and Sp1 with the regulatory regions of the human pro-alpha1(I) collagen gene. Arch Biochem Biophys, 2000,377(1):69-79.
8 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):1743-1751.
9 Bénédicte Fournier, Annie Gravel, David C. et al. Strength and Regulation of the Different Promoters for Chromosomal β-Lactamases of Klebsiella oxytoca. Antimicrob Agents Chemother,1999 April,43(4): 850–855.
10 Cicchillitti L,Jimenez SA,Sala A, et al. B-Myb acts as a repressor of human COL1A1 collagen gene expression by interacting with SP1 and CBF factors in scleroderma fibroblasts. Biochem J,2004,378(Pt 2):609-616.
11 JorgeAlemany,Teresa Borras,Flora de Pablo.Transcriptional stimulation of the б1-crystallin gene by insulin-like growth factorⅠand insulin requires DNAcis elements in chiken.Cell Biology,1990, 87:3353-3357.
12 Morishita R,Higaki J,Tomita N, et al.Application of transcription factor “decoy”strategy as a means of gene therapy and study of gene expression in cardiovascular diseases.Circ Res, 1998,82:1023-1028.
13 伍严安,高春芳,王皓,等.胰岛素样生长因子1 和胰岛素对人α1(Ⅰ)前 胶原启动子活性的影响.中国免疫学杂志,2001,17(11):601-603.
14 pCAT3 Reporter Vectors. TECHNICAL MANUAL,Promega,4/01:6.
15 PCl-neo Mammalian Expression Vector. ECHNICAL MANUAL,Promega, No.215:5.
16 J Sambrook E .Molecular Cloning a laboratory manual 3rd, Cold Spring Harbor Laboratory Press 1998.
17 J.萨姆布鲁克D.W.拉塞尔著黄培堂等译. 分子克隆试验指南(第三 版)北京:科学出版社,2003:1272.
18 曹亚. 实用分子生物学操作指南北京:人民卫生出版社,2003:185-193.
19 J.萨姆布鲁克D.W.拉塞尔著黄培堂等译. 分子克隆试验指南(第三 版)北京:科学出版社,2003:1322-1413.
20 高春芳,陆伦根. 纤维化疾病的基础和临床上海:上海科学技术出版社, 2004:222-225
21 Tokudome T, Horio T, Yoshihara F, et al.Direct effects of high glucose and insulin on protein synthesis in cultured cardiac myocytes and DNA and collagen synthesis in cardiac fibroblasts. Metabolism, 2004 Jun,53(6):710-715.
22 Madibally SV, Solomon V, Mitchell RN, et al.Influence of insulin therapy on burn wound healing in rats. J Surg Res, 2003, 109(2):92-100.
23 Ruiz-TorresA, Melon J, Munoz FJ.Insulin stimulates collagen synthesis in vascular smooth muscle cells from elderly patients. Gerontology, 1998, 44(3):144-148.
24 Marandi S, De Keyser N, SaliezA, et al. Insulin signal transduction in rat small intestine: role of MAP kinases in expression of mucosal hydrolases.Am J Physiol
Gastrointest Liver Physiol, 2001,Feb;280(2):G229-240.
25 Anderson PW, Zhang XY, Tian J, et al.Insulin and angiotensin II are additive in stimulating TGF-beta 1 and matrix mRNAs in mesangial cells. Kidney Int, 1996,Sep;50(3):745-753.
26 Krupsky M, FineA, Kuang PP, et al.Regulation of type I collagen production by insulin and transforming growth factor-beta in human lung fibroblasts.Connect Tissue Res, 1996,34(1):53-62.
27 Skorman SE.Use of injectable collagen to treat chronic diabetic foot ulcers. J Foot Surg,1987 Nov-Dec;26(6):511-515.
28 Kronenwett R,Stridl U,Kirsch M, et al. Oligodeoxyribonucleotide uptake in primary human hematopoetic cells in enhanced by cationic lipids and depends on the hematopoietic cell rubact.J Blood,1998,91,852.
29 Cutroneo KR, Chiu JF.Asense phosphorothioate oligodeoxynucleotide containing the transforming growth factor beta regulatory element acts as a novel local nonsteroidal antifibrotic drug. Wound Repair Regen,2000,8(5):399-404.
30 Kenneth R. Cutroneo1 and Kenneth M, et al. How do Glucocorticoids Compare to Oligo Decoys as Inhibitors of Collagen Synthesis and Potential Toxicity of These Therapeutics? Journal of Cellular Biochemistry,2004 , 92:6–15.
31 Meisler NT,Chiu JF,Cutroneo KR. Promoter competitors as novel antifibrotics that inhibit transforming growth factor-beta induction of collagen and noncollagen protein synthesis in fibroblasts. J Cell Biochem, 1999,75(2):196-205.
32 Yamagiwa H, Yamada Y, Bolander ME,et al.Oligonucleotid decoy mimicking alphaA-crystallin-binding protein 1 binding site on mouse Col2a1 enhancer stimulates transcription from the adjacent Col2a1 promoter in chondrogenic ATDC5 cell. Mol Biotechnol, 2004, Sep;28(1):1-8.
33 高春芳,王皓,徐玲玲,等.人皮肤成纤维细胞中α1( Ⅰ) 前胶原基因转录调 控研究. 中国生物化学与分子生物学报, 2002,18(3):293～298.
34 Inagaki Y, Truter S. Ramirez F. Transforming Growth factorβstimulates α2 ( Ⅰ) collagen gene expression through a cis-acting element that contains an SP-1 binding site. J Biol Chem ,1994 ,269 :14828～14834.