TIMP-1短发夹式RNA体内基因转导及来氟米特对大鼠糖尿病肾病的影响
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摘要
目的:观察TIMP-1短发夹式RNA(TIMP-1shRNA)及来氟米特(leflunomide,LEF)对糖尿病肾病大鼠肾间质纤维化的影响
方法:将雄性Wistar大鼠120只随机分为6组:正常对照组(N组)、糖尿病组(DM组)、糖尿病TIMP-1shRNA转染组(DM-RNAi组)、糖尿病HKshRNA转染组(DM-VE组)、糖尿病来氟米特治疗组(DM-LEF组)、糖尿病羧甲基纤维素组(DM-CMC组)。观察各组大鼠肾功能变化;PAS染色观察大鼠肾间质病理形态改变;RT-PCR方法检测大鼠肾组织中TIMP-1、MCP-1、ICAM-1、VCAM-1、TGF-β1、Fractalkine和RANTES mRNA各时间点基因水平的变化;Western Blot方法检测大鼠肾组织中TIMP-1、MMP-2、MMP-9、TGF-β1、MCP-1、ICAM-1、Fractalkine各组蛋白水平的变化,以及免疫组化检测大鼠肾脏FN、TIMP-1、MMP-2、NF-κB、TGF-β1、MCP-1、α-SMA的表达。
结果:随时间延长,糖尿病大鼠的肾脏功能逐渐受累,DM组尿蛋白/尿肌酐,血肌酐,血尿素氮以及相对肾重(KW/BW)与N组比较均显著升高(P<0.05),DM-RNAi组,DM-LEF组与DM组比较表达下调(P<0.05)。肾组织病理PAS染色可见DM组肾脏呈进行性肾间质纤维化改变,包括肾小管的萎缩、扩张、炎细胞浸润等;而DM-RNAi组和DM-LEF组肾组织病变明显减轻。基因水平检测TIMP-1在10、12、14、16周表达,随时间延长,与N组比较DM组表达升高(P<0.05),DM-RNAi组和DM-LEF组抑制TIMP-1表达(P<0.05)。RT-PCR检测MCP-1、ICAM-1、VCAM-1、TGF-β1、Fractalkine和RANTES mRNA的表达,可见DM组表达升高(P<0.05),DM-RNAi组和DM-LEF组表达下调(P<0.05)。同时,TIMP-1蛋白表达与基因水平表达一致;DM组MMP-2、MMP-9随时间延长蛋白表达降低(P<0.05),而DM-RNAi组和DM-LEF组表达上调(P<0.05);DM组TGF-β1、MCP-1、ICAM-1、Fractalkine表达升高(P<0.05),DM-RNAi组和DM-LEF组表达下调(P<0.05)。免疫组化示DM组TIMP-1、FN、α-SMA、NF-κB、TGF-β1、MCP-1阳性染色表达面积增多(P<0.05),DM-RNAi组和DM-LEF组明显比DM组减少(P<0.05)。
结论:
1.TIMP-1在糖尿病肾病大鼠肾脏中的高表达对糖尿病肾病间质纤维化的发生发展起到重要作用。
2.TIMP-1shRNA在基因水平阻断TIMP-1表达,促进细胞外基质(extracellular material ,ECM)降解增加改善肾间质纤维化;此外,TIMP-1shRNA可能通过抑制炎症反应改善肾小管间质损伤,对糖尿病大鼠肾脏起到保护作用。
3.糖尿病肾病中来氟米特减轻间质炎症反应,抑制炎症因子表达,抑制肌成纤维细胞增殖,从而减轻肾间质纤维化。
Objective: To study the effects of TIMP-1 small hairpin RNA (TIMP-1 shRNA) and leflunomide on the renal interstitial fibrosis caused by diabetic nephropathy in rats.
Methods: 120 healthy Wister rats were randomly divided into 6 groups: normal control group(N group)、diabetes mellitus(DM group)、TIMP-1 shRNA group(DM-RNAi group)、HKshRNA group(DM-VE group)、leflunomide(DM-LEF group)、Carboxymethyl Cellulose group(DM-CMC group). Rats' renal function of each group were monitored;The histological changes of renal interstitial tissues were observed by PAS staining. The mRNAs of TIMP-1、MCP-1、ICAM-1、VCAM-1、TGF-β1、Fractalkine and RANTES were reverse transcribed and quantified by RT-PCR .The proteins of TIMP-1、MMP-2、MMP-9、TGF-β1、MCP-1、ICAM-1 and Fractalkine changes were analyzced by Western Blot analysis.The proteins of FN、TIMP-1、MMP-2、NF-κB、TGF-β1、MCP-1 andα-SMA were assessed by immunohistochemistry analysis.
Results: After the rat model were established ,with the extension of time, kidney function of the diabetic rats' suffered gradually,urine protein/ urine creatinine,serum creatinine, blood urea nitrogen,relative kidney weight(KW/BW) in DM group were higher than N group (P<0.05); compared with DM group,the DM-RNAi group and the DM-LEF group were decrease remarkably (P<0.05).Nephridial tissue pathomorphology mani- fested that:In DM group, interstitial fibrosis including tubular astrophy,loss and dilation,inflammatory cell infiltration were evident.These changes were all improved by TIMP-1shRNA and leflunomide treatment.Respectively analysising DN rat kidney TIMP-1mRNA at 10、12、14、16 weeks by RT-PCR,with the extension of time,the DM group's expression was higher than the N group's(P<0.05),but the DM-RNAi group and the DM-LEF group were decreased conspicuously than DM group(P<0.05).In the gene level,the expressions of MCP-1、ICAM-1、VCAM-1、TGF-β1、Fractalkine and RANTES mRNA were increased in the DM group,but the DM-RNAi group and the DM-LEF group were decreased.Meanwhile,in the protein level rat TIMP-1 was consonanced with the gene level; compared with N group,MMP-2 and MMP-9 were accrescenced in DM group;yet,the DM-RNAi group and the DM-LEF group descented significantly than DM group(P<0.05) ; either TIMP-1shRNA or leflunomide significantly decreased the levels of TGF-β1、MCP-1、ICAM-1 and Fractalkine,in DM group was inceased dramatically(P<0.05).In nephridial tissue,through the immunohistochemistry,the masc dyeing areas of FN、TIMP-1、MMP-2、NF-κB、TGF-β1、MCP-1、α-SMA enhanced in DM group, compared with the DM group,the DM-RNAi group and the DM-LEF group extracellular matrix diminished (P<0.05).
Conclusions:
1. TIMP-1 plays an important role in the development of diabetic nephropathy interstitial fibrosis.
2. TIMP-1shRNA downregulates the gene expression of TIMP-1,promotes the degradation of extracellular material and improves interstitial fibrosis; in addition,TIMP-1shRNA might inhibit inflammation and improve tubules injury ,therefore protects rat renal function.
3. Leflunomide may attenuate interstitial inflammation reaction, suppresse the expressions of inflammatory factors,improve interstitial fibrosis which is probably correlated with myofibroblast proliferation. therefore,it can protect renal function in diabetic nephropathy.
方法:将雄性Wistar大鼠120只随机分为6组:正常对照组(N组)、糖尿病组(DM组)、糖尿病TIMP-1shRNA转染组(DM-RNAi组)、糖尿病HKshRNA转染组(DM-VE组)、糖尿病来氟米特治疗组(DM-LEF组)、糖尿病羧甲基纤维素组(DM-CMC组)。观察各组大鼠肾功能变化;PAS染色观察大鼠肾间质病理形态改变;RT-PCR方法检测大鼠肾组织中TIMP-1、MCP-1、ICAM-1、VCAM-1、TGF-β1、Fractalkine和RANTES mRNA各时间点基因水平的变化;Western Blot方法检测大鼠肾组织中TIMP-1、MMP-2、MMP-9、TGF-β1、MCP-1、ICAM-1、Fractalkine各组蛋白水平的变化,以及免疫组化检测大鼠肾脏FN、TIMP-1、MMP-2、NF-κB、TGF-β1、MCP-1、α-SMA的表达。
结果:随时间延长,糖尿病大鼠的肾脏功能逐渐受累,DM组尿蛋白/尿肌酐,血肌酐,血尿素氮以及相对肾重(KW/BW)与N组比较均显著升高(P<0.05),DM-RNAi组,DM-LEF组与DM组比较表达下调(P<0.05)。肾组织病理PAS染色可见DM组肾脏呈进行性肾间质纤维化改变,包括肾小管的萎缩、扩张、炎细胞浸润等;而DM-RNAi组和DM-LEF组肾组织病变明显减轻。基因水平检测TIMP-1在10、12、14、16周表达,随时间延长,与N组比较DM组表达升高(P<0.05),DM-RNAi组和DM-LEF组抑制TIMP-1表达(P<0.05)。RT-PCR检测MCP-1、ICAM-1、VCAM-1、TGF-β1、Fractalkine和RANTES mRNA的表达,可见DM组表达升高(P<0.05),DM-RNAi组和DM-LEF组表达下调(P<0.05)。同时,TIMP-1蛋白表达与基因水平表达一致;DM组MMP-2、MMP-9随时间延长蛋白表达降低(P<0.05),而DM-RNAi组和DM-LEF组表达上调(P<0.05);DM组TGF-β1、MCP-1、ICAM-1、Fractalkine表达升高(P<0.05),DM-RNAi组和DM-LEF组表达下调(P<0.05)。免疫组化示DM组TIMP-1、FN、α-SMA、NF-κB、TGF-β1、MCP-1阳性染色表达面积增多(P<0.05),DM-RNAi组和DM-LEF组明显比DM组减少(P<0.05)。
结论:
1.TIMP-1在糖尿病肾病大鼠肾脏中的高表达对糖尿病肾病间质纤维化的发生发展起到重要作用。
2.TIMP-1shRNA在基因水平阻断TIMP-1表达,促进细胞外基质(extracellular material ,ECM)降解增加改善肾间质纤维化;此外,TIMP-1shRNA可能通过抑制炎症反应改善肾小管间质损伤,对糖尿病大鼠肾脏起到保护作用。
3.糖尿病肾病中来氟米特减轻间质炎症反应,抑制炎症因子表达,抑制肌成纤维细胞增殖,从而减轻肾间质纤维化。
Objective: To study the effects of TIMP-1 small hairpin RNA (TIMP-1 shRNA) and leflunomide on the renal interstitial fibrosis caused by diabetic nephropathy in rats.
Methods: 120 healthy Wister rats were randomly divided into 6 groups: normal control group(N group)、diabetes mellitus(DM group)、TIMP-1 shRNA group(DM-RNAi group)、HKshRNA group(DM-VE group)、leflunomide(DM-LEF group)、Carboxymethyl Cellulose group(DM-CMC group). Rats' renal function of each group were monitored;The histological changes of renal interstitial tissues were observed by PAS staining. The mRNAs of TIMP-1、MCP-1、ICAM-1、VCAM-1、TGF-β1、Fractalkine and RANTES were reverse transcribed and quantified by RT-PCR .The proteins of TIMP-1、MMP-2、MMP-9、TGF-β1、MCP-1、ICAM-1 and Fractalkine changes were analyzced by Western Blot analysis.The proteins of FN、TIMP-1、MMP-2、NF-κB、TGF-β1、MCP-1 andα-SMA were assessed by immunohistochemistry analysis.
Results: After the rat model were established ,with the extension of time, kidney function of the diabetic rats' suffered gradually,urine protein/ urine creatinine,serum creatinine, blood urea nitrogen,relative kidney weight(KW/BW) in DM group were higher than N group (P<0.05); compared with DM group,the DM-RNAi group and the DM-LEF group were decrease remarkably (P<0.05).Nephridial tissue pathomorphology mani- fested that:In DM group, interstitial fibrosis including tubular astrophy,loss and dilation,inflammatory cell infiltration were evident.These changes were all improved by TIMP-1shRNA and leflunomide treatment.Respectively analysising DN rat kidney TIMP-1mRNA at 10、12、14、16 weeks by RT-PCR,with the extension of time,the DM group's expression was higher than the N group's(P<0.05),but the DM-RNAi group and the DM-LEF group were decreased conspicuously than DM group(P<0.05).In the gene level,the expressions of MCP-1、ICAM-1、VCAM-1、TGF-β1、Fractalkine and RANTES mRNA were increased in the DM group,but the DM-RNAi group and the DM-LEF group were decreased.Meanwhile,in the protein level rat TIMP-1 was consonanced with the gene level; compared with N group,MMP-2 and MMP-9 were accrescenced in DM group;yet,the DM-RNAi group and the DM-LEF group descented significantly than DM group(P<0.05) ; either TIMP-1shRNA or leflunomide significantly decreased the levels of TGF-β1、MCP-1、ICAM-1 and Fractalkine,in DM group was inceased dramatically(P<0.05).In nephridial tissue,through the immunohistochemistry,the masc dyeing areas of FN、TIMP-1、MMP-2、NF-κB、TGF-β1、MCP-1、α-SMA enhanced in DM group, compared with the DM group,the DM-RNAi group and the DM-LEF group extracellular matrix diminished (P<0.05).
Conclusions:
1. TIMP-1 plays an important role in the development of diabetic nephropathy interstitial fibrosis.
2. TIMP-1shRNA downregulates the gene expression of TIMP-1,promotes the degradation of extracellular material and improves interstitial fibrosis; in addition,TIMP-1shRNA might inhibit inflammation and improve tubules injury ,therefore protects rat renal function.
3. Leflunomide may attenuate interstitial inflammation reaction, suppresse the expressions of inflammatory factors,improve interstitial fibrosis which is probably correlated with myofibroblast proliferation. therefore,it can protect renal function in diabetic nephropathy.
引文
1. Ina K, Kitamura H, Tatsukawa S,et al.Transformation of interstitial fibroblasts and tubulointerstitial fibrosis in diabetic nephropathy.Med Electron Microsc, 2002 ,Jun;35(2):87-95.
2. Nyengaard JR,Flyvbjerg A,Rasch R.The impact of renal growth,regression and regrowth in experimental diabetes mellitus on number and size of proximal and distal tubular cells in the rat kidney.Diabetologia,1993,36(11):1126-1131.
3. Chen FE,Huang DB,Chen YQ,et al.Crystal stucture of p50/p65 heterodimer of transcription factor NF-κB bound to DNA.Natrue,1998,391(22):410-413.
4. Tutde KR.Linking metablism and immunology:diabetic nephropatly is an inflammatory disease.J Am Soc Nephrol,2005: 16(6):1537-1538.
5. Richard W,Mankhey,Corinne C.Wells,et al.17β-Estradiol supplementation redu- ces tubulointerstitial fibrosis by increasing MMP activity in the diabetic kidney.Am J Physiol Regul Integr Comp Physiol,2007,292: R769-777.
6. Chow FY, Nikolic-Paterson DJ,Atkins RC,et al.Macrophages in streptozotocin- induced diabetic nephropathy:potential role in renal fibrosis. Nephrol Dial Transplant,2004 Dec;19(12):2987-2996.
7. Fornoni A, Ijaz A, Tejada T, et al.Role of inflammation in diabetic nephropathy. Curr Diabetes Rev,2008 Feb;4(1):10-17.
8. Zeisberg M, Kalluri R.The role of epithelial-to-mesenchymal transition in renal fibrosis.J Mol Med ,2004 ,82 :175-181.
9.孙艳玲,林洪丽,吴泰华,等.基质金属蛋白酶1组织抑制剂短发夹式RNA对肾小球系膜细胞凋亡及细胞外基质的影响.中华肾脏病杂志,2006,22 (6):364-369.
10.董建平,陈香美,师锁柱,等.金属蛋白酶1组织抑制剂在大鼠肾小管间质损害中的表达及其意义.中华肾脏病杂志,2002,18:275-279.
11.邵凤民,尚希瑶,陈香美等.人基质金属蛋白酶1组织抑制剂转基因小鼠肾小管间质炎症的改变.中华肾脏病杂志,2006,22:688-692.
12. Fiore E,Fusco C,Romero P,et al.Matrix metalloproteinase 9 (MMP-9/gelatinase B) proteolytically cleaves ICAM-1 and participates in tumor cell resistance to natural killer cell-mediated cytotoxicity.Oncogene,2002,21:5213-5223.
13. Manna SK,Aggarwal BB.Immunosuppressive leflunomide metabolite (A771726) blocks TNF-dependent nuclear factor-B activation and gene expression.J Immunol,1999,162:2095.
14.刘维萍,沈晓玉,彭勃,等.来氟米特对肾大部切除大鼠肾皮质α-平滑肌肌动蛋白的影响.中国药物与临床,2005,5:112-114.
15.陈琳,苟兴化,严律南,等.携带反义多药耐药相关蛋白的重组腺病毒载体的构建.四川大学学报(医学版),2003,34(1):131.
16.雷作熹,罗仁,董晓蕾,等.STZ诱导糖尿病肾病大鼠模型的建立.中国实验动物学报,2005,13(3): 163-166.
17. Arenz C,Schepers U.RNA interference:from an ancient mechanism to a statc of the art therapeutic application?Naturwissenschaften,2003,90:345-359.
18. 18Ichim TE,Li M,Qian H,et al.RNA interference:a potent tool for gene-specific therapcutics.Am J Transplant,2004,4:1227-1236.
19. Urushibara M,Takayanagi H,Koga T,et al.The antirheumatic drug leflunomide inhibits osteoclastogenesis by interfering with recepto ractivator of NF-kappa B ligand-stimulated induction of nuclear factor of activated T cells c1.Arthritis Rheum,2004,50(3):794-804.
20. Zeisberg M,Maeshima Y,Mosterman B,et al.Renal fibrosis:Extracellular matrix microenvironment regulates migratory behavior of activated tubular epithelial cells.Am J Pathol, 2002, 160 (6) : 2001-2008.
21. Lenz O,Elliot SJ,Stetler-Stevenson WG.Matrix metalloproteinase in renal development and disease.J Am Soc Nephrol,2000,11:574-581.
22. Beaudeux JL,Giral P,Bruckert E,et al.Matrix metalloproteinase, inflammation and atherosclerosis:therapeutic perspective.Clin Chem Lab Med,2004,42:121- 131.
23. Lam S,van der Geest RN,Verhagen NA,et al.Secretion of collagen typeⅣby human renal fibroblasts is increased by high glucose viaa TGF-beta-independent pathway.Nephrol Dial Transplant,2004,19(7):1694-1701.
24. Oh JH,Ha H,Yu MR,et al.Sequential effects of high glucose on mesangial cell transforming growth factor-beta1 and fibronectin synthesis.Kidney Int,1998,54 (6):1872-1878.
25. Parks WC,Wilson CL,Lopez-Boado YS.Matrix metalloproteinases as modulators of inflammation and innate immunity.Nat Rev Immunol,2004,4:617-629.
26. Kridel SJ,Chen E,Kortra LP,et al.Substrate hydrolysis by matrix metallo- proteinase-9.J Biol Chem,2001,276:20572-20578.
27. Cohen MP,Shea E,Chen S et al.Glycated albumin increases oxidative stress, activates NF-kappa B and extracellular signal-regulated kinase(ERK),andstimulates ERK-dependent transforming growth factor-beta 1 production in macrophage R A W cells.J Lab Clin Med,2003,141(4):242-249.
28.丁鹤林,王佑民,徐明彤,等.抑制NF-κB活性对糖尿病大鼠肾脏TGF-β1mRNA表达影响.中国药理学通报,2004,20(9):1020-1025.
29.刘维萍,林海英,田李清,等.来氟米特对肾大部切除大鼠肾皮质TGF-β1表达的影响.中原医刊,2004,31(23):1-2.
30. Goumenos D S,Brown CB,Shortland J,et al.Myofibroblasts,predictors of progression of mesangial IgA nephropathy.Nephrol Dial Transplant,1994,9: 1418.
31. Yang N,Wu L L,Nicolic-Paterson D J,et al.Local macrophage and fibroblast proliferation in progressive renal injury in the rat remnant kidney.Nephrol Dial Transplant,1998,13(8):1967.
32. Sharma k,Morris RE,Huang X,et al.Inhibition of arterial intimal thickening by leflunomide:in vivo efficacy and molecular mechanisms.Am Soc Trans Surg, 1994,1:97-105.
1. Eddy AA.Molecular casis of renal fibrosis.Pediatr.Nephrol,2000,15(3-4): 290- 301.
2. Liu,Y.Renal fibrosis:new insights into the pathogenesis and therapeutics.Kidney International,2006,69(2):213-217.
3.罗海清,梁东,刘华锋.肾间质纤维化的形成机制及中药防治作用.中国中西医结合肾病杂志,2004,5(7):432-434.
4. Kuncio GS,Neilson EG.Haverty T.Mechanisms of tubulointerstitial fibrosis. Kidney Int,1991,39(3):550-556.
5.陈荣权,陈香美,崔世维,等.组织金属蛋白酶抑制物在老年大鼠肾小管间质病理损害中的动态变化.中华老年医学杂志,2004,23(4):253-258.
6. Lenz O Elliot SJ,Stetler-Stevenson WG.Matrix metalloproteinase in renal development and disease.J Am Soc Nephrol,2000,11:574-581.
7. Gonzalez-AvilaG,Iturria C,Vadillo-Ortega F,et al.Changes in matrix metallo- proteinase during the evolution of interstitial renal fibrosis in a rat experimental model.Pathorbiology,1998,66(5):196-204.
8. Sharma AK,Mauer SM,Kim Y,et al.Altered expression of matrix metallo- proteinase-2,TIMP-1 and TIMP-2 obstructive nephropathy.J Lab Clin Med, 1995, 125(6):754-761.
9. Johnson TS,Haylor JL,Thomas GL,et al.Matrix metallor proteinases and their inhibitions in experimental renal scarring.Exp Nephrol,2002,10(3):182-195.
10. Duymelinck C,Deng JT,Dauwe SE,et al.Inhibition of the matrix metallo -proteinase system in a rat model of chronic cyclosporine nephropahy.Kidney Int,1998,54(3):804-818
11. Nakamura T,Ushiyama C,Suzuki S,et al.Elevation of serum levels of metalloproteinase-1,tissue inhibitor of melalloproteinase-1 and typeⅣcollagen and plasma levels of metalloproteinase-9 in polycystic kidney disease.Am J Nephrol,2000,20(1):32-36.
12. Ishidoya S,Morrissey J,McCracken R,et al.Delayedt reatment with enalapril halts tubulointerst itial fibrosis in rats with obstructive nephropathy.Kidney Int, 1996,49(4):1110-1119.
13. Tang WW,Feng L,XiaY,et al.Extracellular matrix accumulation in immune- mediated tubulointerstitial injury.Kidney Int,1994,45(4):1077-1084.
14.董健平,陈香美,师锁柱,等.金属蛋白酶组织抑制剂1在大鼠肾小管间质损害中的表达及其意义.中华肾脏病杂志,2002,18(4):275-279.
15.董健平,陈香美,冯全洲,等.反义金属蛋白酶组织抑制剂-1对大鼠肾小管间质损害的治疗作用.中华医学杂志,2002,82(9):617-621.
16. Taube ME,Liu XW,Fridman R,et al.TIMP-1 regulation of cell cycleinhuman breast epithelial cells via stabilization of p27(KIP1) protein. Oncogene, 2006, 25 (21):3041-3048.
17.刘书馨,吕杨,陈香美,等.体外瞬时转染TIMP-1基因促进成纤维细胞增殖.中国中西医结合肾病杂志.2007,8(2):273-76.
18. Frazier K,Williams S,Kothapalli D,et al.Stimulation of fibroblast cell growth,matrix production,and granulation tissue formation by connective tissue growth factor.J Invest Dermatol,1996,107(3):404-411.
19. Beaudeux JL,Giral P,Bruckert E,et al. Matrix metalloproteinase, inflammation and atherosclerosis:therapeutic perspective.Clin Chem Lab Med,2004,42:121- 131.
20. Chromek M,Tullus K,Lundahl J,et al.Tissue inhibitor of metalloproteinase 1 activates normal human granulocytes,protects them from apoptosis,and blocks their transmigration during inflammation.Infect Immun,2004,72:82-88.
21.邵凤民,尚希瑶,陈香美,等.人基质金属蛋白酶1组织抑制剂转基因小鼠肾小管间质炎症的改变.中华肾脏病杂志,2006,22:688-692.
22. Li JH,Zhu HJ,Huang XR,et al.Smad7 inhibits fibrotic effect of TGF-Beta on renal tubular epithelial cells by blocking Smad2 activation.J Am Soc Nephrol, 2002,13(6):1464-1472.
23.张雪光,陈香美,洪权,等.人TIMP-1转基因小鼠随年龄肾组织炎症反应变化的意义.中华老年医学杂志,2005,25:916-921.
24. Verrecchia F,Chu ML,Mauviel A.Identification of novel TGF-beta/Smad gene targets in dermal fibroblasts using a combined cDNA microarray/promoter transactivation approach.J Biol Chem,2001,276,17058-17062.
25. Hall MC,Young DA,Waters JG,et al.The comparative role of activator protein 1 and Smad factors in the regulation of TIMP-1 gene expression by transforming growth factor-beta 1.J Biol Chem,2003,278:10304-10313.
26. Guangyan Cai, Xueguang Zhang, Quan Hong,et al. Tissue inhibitor of metalloproteinase-1 exacerbated renal interstitial fibrosis through enhancing inflammation.Nephrology Dialysis Transplantation 2008,23(6):1861-1875.
27. Zhang X,Chen X,Hong Q,et al.TIMP-1 promotes age-related renal fibrosis through upregulating ICAM-1 in human TIMP-1 transgenic mice.J Gerontol A Biol Sci Med Sci. 2006,61(11):1130-43.
28. Ritter LM,Garfield SH,Thorgeirsson UP.Tisue inhibitor of metalloproteinases-1 (TIMP-1) binds to the cell suface and translocates to the nucleus of human MCF-7 breast carcinoma cells.Biochem Biophys Res Commun,1999,257:494- 499.
29. Parks WC,Wilson CL,Lopez-Boado YS.Matrix metalloproteinases as modulators of inflammation and innate immunity.Nat Rev Immunol.2004,4:617-629.
30. Fiore E,Fusco C,Romero P,et al.Matrix metalloproteinase 9(MMP-9/gelatinase B) proteolytically cleaves ICAM-1 and participates in tumor cell resistance to natural killer cellmediated cytotoxicity.Oncogene,2002,21:5213-5223.
31. Kridel SJ,Chen E,Kortra LP,et al.Substrate hydrolysis by matrix metallo- proteinase-9.J Biol Chem,2001,276:20572-20578.
2. Nyengaard JR,Flyvbjerg A,Rasch R.The impact of renal growth,regression and regrowth in experimental diabetes mellitus on number and size of proximal and distal tubular cells in the rat kidney.Diabetologia,1993,36(11):1126-1131.
3. Chen FE,Huang DB,Chen YQ,et al.Crystal stucture of p50/p65 heterodimer of transcription factor NF-κB bound to DNA.Natrue,1998,391(22):410-413.
4. Tutde KR.Linking metablism and immunology:diabetic nephropatly is an inflammatory disease.J Am Soc Nephrol,2005: 16(6):1537-1538.
5. Richard W,Mankhey,Corinne C.Wells,et al.17β-Estradiol supplementation redu- ces tubulointerstitial fibrosis by increasing MMP activity in the diabetic kidney.Am J Physiol Regul Integr Comp Physiol,2007,292: R769-777.
6. Chow FY, Nikolic-Paterson DJ,Atkins RC,et al.Macrophages in streptozotocin- induced diabetic nephropathy:potential role in renal fibrosis. Nephrol Dial Transplant,2004 Dec;19(12):2987-2996.
7. Fornoni A, Ijaz A, Tejada T, et al.Role of inflammation in diabetic nephropathy. Curr Diabetes Rev,2008 Feb;4(1):10-17.
8. Zeisberg M, Kalluri R.The role of epithelial-to-mesenchymal transition in renal fibrosis.J Mol Med ,2004 ,82 :175-181.
9.孙艳玲,林洪丽,吴泰华,等.基质金属蛋白酶1组织抑制剂短发夹式RNA对肾小球系膜细胞凋亡及细胞外基质的影响.中华肾脏病杂志,2006,22 (6):364-369.
10.董建平,陈香美,师锁柱,等.金属蛋白酶1组织抑制剂在大鼠肾小管间质损害中的表达及其意义.中华肾脏病杂志,2002,18:275-279.
11.邵凤民,尚希瑶,陈香美等.人基质金属蛋白酶1组织抑制剂转基因小鼠肾小管间质炎症的改变.中华肾脏病杂志,2006,22:688-692.
12. Fiore E,Fusco C,Romero P,et al.Matrix metalloproteinase 9 (MMP-9/gelatinase B) proteolytically cleaves ICAM-1 and participates in tumor cell resistance to natural killer cell-mediated cytotoxicity.Oncogene,2002,21:5213-5223.
13. Manna SK,Aggarwal BB.Immunosuppressive leflunomide metabolite (A771726) blocks TNF-dependent nuclear factor-B activation and gene expression.J Immunol,1999,162:2095.
14.刘维萍,沈晓玉,彭勃,等.来氟米特对肾大部切除大鼠肾皮质α-平滑肌肌动蛋白的影响.中国药物与临床,2005,5:112-114.
15.陈琳,苟兴化,严律南,等.携带反义多药耐药相关蛋白的重组腺病毒载体的构建.四川大学学报(医学版),2003,34(1):131.
16.雷作熹,罗仁,董晓蕾,等.STZ诱导糖尿病肾病大鼠模型的建立.中国实验动物学报,2005,13(3): 163-166.
17. Arenz C,Schepers U.RNA interference:from an ancient mechanism to a statc of the art therapeutic application?Naturwissenschaften,2003,90:345-359.
18. 18Ichim TE,Li M,Qian H,et al.RNA interference:a potent tool for gene-specific therapcutics.Am J Transplant,2004,4:1227-1236.
19. Urushibara M,Takayanagi H,Koga T,et al.The antirheumatic drug leflunomide inhibits osteoclastogenesis by interfering with recepto ractivator of NF-kappa B ligand-stimulated induction of nuclear factor of activated T cells c1.Arthritis Rheum,2004,50(3):794-804.
20. Zeisberg M,Maeshima Y,Mosterman B,et al.Renal fibrosis:Extracellular matrix microenvironment regulates migratory behavior of activated tubular epithelial cells.Am J Pathol, 2002, 160 (6) : 2001-2008.
21. Lenz O,Elliot SJ,Stetler-Stevenson WG.Matrix metalloproteinase in renal development and disease.J Am Soc Nephrol,2000,11:574-581.
22. Beaudeux JL,Giral P,Bruckert E,et al.Matrix metalloproteinase, inflammation and atherosclerosis:therapeutic perspective.Clin Chem Lab Med,2004,42:121- 131.
23. Lam S,van der Geest RN,Verhagen NA,et al.Secretion of collagen typeⅣby human renal fibroblasts is increased by high glucose viaa TGF-beta-independent pathway.Nephrol Dial Transplant,2004,19(7):1694-1701.
24. Oh JH,Ha H,Yu MR,et al.Sequential effects of high glucose on mesangial cell transforming growth factor-beta1 and fibronectin synthesis.Kidney Int,1998,54 (6):1872-1878.
25. Parks WC,Wilson CL,Lopez-Boado YS.Matrix metalloproteinases as modulators of inflammation and innate immunity.Nat Rev Immunol,2004,4:617-629.
26. Kridel SJ,Chen E,Kortra LP,et al.Substrate hydrolysis by matrix metallo- proteinase-9.J Biol Chem,2001,276:20572-20578.
27. Cohen MP,Shea E,Chen S et al.Glycated albumin increases oxidative stress, activates NF-kappa B and extracellular signal-regulated kinase(ERK),andstimulates ERK-dependent transforming growth factor-beta 1 production in macrophage R A W cells.J Lab Clin Med,2003,141(4):242-249.
28.丁鹤林,王佑民,徐明彤,等.抑制NF-κB活性对糖尿病大鼠肾脏TGF-β1mRNA表达影响.中国药理学通报,2004,20(9):1020-1025.
29.刘维萍,林海英,田李清,等.来氟米特对肾大部切除大鼠肾皮质TGF-β1表达的影响.中原医刊,2004,31(23):1-2.
30. Goumenos D S,Brown CB,Shortland J,et al.Myofibroblasts,predictors of progression of mesangial IgA nephropathy.Nephrol Dial Transplant,1994,9: 1418.
31. Yang N,Wu L L,Nicolic-Paterson D J,et al.Local macrophage and fibroblast proliferation in progressive renal injury in the rat remnant kidney.Nephrol Dial Transplant,1998,13(8):1967.
32. Sharma k,Morris RE,Huang X,et al.Inhibition of arterial intimal thickening by leflunomide:in vivo efficacy and molecular mechanisms.Am Soc Trans Surg, 1994,1:97-105.
1. Eddy AA.Molecular casis of renal fibrosis.Pediatr.Nephrol,2000,15(3-4): 290- 301.
2. Liu,Y.Renal fibrosis:new insights into the pathogenesis and therapeutics.Kidney International,2006,69(2):213-217.
3.罗海清,梁东,刘华锋.肾间质纤维化的形成机制及中药防治作用.中国中西医结合肾病杂志,2004,5(7):432-434.
4. Kuncio GS,Neilson EG.Haverty T.Mechanisms of tubulointerstitial fibrosis. Kidney Int,1991,39(3):550-556.
5.陈荣权,陈香美,崔世维,等.组织金属蛋白酶抑制物在老年大鼠肾小管间质病理损害中的动态变化.中华老年医学杂志,2004,23(4):253-258.
6. Lenz O Elliot SJ,Stetler-Stevenson WG.Matrix metalloproteinase in renal development and disease.J Am Soc Nephrol,2000,11:574-581.
7. Gonzalez-AvilaG,Iturria C,Vadillo-Ortega F,et al.Changes in matrix metallo- proteinase during the evolution of interstitial renal fibrosis in a rat experimental model.Pathorbiology,1998,66(5):196-204.
8. Sharma AK,Mauer SM,Kim Y,et al.Altered expression of matrix metallo- proteinase-2,TIMP-1 and TIMP-2 obstructive nephropathy.J Lab Clin Med, 1995, 125(6):754-761.
9. Johnson TS,Haylor JL,Thomas GL,et al.Matrix metallor proteinases and their inhibitions in experimental renal scarring.Exp Nephrol,2002,10(3):182-195.
10. Duymelinck C,Deng JT,Dauwe SE,et al.Inhibition of the matrix metallo -proteinase system in a rat model of chronic cyclosporine nephropahy.Kidney Int,1998,54(3):804-818
11. Nakamura T,Ushiyama C,Suzuki S,et al.Elevation of serum levels of metalloproteinase-1,tissue inhibitor of melalloproteinase-1 and typeⅣcollagen and plasma levels of metalloproteinase-9 in polycystic kidney disease.Am J Nephrol,2000,20(1):32-36.
12. Ishidoya S,Morrissey J,McCracken R,et al.Delayedt reatment with enalapril halts tubulointerst itial fibrosis in rats with obstructive nephropathy.Kidney Int, 1996,49(4):1110-1119.
13. Tang WW,Feng L,XiaY,et al.Extracellular matrix accumulation in immune- mediated tubulointerstitial injury.Kidney Int,1994,45(4):1077-1084.
14.董健平,陈香美,师锁柱,等.金属蛋白酶组织抑制剂1在大鼠肾小管间质损害中的表达及其意义.中华肾脏病杂志,2002,18(4):275-279.
15.董健平,陈香美,冯全洲,等.反义金属蛋白酶组织抑制剂-1对大鼠肾小管间质损害的治疗作用.中华医学杂志,2002,82(9):617-621.
16. Taube ME,Liu XW,Fridman R,et al.TIMP-1 regulation of cell cycleinhuman breast epithelial cells via stabilization of p27(KIP1) protein. Oncogene, 2006, 25 (21):3041-3048.
17.刘书馨,吕杨,陈香美,等.体外瞬时转染TIMP-1基因促进成纤维细胞增殖.中国中西医结合肾病杂志.2007,8(2):273-76.
18. Frazier K,Williams S,Kothapalli D,et al.Stimulation of fibroblast cell growth,matrix production,and granulation tissue formation by connective tissue growth factor.J Invest Dermatol,1996,107(3):404-411.
19. Beaudeux JL,Giral P,Bruckert E,et al. Matrix metalloproteinase, inflammation and atherosclerosis:therapeutic perspective.Clin Chem Lab Med,2004,42:121- 131.
20. Chromek M,Tullus K,Lundahl J,et al.Tissue inhibitor of metalloproteinase 1 activates normal human granulocytes,protects them from apoptosis,and blocks their transmigration during inflammation.Infect Immun,2004,72:82-88.
21.邵凤民,尚希瑶,陈香美,等.人基质金属蛋白酶1组织抑制剂转基因小鼠肾小管间质炎症的改变.中华肾脏病杂志,2006,22:688-692.
22. Li JH,Zhu HJ,Huang XR,et al.Smad7 inhibits fibrotic effect of TGF-Beta on renal tubular epithelial cells by blocking Smad2 activation.J Am Soc Nephrol, 2002,13(6):1464-1472.
23.张雪光,陈香美,洪权,等.人TIMP-1转基因小鼠随年龄肾组织炎症反应变化的意义.中华老年医学杂志,2005,25:916-921.
24. Verrecchia F,Chu ML,Mauviel A.Identification of novel TGF-beta/Smad gene targets in dermal fibroblasts using a combined cDNA microarray/promoter transactivation approach.J Biol Chem,2001,276,17058-17062.
25. Hall MC,Young DA,Waters JG,et al.The comparative role of activator protein 1 and Smad factors in the regulation of TIMP-1 gene expression by transforming growth factor-beta 1.J Biol Chem,2003,278:10304-10313.
26. Guangyan Cai, Xueguang Zhang, Quan Hong,et al. Tissue inhibitor of metalloproteinase-1 exacerbated renal interstitial fibrosis through enhancing inflammation.Nephrology Dialysis Transplantation 2008,23(6):1861-1875.
27. Zhang X,Chen X,Hong Q,et al.TIMP-1 promotes age-related renal fibrosis through upregulating ICAM-1 in human TIMP-1 transgenic mice.J Gerontol A Biol Sci Med Sci. 2006,61(11):1130-43.
28. Ritter LM,Garfield SH,Thorgeirsson UP.Tisue inhibitor of metalloproteinases-1 (TIMP-1) binds to the cell suface and translocates to the nucleus of human MCF-7 breast carcinoma cells.Biochem Biophys Res Commun,1999,257:494- 499.
29. Parks WC,Wilson CL,Lopez-Boado YS.Matrix metalloproteinases as modulators of inflammation and innate immunity.Nat Rev Immunol.2004,4:617-629.
30. Fiore E,Fusco C,Romero P,et al.Matrix metalloproteinase 9(MMP-9/gelatinase B) proteolytically cleaves ICAM-1 and participates in tumor cell resistance to natural killer cellmediated cytotoxicity.Oncogene,2002,21:5213-5223.
31. Kridel SJ,Chen E,Kortra LP,et al.Substrate hydrolysis by matrix metallo- proteinase-9.J Biol Chem,2001,276:20572-20578.