两种不同肝纤维化相关细胞因子shRNA表达载体的构建
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摘要
目的:结缔组织生长因子(CTGF)是新近发现的一种促肝纤维化的重要细胞因子,是转化生长因子β(transforming growth factorβ,TGF-β)信号传导途径的下游效应介质。CTGF可直接介导原代肝星状细胞(hepatic stellate cell,HSC)活化、增殖及迁移,还能促进活化HSC的合成及分泌细胞外基质(extracellular matrix,ECM)。金属蛋白酶组织抑制因子1(TIMP-1)也是TGF-β信号传导途径的下游效应介质,在肝纤维化发生时,它主要由激活的HSC表达。TIMP-1不仅抑制基质金属蛋白酶(matrix metalloproteinase, MMPs)活性阻止胶原降解,也通过抑制HSC凋亡、促进胶原分泌而在肝纤维化病情进展中发挥关键作用。所以,减少CTGF和TIMP-1的表达,可以减轻肝纤维化程度。本研究旨在构建以大鼠CTGF或TIMP-1基因为靶点的短发夹RNA(short hairpin RNA, shRNA)表达载体,为进一步探索肝纤维化的基因治疗提供有力工具。方法:根据前期筛选出的对CTGF和TIMP-1基因最有效的RNA干扰靶位,即CTGF基因1560～1580nt和TIMP-1基因412～432nt ,按照RNA干扰(RNA interference,RNAi)序列设计原则,各设计1对含有短发夹结构的RNAi靶点序列,退火形成双链DNA,分别克隆到经双酶切后的质粒载体psiRNA-h7SKGFPzeo,构建成含目的靶基因片段的重组质粒载体psiRNA-GFP-CTGF和psiRNA-GFP-TIMP-1,并对重组质粒进行双酶切琼脂糖电泳和测序鉴定。结果:酶切证实以大鼠CTGF或TIMP-1基因为靶点的表达shRNA的目的基因片段分别被成功的克隆到质粒载体psiRNA-h7SKGFPzeo中,测序结果证明重组质粒载体的插入序列与设计的靶基因片段完全一致。结论:成功构建靶向大鼠CTGF和TIMP-1最有效的RNA干扰靶位的shRNA表达质粒重组体,为进一步探索肝纤维化基因治疗的新途径打下了实验基础。
objective: Connective tissue growth factor (CTGF) is a recently discovered important cytokine of promoting hepatic fibrosis,which is transforming growth factor-β(TGF-β) signal transduction pathway downstream effect factor, which not only can directly mediate primary hepatic stellate cell (HSC) activation, proliferation and migration, but also can promote the HSC to synthesize and secret extracellular matrix. Another effect factor of TGF-βsignal transduction pathway downstream, Tissue inhibitor of metalloproteinase 1 (TIMP-1) is mainly expressed by activated HSC during the progress of hepatic fibrosis, which not only inhibits matrix metalloproteinase (matrix metalloproteinase, MMPs) activity to prevent collagen degradation, but also inhibits HSC apoptosis and promote collagen secreting. In the progress of hepatic fibrosis disease, TIMP-1 plays a key role. Therefore, reduced CTGF and TIMP-1 expression maybe could lessen hepatic fibrosis. The purpose of this study is to construct shRNA (short hairpin RNA) expression plasmid vectors targeting to rat connective tissue growth factor (CTGF) or tissue inhibitor of metalloproteinase 1 (TIMP-1). This study provides a powerful tool for further exploring a new gene therapy way of hepatic fibrosis in the future. Methods: According to the RNA interference sequences of rat CTGF gene (1560～1580nt) and rat TIMP-1 (412～432nt) , the targeted sequences had been screened out in the primary experiments, and based on the RNA interference (RNAi) sequence of design principles, two pairs of oligonucleotides were synthesized chemically, and then subjected to be annealed to form two double-stranded DNA fragments, respectively. The two double-stranded DNA fragments were then cloned into the plasmid vector, psiRNA-h7SKGFPzeo, respectively. The recombinant plasmids were named as psiRNA-GFP-CTGF or psiRNA-GFP-TIMP-1, which shoμld product shRNA in eukaryocytes. The recombinant plasmids psiRNA-GFP-CTGF or psiRNA-GFP-TIMP-1 was verified by Agarose gel electrophoresis and sequencing. Resμlts:By Agarose gel electrophoresis and sequencing, the two double-stranded DNA fragments were inserted correctly in psiRNA-h7SKGFPzeo vectors as expected, respectively. Conclusions: Two recombinant plasmids, psiRNA-GFP-CTGF and psiRNA-GFP-CTGF were successfμlly constructed. These new plasmids targeting to the rat CTGF and TIMP-1 for RNA interference shRNA expression woμld contribute to further explore new ways of gene therapy experiments for hepatic fibrosis.
objective: Connective tissue growth factor (CTGF) is a recently discovered important cytokine of promoting hepatic fibrosis,which is transforming growth factor-β(TGF-β) signal transduction pathway downstream effect factor, which not only can directly mediate primary hepatic stellate cell (HSC) activation, proliferation and migration, but also can promote the HSC to synthesize and secret extracellular matrix. Another effect factor of TGF-βsignal transduction pathway downstream, Tissue inhibitor of metalloproteinase 1 (TIMP-1) is mainly expressed by activated HSC during the progress of hepatic fibrosis, which not only inhibits matrix metalloproteinase (matrix metalloproteinase, MMPs) activity to prevent collagen degradation, but also inhibits HSC apoptosis and promote collagen secreting. In the progress of hepatic fibrosis disease, TIMP-1 plays a key role. Therefore, reduced CTGF and TIMP-1 expression maybe could lessen hepatic fibrosis. The purpose of this study is to construct shRNA (short hairpin RNA) expression plasmid vectors targeting to rat connective tissue growth factor (CTGF) or tissue inhibitor of metalloproteinase 1 (TIMP-1). This study provides a powerful tool for further exploring a new gene therapy way of hepatic fibrosis in the future. Methods: According to the RNA interference sequences of rat CTGF gene (1560～1580nt) and rat TIMP-1 (412～432nt) , the targeted sequences had been screened out in the primary experiments, and based on the RNA interference (RNAi) sequence of design principles, two pairs of oligonucleotides were synthesized chemically, and then subjected to be annealed to form two double-stranded DNA fragments, respectively. The two double-stranded DNA fragments were then cloned into the plasmid vector, psiRNA-h7SKGFPzeo, respectively. The recombinant plasmids were named as psiRNA-GFP-CTGF or psiRNA-GFP-TIMP-1, which shoμld product shRNA in eukaryocytes. The recombinant plasmids psiRNA-GFP-CTGF or psiRNA-GFP-TIMP-1 was verified by Agarose gel electrophoresis and sequencing. Resμlts:By Agarose gel electrophoresis and sequencing, the two double-stranded DNA fragments were inserted correctly in psiRNA-h7SKGFPzeo vectors as expected, respectively. Conclusions: Two recombinant plasmids, psiRNA-GFP-CTGF and psiRNA-GFP-CTGF were successfμlly constructed. These new plasmids targeting to the rat CTGF and TIMP-1 for RNA interference shRNA expression woμld contribute to further explore new ways of gene therapy experiments for hepatic fibrosis.
引文
[1] Liu X, Hu H, Yin JQ.Therapeutic strategies against TGF-beta signaling pathway in hepatic fibrosis.Liver Int. 2006 Feb;26(1):8-22.
[2] Chen MH, Chen JC, Tsai CC, et al.The role of TGF-beta 1 and cytoki- nes in the modulation of liver fibrosis by Sho-saiko-to in rat's bile duct ligated model. J Ethnopharmacol. 2005 Feb, 10;97(1):7-13.
[3] Sentíes-Gómez MD, Gálvez-Gastélum FJ, Meza-García E , et a1.Hepatic fibrosis:role of matrix metaloproteases and TGF beta.Gac Med Mex,2005 Jul-Aug,l4l(4):315-322.
[4] Nakamuta M,Morizono S,Tsuruta S,Kohjima M, et a1.Remote delivery and expression of soluble type II TGF-βreceptor in muscle prevents hepatic fibrosis in rats.Int .J. Mol Med,2005Jul,16(1):59-64.
[5]徐新保,冷希圣.阻断TGF-β信号传导在减缓四氯化碳乙醇诱导的小鼠肝细胞癌发展中的作用.中华医学杂志,2004,84(13):l122 -1125.
[6] Letterio JJ.Bottinger EP.TGF-beta knockout and dominant- nega -tive receptor transgenic mice.Miner ElectrolyteMetab,1998,24(2-3):161-167.
[7] Wang JF, Olson ME, Ma L,et al. Connective tissue growth factor siRNA modulates mRNA levels for a subset of molecules in normal and TGF– beta1-stimulated porcine skin fibroblasts. Wound Repair Regen. 2004 Mar -Apr;12(2):205-216.
[8]李光明,谢青,李定国等.抗结缔组织生长因子小干扰RNA对鼠原代肝星状细胞增殖的影响.中华肝脏病杂志,2006,14(3):224-225.
[9]主余华,任万华,张春清,等.结缔组织生长因子短发夹状RNA表达质粒的构建及其对肝星状细胞结缔组织生长因子表达的影响.中华肝脏病杂志,2006,14(3):228-229.
[10] Li G, Xie Q, Shi Y,et at. Inhibition of connective tissue growth factor by siRNA prevents liver fibrosis in rats.J GeneMed.2006 Jul, 8(7): 889-900.
[11] JIANG Wei,WANG Ji-yao,YANG Chang-qing,et al. Effects of a plasmid expressing antisense tissue inhibitor of metalloproteinase-1 on liver fibrosis in rats. Chin Med J, 2005 Feb,118(3):192-197.
[12] Iimuro Y,Nishio T,Morimoto T,et a1.Delivery of matrix metallopro- teinase-1 attenuates established liver fibrosis in the rat . Gastroenter- ology,2003 Feb,124(2):445-458.
[13] Kothapalli D,Grotendorst GR.CTGF modulastes cell cycle progression in cAMP-arrested NRK fibrosis. J Cell Physiol,2000 Jan,182(1):119-126.
[14] It0 Y,Goldschmeding R,Bende R,Claessen N, et a1.Kinetics of connective tissue growth factor expression during experimental proliferative Glomerulonephritis.J Am Soc Nephrol,2001 Mar,12(3):472-484.
[15] Murphy FR,Issa R, Zhou X, et a1.Inhibition of apoptosis of activated hepatic stellate cells by tissue inhibitor of metalloprofejnase一1 is mediated via effects of matrix metalloproteinase inhibition : implications for reversibility of liver fibrosis.J Biol Chem.2002,277 (13):11069 -11076.
[16] Farrel GC.Drugs and steatohepatitis.Semin Liver Dis,2002,22(2):185-194.
[17] Shin JY, Hur W, Wang JS,et al. HCV core protein promotes liverfibrogenesis via up-regulation of CTGF with TGF-beta1. Exp Mol Med. 2005 Apr 30;37(2):138-145.
[18] Kobayashi H, Hayashi N, Hayashi K, et al. Connective tissue growth factor and progressive fibrosis in biliary atresia. Pediatr Surg Int. 2005 Jan;21(1):12-16.
[19] Sedlaczek N,Jia JD,Bauer M,et al.Proliferating bile duct epithelial cells are a major source of connective tissue growth factor in rat biliary fibrosis. Am J Pathol.2001,158(4):1239-1244.
[20] Zhang BB,Cai WM ,W eng HL,et a1.Diagnostic value of platelet derived growth factor.BB.transforming growth factor-be ta1.matrix metalloproteinase-1 and tissue inhibitor of matrix metalloproteinase-l in serum and peripheral blood mononuclear cells for hepatic fibrosis.World J Gastroenterol,2003,9(11):2490-2496.
[21] Arthur MJ.FibrogenesisII.metalloproteinases and their inhibitors in liver fibrosis.Am J Physiol Gastrointest Liver Physiol,2000,279(2):G245-249.
[22] Eickelbe rg O.Kohler E,Reichenberger F,et al.Extracellular matrix deposition by primary human lung fibroblasts in response to TGF-beta1 and TGF-beta3.Am J Physiol,1999,276(5 Pt 1):L814-824.
[23]中华医学会传染病与寄生虫病学分会、肝病学分会联合修订病毒性肝炎防治方案.中华传染病杂志,2001,19(1):56—62.
[24] Elbashir SM,Harborth J,Weber K,et a1.Analysis of gene function in somatic mammalian cells using small interfering RNAs.Methods,2002,26:199-213.
[25] Tuschl T,Zamore PD,Lehmann R,et a1.Targeted mRNA degradationby double-stranded RNA in vitro.Genes Dev,1999,13:3l9l-3197.
[26] Bemstein E,Caudy AA,Hammond SM,et a1.Role for a bidentate ribonuclease in the initiation step of RNA interference.Nature,2001,409 (6818):363-366.
[27]张春,朱忠华,邓安国.结缔组织生长因子反义寡核苷酸对肾小管上皮细胞纤溶酶原激活物抑制物1表达的影响[J].中华肾脏病杂志,2004,20(4):264-267.
[28]张海燕,李幼姬,杜勇等.结缔组织生长因子反义寡核苷酸对肾小管上皮细胞胶原分泌的影响[J].中华肾脏病杂志,2004,20(2):122-126.
[1] Sentíes-Gómez MD, Gálvez-Gastélum FJ, Meza-García E,et a1. Hepatic fibrosis:role of matrix metaloproteases and TGF beta.Gac Med Mex,2005,l4l(4):315-322.
[2] Kopp JB, Factor VM, Mozes M, et a1.Transgenic mice with increased plasma levels of TGF-beta 1 develop progressive renal disease .Lab Invest,1996,74(6):991-1003.
[3]蒋炜,王吉耀.反义转化生长因子βⅠ型受体表达质粒对实验性肝纤维化的影响.中华医学杂志,2002,82(17):l 160-1164.
[4]蒋炜,王吉耀.反义转化生长因子βII型受体表达质粒对实验性肝纤维化的影响.中华肝脏病杂志,2004,12(3):137-140.
[5] Nakamuta M,Morizono S,Tsuruta S,et a1.Remote delivery and expression of soluble type II TGF-βreceptor in muscle prevents hepatic fibrosis in rats.Int .J. Mol Med,2005,16(1):59-64.
[6]徐新保,冷希圣.阻断TGF-β信号传导在减缓四氯化碳乙醇诱导的小鼠肝细胞癌发展中的作用.中华医学杂志,2004,84(13):l122-1125.
[7] Inagaki Y, Kushida M, Higashi K, et a1. Cell type-specific intervention of transforming growth factor-β/ Smad signaling suppresses collagen gene expression and hepatic fibrosis in mice. Gastroenter- ology, 2005, 129(1):259-268.
[8] Letterio JJ.Bottinger EP.TGF-beta knockout and dominant- negative receptor transgenic mice.Miner ElectrolyteMetab,1998,24(2-3):161-167.
[9] Kim KH,Park GT,Lim YB,Reu SW,et a1.Expression of connective tissue growth factor,a biomarker in senescence of human diploid fibroblasts,is-up-regulated by a transforming growth factor-beta- mediated signaling pathway.Biochem Biophys Res Commun,2004,318(4):819-825.
[10] Kasaragod AB,Lucia MS,Cabirac G,et al.Connective tissue growth factor expression in pediatric myofibroblastic tumors. Pediatr Dev Pathol,2001,4(1):37 -45.
[11] Lipardi C,Wei Q,Paterson BM.RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs.Cell,2001,107(3):297-307.
[12] Ingrid E. Blom, Roel Goldschmeding and Andrew Leask.Gene regulation of connective tissue growth factor: new targets for anti-fibrotic therapy?Marx Biol,2002,21(6):473-482.
[13] Leask A,Abraham DJ.The role of connective tissue growth factor,a multifunctional matricellular protein, in fibroblast biology.Biochem Cell Biol,2003,81(6):355-363.
[14] Wang JF,Olson ME,Ma L,et a1.Connective tissue growth factor siRNA modulates mRNA levels for a subset of molecules in normal and TGF-beta 1-stimulated porcine skin fibroblasts[J ].Wound Repair and Regen,2004,12(2):205-216.
[15] Abraham D, Ponticos M, Nagase H.Connective tissue remodeling:cross-talk between endothelins and matrix metalloproteinases.Curr Vasc Pharmaco1.2005,3(4):369-379.
[16] Schuppan D,RueM M,Somasundaram R,et a1.Matrix as a Modulatorof hepatic fibrogenesis.Semin Liver Dis,200l,21(3):351-372.
[17] Murawaki Y,Ikuta Y,Idobe Y,et a1.Tissue inhibitor of metallopro- teinase-l in the liver of patients with chronic liver disease.J Hepatol,1997,26(6):12l3-1219.
[18] Nie QH,Duan GR,LuoXD,et a1.Expression of TIMP-l and TIMP-2 in rats with hepatic fibrosis.World J Gastroenterol,2004,l0(1):86-90.
[19] Iimuro Y , Nishio T , Morimoto T , et a1.Delivery of matrix metalloproteinase-1 attenuates established liver fibrosis in the rat.Gastroenterology,2003,124(2):445-458.
[20] Nie Q H,Cheng Y Q,Xie Y M,et a1.Inhibiting effect of antisense oligonucleotides phosphorthioate on gene expression of TIMP-l in rat liver fibrosis.World J Gastroenterol.200l,7(3):363-369.
[21] Siller Lopez F,Sandoval A,Salgado S,et a1.Treatment With Human Metalloproteinase-8 Gene Delivery Ameliorates Experimental Rat Liver Cirrhosis[J].Gastroenterology,2004,126(4):1122-1133.
[22] Roderfeld M,Weiskirchen R,Wagner S,et a1.Inhibition of hepatic fibrogenesis by matrix metlloproteinase-9 mutants in mice[J].FASEB J,2006,20(3):444-454.
[2] Chen MH, Chen JC, Tsai CC, et al.The role of TGF-beta 1 and cytoki- nes in the modulation of liver fibrosis by Sho-saiko-to in rat's bile duct ligated model. J Ethnopharmacol. 2005 Feb, 10;97(1):7-13.
[3] Sentíes-Gómez MD, Gálvez-Gastélum FJ, Meza-García E , et a1.Hepatic fibrosis:role of matrix metaloproteases and TGF beta.Gac Med Mex,2005 Jul-Aug,l4l(4):315-322.
[4] Nakamuta M,Morizono S,Tsuruta S,Kohjima M, et a1.Remote delivery and expression of soluble type II TGF-βreceptor in muscle prevents hepatic fibrosis in rats.Int .J. Mol Med,2005Jul,16(1):59-64.
[5]徐新保,冷希圣.阻断TGF-β信号传导在减缓四氯化碳乙醇诱导的小鼠肝细胞癌发展中的作用.中华医学杂志,2004,84(13):l122 -1125.
[6] Letterio JJ.Bottinger EP.TGF-beta knockout and dominant- nega -tive receptor transgenic mice.Miner ElectrolyteMetab,1998,24(2-3):161-167.
[7] Wang JF, Olson ME, Ma L,et al. Connective tissue growth factor siRNA modulates mRNA levels for a subset of molecules in normal and TGF– beta1-stimulated porcine skin fibroblasts. Wound Repair Regen. 2004 Mar -Apr;12(2):205-216.
[8]李光明,谢青,李定国等.抗结缔组织生长因子小干扰RNA对鼠原代肝星状细胞增殖的影响.中华肝脏病杂志,2006,14(3):224-225.
[9]主余华,任万华,张春清,等.结缔组织生长因子短发夹状RNA表达质粒的构建及其对肝星状细胞结缔组织生长因子表达的影响.中华肝脏病杂志,2006,14(3):228-229.
[10] Li G, Xie Q, Shi Y,et at. Inhibition of connective tissue growth factor by siRNA prevents liver fibrosis in rats.J GeneMed.2006 Jul, 8(7): 889-900.
[11] JIANG Wei,WANG Ji-yao,YANG Chang-qing,et al. Effects of a plasmid expressing antisense tissue inhibitor of metalloproteinase-1 on liver fibrosis in rats. Chin Med J, 2005 Feb,118(3):192-197.
[12] Iimuro Y,Nishio T,Morimoto T,et a1.Delivery of matrix metallopro- teinase-1 attenuates established liver fibrosis in the rat . Gastroenter- ology,2003 Feb,124(2):445-458.
[13] Kothapalli D,Grotendorst GR.CTGF modulastes cell cycle progression in cAMP-arrested NRK fibrosis. J Cell Physiol,2000 Jan,182(1):119-126.
[14] It0 Y,Goldschmeding R,Bende R,Claessen N, et a1.Kinetics of connective tissue growth factor expression during experimental proliferative Glomerulonephritis.J Am Soc Nephrol,2001 Mar,12(3):472-484.
[15] Murphy FR,Issa R, Zhou X, et a1.Inhibition of apoptosis of activated hepatic stellate cells by tissue inhibitor of metalloprofejnase一1 is mediated via effects of matrix metalloproteinase inhibition : implications for reversibility of liver fibrosis.J Biol Chem.2002,277 (13):11069 -11076.
[16] Farrel GC.Drugs and steatohepatitis.Semin Liver Dis,2002,22(2):185-194.
[17] Shin JY, Hur W, Wang JS,et al. HCV core protein promotes liverfibrogenesis via up-regulation of CTGF with TGF-beta1. Exp Mol Med. 2005 Apr 30;37(2):138-145.
[18] Kobayashi H, Hayashi N, Hayashi K, et al. Connective tissue growth factor and progressive fibrosis in biliary atresia. Pediatr Surg Int. 2005 Jan;21(1):12-16.
[19] Sedlaczek N,Jia JD,Bauer M,et al.Proliferating bile duct epithelial cells are a major source of connective tissue growth factor in rat biliary fibrosis. Am J Pathol.2001,158(4):1239-1244.
[20] Zhang BB,Cai WM ,W eng HL,et a1.Diagnostic value of platelet derived growth factor.BB.transforming growth factor-be ta1.matrix metalloproteinase-1 and tissue inhibitor of matrix metalloproteinase-l in serum and peripheral blood mononuclear cells for hepatic fibrosis.World J Gastroenterol,2003,9(11):2490-2496.
[21] Arthur MJ.FibrogenesisII.metalloproteinases and their inhibitors in liver fibrosis.Am J Physiol Gastrointest Liver Physiol,2000,279(2):G245-249.
[22] Eickelbe rg O.Kohler E,Reichenberger F,et al.Extracellular matrix deposition by primary human lung fibroblasts in response to TGF-beta1 and TGF-beta3.Am J Physiol,1999,276(5 Pt 1):L814-824.
[23]中华医学会传染病与寄生虫病学分会、肝病学分会联合修订病毒性肝炎防治方案.中华传染病杂志,2001,19(1):56—62.
[24] Elbashir SM,Harborth J,Weber K,et a1.Analysis of gene function in somatic mammalian cells using small interfering RNAs.Methods,2002,26:199-213.
[25] Tuschl T,Zamore PD,Lehmann R,et a1.Targeted mRNA degradationby double-stranded RNA in vitro.Genes Dev,1999,13:3l9l-3197.
[26] Bemstein E,Caudy AA,Hammond SM,et a1.Role for a bidentate ribonuclease in the initiation step of RNA interference.Nature,2001,409 (6818):363-366.
[27]张春,朱忠华,邓安国.结缔组织生长因子反义寡核苷酸对肾小管上皮细胞纤溶酶原激活物抑制物1表达的影响[J].中华肾脏病杂志,2004,20(4):264-267.
[28]张海燕,李幼姬,杜勇等.结缔组织生长因子反义寡核苷酸对肾小管上皮细胞胶原分泌的影响[J].中华肾脏病杂志,2004,20(2):122-126.
[1] Sentíes-Gómez MD, Gálvez-Gastélum FJ, Meza-García E,et a1. Hepatic fibrosis:role of matrix metaloproteases and TGF beta.Gac Med Mex,2005,l4l(4):315-322.
[2] Kopp JB, Factor VM, Mozes M, et a1.Transgenic mice with increased plasma levels of TGF-beta 1 develop progressive renal disease .Lab Invest,1996,74(6):991-1003.
[3]蒋炜,王吉耀.反义转化生长因子βⅠ型受体表达质粒对实验性肝纤维化的影响.中华医学杂志,2002,82(17):l 160-1164.
[4]蒋炜,王吉耀.反义转化生长因子βII型受体表达质粒对实验性肝纤维化的影响.中华肝脏病杂志,2004,12(3):137-140.
[5] Nakamuta M,Morizono S,Tsuruta S,et a1.Remote delivery and expression of soluble type II TGF-βreceptor in muscle prevents hepatic fibrosis in rats.Int .J. Mol Med,2005,16(1):59-64.
[6]徐新保,冷希圣.阻断TGF-β信号传导在减缓四氯化碳乙醇诱导的小鼠肝细胞癌发展中的作用.中华医学杂志,2004,84(13):l122-1125.
[7] Inagaki Y, Kushida M, Higashi K, et a1. Cell type-specific intervention of transforming growth factor-β/ Smad signaling suppresses collagen gene expression and hepatic fibrosis in mice. Gastroenter- ology, 2005, 129(1):259-268.
[8] Letterio JJ.Bottinger EP.TGF-beta knockout and dominant- negative receptor transgenic mice.Miner ElectrolyteMetab,1998,24(2-3):161-167.
[9] Kim KH,Park GT,Lim YB,Reu SW,et a1.Expression of connective tissue growth factor,a biomarker in senescence of human diploid fibroblasts,is-up-regulated by a transforming growth factor-beta- mediated signaling pathway.Biochem Biophys Res Commun,2004,318(4):819-825.
[10] Kasaragod AB,Lucia MS,Cabirac G,et al.Connective tissue growth factor expression in pediatric myofibroblastic tumors. Pediatr Dev Pathol,2001,4(1):37 -45.
[11] Lipardi C,Wei Q,Paterson BM.RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs.Cell,2001,107(3):297-307.
[12] Ingrid E. Blom, Roel Goldschmeding and Andrew Leask.Gene regulation of connective tissue growth factor: new targets for anti-fibrotic therapy?Marx Biol,2002,21(6):473-482.
[13] Leask A,Abraham DJ.The role of connective tissue growth factor,a multifunctional matricellular protein, in fibroblast biology.Biochem Cell Biol,2003,81(6):355-363.
[14] Wang JF,Olson ME,Ma L,et a1.Connective tissue growth factor siRNA modulates mRNA levels for a subset of molecules in normal and TGF-beta 1-stimulated porcine skin fibroblasts[J ].Wound Repair and Regen,2004,12(2):205-216.
[15] Abraham D, Ponticos M, Nagase H.Connective tissue remodeling:cross-talk between endothelins and matrix metalloproteinases.Curr Vasc Pharmaco1.2005,3(4):369-379.
[16] Schuppan D,RueM M,Somasundaram R,et a1.Matrix as a Modulatorof hepatic fibrogenesis.Semin Liver Dis,200l,21(3):351-372.
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