整合素连接激酶及相关因子在肾间质纤维化大鼠模型中的表达及意义
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摘要
目的:肾间质纤维化(RIF)是由多种原因引起的细胞外基质(ECM)在肾间质内过度沉积和间质成纤维细胞的增生而形成,是各种肾脏疾病进展到终末期的共同途径和主要病理基础。表达α-SMA的肌成纤维细胞(MyoF)是合成ECM的主要细胞,已有研究表明在RIF过程中约36%的MyoF是由小管上皮细胞转化而来,这过程称为小管上皮细胞转分化(EMT)。TGF-β1是公认的致纤维化因子,能通过多种信号传导途径诱导EMT的发生和发展。ILK是其信号传导途径的效应分子,介导了TGF-β1诱导肾小管EMT的各个关键步骤,最终导致肾间质纤维化的发生。本研究主要通过组织病理学观察、免疫组织化学和RT-PCR方法,了解ILK、α-SMA及TGF-β1在肾间质纤维化动物模型中的表达,探讨它们与肾间质纤维化形成的关系。
方法:1.肾间质纤维化动物模型的建立:选取清洁级SD大鼠72只,随机分为三组:正常组、手术组、假手术组,手术组采用单侧输尿管结扎方法,建立肾间质纤维化模型,即UUO模型,手术组大鼠常规麻醉后在近左肾下极处游离并结扎左输尿管;假手术组大鼠仅行开腹术游离左输尿管;正常组不做任何处理。各组大鼠分别于术后1、3、7、14天处死,取其左肾,观察左肾大体形态,光镜下观察肾脏病理改变及间质纤维化程度。2.用免疫组织化学技术检测肾间质中的ILK和TGF-β1、α-SMA的表达情况,进一步分析各组之间ILK和TGF-β1、α-SMA表达量与肾间质损伤程度之间的关系,以及ILK和TGF-β1、α-SMA彼此之间的相关关系。3.用逆转录方法半定量测定ILKmRNA在UUO模型中的表达量,并分析其与肾间质损害程度的关系。
结果:1.采用单侧输尿管结扎术构建肾间质纤维化动物模型成功,未见大鼠死亡。2.肾组织形态学改变:与对照组相、假手术组比,UUO组大鼠肾脏明显肿大,肾皮质变薄,肾盂积水,并随着时限的延长而加重。3.免疫组化分析显示:ILK在正常肾组织中几乎无阳性表达,UUO模型中,ILK主要表达在肾间质,随着肾间质病变程度加重,其阳性表达面积显著增加,各组之间表达有显著性差异(P<0.001),与肾间质病变程度呈显著正相关关系(r=0.842,P<0.001);TGF-β1在正常肾间质中仅有微量的表达,随着肾间质病变程度的加重表达逐渐增加,各组之间TGF-β1的表达有显著性差异(P<0.001),和肾间质病变程度呈显著正相关关系(r=0.892,P<0.001) ;α-SMA在正常肾组织中,仅表达于血管平滑肌肌层,在肾小管上皮细胞胞浆有微量表达,随肾间质病变加重表达增强各组之间α-SMA的表达有显著性差异(P<0.001),和肾间质病变程度之间有正相关关系(r=0.854,P<0.001)。
结论:1.ILK作为TGF-β1的下游信号因子参与EMT,EMT可能是致肾间质纤维化的一个重要方式。2.TGF-β1可能在RIF的发生中起重要作用,EMT可能是TGF-β1致肾间质纤维化的一个重要途径。3.α-SMA作为MyoF的标志蛋白参与EMT,在肾间质纤维化发生中起重要作用。4.在各种病变程度的肾小管间质中,ILK和TGF-β1、α-SMA的表达有正相关关系,推测ILK可能在TGF-β1介导的EMT过程中起到重要的作用。
Objective: Renal interstitial fibrosis(RIF) is caused and formed by many kinds of reasons that extra cellular matrix(ECM) is excessive accumulation and myofibroblast(Myof) is excessive proliferation in renal interstitium. Renal interstitial fibrosis is common pathway and major pathology foundation which is various kinds of renal diseases to evolve the end stage of renal disease. Myofibroblast that expressα- smooth muscle actin is major effective cell to compose extra cellular matrix. Emerging evidence suggests that in the process of renal interstitial fibrosis, about thirty six percentage of myofibroblast come from tubular epithelial cell to change myofibroblast. This process is named epithelial to myofibroblast transdifferentiation(EMT). Transform growth factor-β1(TGF-β1) is generally acknowledged to cause fibrosis factor. It may induce that extra cellular matrix happen and develop through many signal conduct path. Integrin -linked kinase(ILK) is major effective cell of signal conduct path of transform growth factor-β1.It participate kinds of committed step that transform growth factor-β1 induce epithelial to myofibroblast transdifferentiation.To cause renal interstitial fibrosis happening.To observe histopathology and immunohistochemistry ,to do RT-PCR method ,we can comprehend that expression of ILK and TGF-β1、α- smooth muscle actin in the animal modelof renal interstitial fibrosis,and investigate relationship between them and renal interstitial fibrosis. Methods:
1. Construction of the animal modelof renal interstitial fibrosis: to select clearing SD rat seventy two,to divide randomly thrid group:control group, sham operated group, operated goup. Operated goup use unilateral ureter obstruction experiment method ,named UUO. operated goup. ligate left ureter which adjoin left renal anus perineum ;sham operated group only open abdominal cavity,and dissociation left ureter. Thrid group rat was sacrificed respectively after operatation in ond day ,thrid day, seven day , fourteen day .we take them left renal.To observe the left renal shap by our eye and renal histopathology and the degree of renal interstitial fibrosis
2.Using immunohistochemistry method to detect expression of ILK and correlation factor in the animal model of renal interstitial fibrosis and to analyze relationship between ILK and TGF-β1、α- smooth muscle actin and renal interstitial fibrosis.
3. Using RT-PCR method to detect expression of ILK mRNA in the animal model of renal interstitial fibrosis and to analyze relationship between ILK mRNA and renal interstitial fibrosis.
Results:
1.Construction of the animal model of renal interstitial fibrosis is success by ureter obstruction experiment method.
2. Renal histopathology change : to compare control group , the renal of sham operated group and operated.goup is significantly intumescence and thinningz renal cortex .There are a great quantity urine to exist in the renal pelvis and to accumulate more and more with time prolong.
3.Using immunohistochemistry method to detect expression of ILK and TGF-β1、α- smooth muscle actin: ILK was rarely detected in control group, and the expression of ILK increased gradually in other groups with the degree of renal interstitial pathological injury (r=0.842,P<0.001). A significant positive correlation was found between the expression of ILK in renal tubulointerstitial tissue and the degree of renal tubulointerstitial pathological injury ( P<0.001). (2) TGF-β1 was rarely detected in control group, and the expression of TGF-β1 increased gradually in other groups with the degree of renal interstitial pathological injury (P<0.001). A significant positive correlation was found between the expression of TGF-β1 in renal tubulointerstitial tissue and the degree of renal tubulointerstitial pathological injury (r=0.892, P<0.001). (3) At normal control group, Only expressesα-SMA in the blood vessel smooth muscle has very few expression the renal interrenal .Aggravates the expression enhancement along with the renal interrenal pathological change, A significant positive correlation was found between the expression ofα- SMA in renal tubulointerstitial tissue and the degree of renal tubulointerstitial pathological injury (r=0.854,P<0.001).
Conclusion:
1.ILK as major effective cell of signal conduct path of transform growth factor-β1 participate kinds of committed step that transform growth factor-β1 induce epithelial to myofibroblast transdifferentiation.EMT may be a important way to lead to renal interstitial fibrosis(RIF).
2.TGF-β1 appear to be important in the genesis of renal interstitial fibrosis. TGF-β1 might induces renal interstitial fibrosis by EMT.
3.α-SMA as labelling proteinum of myofibroblast participate kinds of committed step that transform growth factor-β1 induce epithelial to myofibroblast transdifferentiation.α-SMA play an important role in ithe process of renal interstitial fibrosis.
4.In all degrees of renal interstitial pathological injury, A significant positive correlation was found between the expression of ILK and TGF-β1α-SMA. ILK may be participate in the formation of renal interstitial fibrosis, via the induction renal tubular epithelial to myofibroblast transformation (EMT).
方法:1.肾间质纤维化动物模型的建立:选取清洁级SD大鼠72只,随机分为三组:正常组、手术组、假手术组,手术组采用单侧输尿管结扎方法,建立肾间质纤维化模型,即UUO模型,手术组大鼠常规麻醉后在近左肾下极处游离并结扎左输尿管;假手术组大鼠仅行开腹术游离左输尿管;正常组不做任何处理。各组大鼠分别于术后1、3、7、14天处死,取其左肾,观察左肾大体形态,光镜下观察肾脏病理改变及间质纤维化程度。2.用免疫组织化学技术检测肾间质中的ILK和TGF-β1、α-SMA的表达情况,进一步分析各组之间ILK和TGF-β1、α-SMA表达量与肾间质损伤程度之间的关系,以及ILK和TGF-β1、α-SMA彼此之间的相关关系。3.用逆转录方法半定量测定ILKmRNA在UUO模型中的表达量,并分析其与肾间质损害程度的关系。
结果:1.采用单侧输尿管结扎术构建肾间质纤维化动物模型成功,未见大鼠死亡。2.肾组织形态学改变:与对照组相、假手术组比,UUO组大鼠肾脏明显肿大,肾皮质变薄,肾盂积水,并随着时限的延长而加重。3.免疫组化分析显示:ILK在正常肾组织中几乎无阳性表达,UUO模型中,ILK主要表达在肾间质,随着肾间质病变程度加重,其阳性表达面积显著增加,各组之间表达有显著性差异(P<0.001),与肾间质病变程度呈显著正相关关系(r=0.842,P<0.001);TGF-β1在正常肾间质中仅有微量的表达,随着肾间质病变程度的加重表达逐渐增加,各组之间TGF-β1的表达有显著性差异(P<0.001),和肾间质病变程度呈显著正相关关系(r=0.892,P<0.001) ;α-SMA在正常肾组织中,仅表达于血管平滑肌肌层,在肾小管上皮细胞胞浆有微量表达,随肾间质病变加重表达增强各组之间α-SMA的表达有显著性差异(P<0.001),和肾间质病变程度之间有正相关关系(r=0.854,P<0.001)。
结论:1.ILK作为TGF-β1的下游信号因子参与EMT,EMT可能是致肾间质纤维化的一个重要方式。2.TGF-β1可能在RIF的发生中起重要作用,EMT可能是TGF-β1致肾间质纤维化的一个重要途径。3.α-SMA作为MyoF的标志蛋白参与EMT,在肾间质纤维化发生中起重要作用。4.在各种病变程度的肾小管间质中,ILK和TGF-β1、α-SMA的表达有正相关关系,推测ILK可能在TGF-β1介导的EMT过程中起到重要的作用。
Objective: Renal interstitial fibrosis(RIF) is caused and formed by many kinds of reasons that extra cellular matrix(ECM) is excessive accumulation and myofibroblast(Myof) is excessive proliferation in renal interstitium. Renal interstitial fibrosis is common pathway and major pathology foundation which is various kinds of renal diseases to evolve the end stage of renal disease. Myofibroblast that expressα- smooth muscle actin is major effective cell to compose extra cellular matrix. Emerging evidence suggests that in the process of renal interstitial fibrosis, about thirty six percentage of myofibroblast come from tubular epithelial cell to change myofibroblast. This process is named epithelial to myofibroblast transdifferentiation(EMT). Transform growth factor-β1(TGF-β1) is generally acknowledged to cause fibrosis factor. It may induce that extra cellular matrix happen and develop through many signal conduct path. Integrin -linked kinase(ILK) is major effective cell of signal conduct path of transform growth factor-β1.It participate kinds of committed step that transform growth factor-β1 induce epithelial to myofibroblast transdifferentiation.To cause renal interstitial fibrosis happening.To observe histopathology and immunohistochemistry ,to do RT-PCR method ,we can comprehend that expression of ILK and TGF-β1、α- smooth muscle actin in the animal modelof renal interstitial fibrosis,and investigate relationship between them and renal interstitial fibrosis. Methods:
1. Construction of the animal modelof renal interstitial fibrosis: to select clearing SD rat seventy two,to divide randomly thrid group:control group, sham operated group, operated goup. Operated goup use unilateral ureter obstruction experiment method ,named UUO. operated goup. ligate left ureter which adjoin left renal anus perineum ;sham operated group only open abdominal cavity,and dissociation left ureter. Thrid group rat was sacrificed respectively after operatation in ond day ,thrid day, seven day , fourteen day .we take them left renal.To observe the left renal shap by our eye and renal histopathology and the degree of renal interstitial fibrosis
2.Using immunohistochemistry method to detect expression of ILK and correlation factor in the animal model of renal interstitial fibrosis and to analyze relationship between ILK and TGF-β1、α- smooth muscle actin and renal interstitial fibrosis.
3. Using RT-PCR method to detect expression of ILK mRNA in the animal model of renal interstitial fibrosis and to analyze relationship between ILK mRNA and renal interstitial fibrosis.
Results:
1.Construction of the animal model of renal interstitial fibrosis is success by ureter obstruction experiment method.
2. Renal histopathology change : to compare control group , the renal of sham operated group and operated.goup is significantly intumescence and thinningz renal cortex .There are a great quantity urine to exist in the renal pelvis and to accumulate more and more with time prolong.
3.Using immunohistochemistry method to detect expression of ILK and TGF-β1、α- smooth muscle actin: ILK was rarely detected in control group, and the expression of ILK increased gradually in other groups with the degree of renal interstitial pathological injury (r=0.842,P<0.001). A significant positive correlation was found between the expression of ILK in renal tubulointerstitial tissue and the degree of renal tubulointerstitial pathological injury ( P<0.001). (2) TGF-β1 was rarely detected in control group, and the expression of TGF-β1 increased gradually in other groups with the degree of renal interstitial pathological injury (P<0.001). A significant positive correlation was found between the expression of TGF-β1 in renal tubulointerstitial tissue and the degree of renal tubulointerstitial pathological injury (r=0.892, P<0.001). (3) At normal control group, Only expressesα-SMA in the blood vessel smooth muscle has very few expression the renal interrenal .Aggravates the expression enhancement along with the renal interrenal pathological change, A significant positive correlation was found between the expression ofα- SMA in renal tubulointerstitial tissue and the degree of renal tubulointerstitial pathological injury (r=0.854,P<0.001).
Conclusion:
1.ILK as major effective cell of signal conduct path of transform growth factor-β1 participate kinds of committed step that transform growth factor-β1 induce epithelial to myofibroblast transdifferentiation.EMT may be a important way to lead to renal interstitial fibrosis(RIF).
2.TGF-β1 appear to be important in the genesis of renal interstitial fibrosis. TGF-β1 might induces renal interstitial fibrosis by EMT.
3.α-SMA as labelling proteinum of myofibroblast participate kinds of committed step that transform growth factor-β1 induce epithelial to myofibroblast transdifferentiation.α-SMA play an important role in ithe process of renal interstitial fibrosis.
4.In all degrees of renal interstitial pathological injury, A significant positive correlation was found between the expression of ILK and TGF-β1α-SMA. ILK may be participate in the formation of renal interstitial fibrosis, via the induction renal tubular epithelial to myofibroblast transformation (EMT).
引文
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[6] Kondo S ,Kagami S ,Kido H ,et al. Role of mast cell tryptase in renal interstitial fibrosis.[J] Am Soc Nephol ,2001,12(8) :1668 - 1976.
[7] Okada H , Inoue T , Suzuki H , et al. Epithelial-mesenchymal transformation of renal tubular epithelial cells in vitro and in vivo [J] Nephrol Dial Transplant ,2000 ,15 (suppl6):44 - 46.
[8] Badid C.Mounier N.Costa AM. Desmoulere.Role of myofibroblasts during normal tissue repair and excessive scarring: interest of their assessment in nephropathies. [J] Histol Histopathol 2000;l5(1),269—280
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[10] Liu YH. Epithelial to mesenchymal transition in renal fibrogenesis : pathologic significance , molecular mechanism , andtherapeutic intervention.[ J ]Am Soc Nephrol ,2004 ,15 (1):1- 12.
[11]Yang J , Liu Y. Dissection of key events in tubular epithelial to myofibroblast transition and its implications in renal interstitial fibrosis. [J] Am Pat hol , 2001 ,159 (4) :1465-1475
[12] Rastaldi MP. Epithelial-mesenchymal transition and its implications for the development of renal tubulointerstitial fibrosis. [ J] Nephrol , 2006, 19(4):407-412.
[13]Li JH, Zhu HJ, Huang XR, et al Smad7 inhibits fibrotic effect of TGF-βon renal Tubular epithelial cells by blocking Smad2 activation [J] Am Soc Nephrol 2003,14(9):1464-1472
[14] Liu Y. Epithelial to mesenchymal transition in renal fibrogenesis pathologic significance , molecular mechanism , and therapeutic intervention.[ J ]Am Soc Nephrol , 2004,15(1):1-12
[15] Wang W,Huang XR ,et al. Signaling mechanism of TGF-beta1 in prevention of renal inflammation: role of Smad7.[ J ] Am Soc Nephrol ,2005 ,16(5):1371 - 1383.
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[1] Tang WW,Van GY, Q IM Myofilbroblast and alphale 1 collagen expression in experimental tubulointersititial nephritis Kidney Int. 1999,51(4):926-930
[2] 何玉华、冯梅、梁勇等 肾小管上皮细胞表型转化在肾小管间质纤维化中的作用 [J] 中国中西医结合肾病杂志 2005 5(6)308-311
[3]Klahr S,Morrissey J,Hruska K,et a1.New approaches to delay the progression of chronic renal failure.[J]Kidney Int.2002,80(supp 1):23—26
[4] Eddy AA. Molecular basis renal fibrosis. Pediatr [J] Nephrol , 2000 ,15(34) :290 - 301.
[5]罗海清、梁冬、刘华锋 肾间质纤维化的形成机制及中药防治作用 [J] 中国中西医结合肾病杂志 2004 5(7)432-434
[6] Kondo S ,Kagami S ,Kido H ,et al. Role of mast cell tryptase in renal interstitial fibrosis.[J] Am Soc Nephol ,2001,12(8) :1668 - 1976.
[7] Okada H , Inoue T , Suzuki H , et al. Epithelial-mesenchymal transformation of renal tubular epithelial cells in vitro and in vivo [J] Nephrol Dial Transplant ,2000 ,15 (suppl6):44 - 46.
[8] Badid C.Mounier N.Costa AM. Desmoulere.Role of myofibroblasts during normal tissue repair and excessive scarring: interest of their assessment in nephropathies. [J] Histol Histopathol 2000;l5(1),269—280
[9] Zeisberq M , Bonner G, Maeshima Y, et al. Renal Fibrosis : Collagen Composition and Assembly Regulates Epithelial - Mesenchymal Transdifferentiation. [J] Am Pathol ,2001 ,159(4) :1313 - 1321.
[10] Liu YH. Epithelial to mesenchymal transition in renal fibrogenesis : pathologic significance , molecular mechanism , andtherapeutic intervention.[ J ]Am Soc Nephrol ,2004 ,15 (1):1- 12.
[11]Yang J , Liu Y. Dissection of key events in tubular epithelial to myofibroblast transition and its implications in renal interstitial fibrosis. [J] Am Pat hol , 2001 ,159 (4) :1465-1475
[12] Rastaldi MP. Epithelial-mesenchymal transition and its implications for the development of renal tubulointerstitial fibrosis. [ J] Nephrol , 2006, 19(4):407-412.
[13]Li JH, Zhu HJ, Huang XR, et al Smad7 inhibits fibrotic effect of TGF-βon renal Tubular epithelial cells by blocking Smad2 activation [J] Am Soc Nephrol 2003,14(9):1464-1472
[14] Liu Y. Epithelial to mesenchymal transition in renal fibrogenesis pathologic significance , molecular mechanism , and therapeutic intervention.[ J ]Am Soc Nephrol , 2004,15(1):1-12
[15] Wang W,Huang XR ,et al. Signaling mechanism of TGF-beta1 in prevention of renal inflammation: role of Smad7.[ J ] Am Soc Nephrol ,2005 ,16(5):1371 - 1383.
[16] Sato M,Muragaki Y, Saika S, Targeted disruption of TGF-beta1/Smad3 signaling protects against renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction. [J]clin Invest 2003 112(10):1486-1494
[17] 秦晓华, 涂卫平 TGF2β/ Smad 信号通路与肾脏疾病 [J ] 江西医学院学报2005, 45(6):191-194
[18] Yang Y, Guo L,Wu C ,et al Formation and Phosphorylation of the PINCH-1–Integrin Linked Kinase–Parvin Complex Are Important for Regulation of Renal Glomerular Podocyte Adhesion,Architecture, and Survival [J] Am Soc Nephrol 2005 16(7): 1966–1976,
[19] Miller MG, Naruszewicz I,Kumar AS,et al Integrin-linked kinase is a positive mediator of L6 myoblast differentiation [J] Elsevier Inc. 2003 310(3):796–803
[20]Li Y, Yang J, Dai C, et al. Role for integrin-linked kinase in mediating tubular epithelial to mesenchymal transition and renal interstitial fibrogenesis[J]. Clin Invest, 2003, 112(4):503 - 516.
[21] 陈秋, 张璟, 敖绪军 整合素连接激酶在人肾间质纤维化组织中的表达及意义[J] 第三军医大学学报 2005(11):2262-2266 [ 22] Grashoff C, Thievessen I, et al Integrin-linked kinase: integrin’s mysterious partner[J] Curr Opin Cell Biol 2004, 16(5) :565–571
[23]Geleilete TJ,Melo CC,Costa RS,et a1.Role of myofibroblasts,macrophages transforming growth factor—β,endothelin,angioterrsin—II,and fibronectin in the progression of tubulointerstitial nephritis induced by gentamicin.[J] Nephrol,2002,15(6):633—642.
[24] Ogata Y,lshidoya S,Fukuzaki A,et a1.Upregulated expression transforming growth factor—beta,type IV collagen,and plasminogen activator inhibitor一1 mRNA are decreased after release of unilateral ureteral obstruction.[J ]Exp Med,2002,197(3):159—168.