肝再生增强因子对大鼠肾小管上皮细胞转分化影响的研究
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摘要
目的:利用已构建的大鼠肝再生增强因子(rALR)毕赤酵母(pichia pastoris)重组表达质粒pPIC9K-rALR,在酵母菌GS115中诱导表达rALR,并进行纯化和体外生物学活性鉴定,为进一步研究rALR的生物学功能提供基础。
方法:经PCR鉴定的重组质粒pPIC9K-rALR酶切线性化后转入宿主菌GS115中,在甲醇诱导下进行表达。表达产物rALR经超滤方法纯化后,用15% SDS-PAGE和Western blot印迹鉴定。采用3H-TdR掺入法,观察rALR在体外对人肝癌细胞株QGY的促增殖作用以鉴定其活性。
结果:目的蛋白rALR以外分泌方式在宿主菌GS115中获得高效表达,其分子量约为15KD,与预计理论值相符合,超滤纯化得到的目的蛋白经SDS-PAGE和Western bolt鉴定为单一条带,在体外rALR确能刺激QGY增殖,而且这种刺激作用存在着量效关系。
结论:rALR在毕赤酵母菌中被高效表达和成功纯化,在体外rALR确能以剂量依赖关系刺激人肝癌细胞株QGY增殖。
目的:探讨重组大鼠肝再生因子(rrALR)对转化生长因子β1(TGF-β1)诱导的肾小管上皮细胞转分化的影响。
方法:观察细胞形态学变化,检测α平滑肌肌动蛋白(α-SMA)、E-钙粘蛋白(E-cadherin)和结缔组织生长因子(CTGF)蛋白及mRNA的表达水平。
结果:NRK-52E细胞在加入TGF-β1刺激培养后,细胞形态由方形或多角形变为梭形成纤维细胞;α-SMA和CTGF的mRNA及蛋白表达增加,E-cadherin mRNA及蛋白表达明显降低。而TGF-β1和rrALR共培养组,多数细胞保持正常上皮细胞形态,NRK52E细胞转分化程度减轻,α-SMA和CTGF蛋白及mRNA表达增高程度高于正常对照组,但明显低于TGF-β1刺激组(PO.05)。
结论:rrALR能抑制TGF-β1诱导的NRK52E2细胞转分化,减少细胞CTGF表达。
Objective: To express rALR in GS115 with constructed pichia pastoris expression plasmid of rat augmenter of liver regeneration (rALR) and purify it by ultrafiltration and identify its bioactivity in vitro, for studying its biological functions.
Methods: The recombinant plasmid pPIC9K-rALR was transformed into GS115 by electroporation. The rALR was expressed in GS115 under the induction of methanol and purified with ultrafiltration. The purified product was identified by 15% SDS-PAGE and Western blot. The effects of rALR on the proliferations of QGY were evaluated by 3H-TdR methods in vitro.
Results: The rALR was efficiently expressed as a secretive protein in GS115. Its molecular weight(1.5×104 dalton) was in correspondance with theoretic values. Single band of rALR was observed in SDS-PAGE electrophoresis and in western blot after ultrafiltration. The rALR promoted the proliferation of QGY by a dose-dependent way in vitro.
Conclusion: The rALR was efficiently expressed in GS115 and successfully purified by ultrafiltration. The proliferation of QGY was stimulated by rALR in vitro with a dose-dependent way.
Objective: To investigate the effect of recombination rat augmenter of liver regeneration(rrALR) on TGF-β1-induced renal tubular epithelial cells transdifferentiation(TEMT). The cells were cultured for 48 hours. Morphological change of cells
Methods: The cells of normal rat kidney tubular epithelial cell line(NRK-52E) were divided into four groups, normal control cells, rrALR treated cells, TGF-β1 treated cells, TGF-β1 plus rrALR treated cells. was observed and expressions ofα-SMA, CTGF, E-cadherin protein and mRNA were analysed.
Results: The results showed that TGF-β1 induced cell morphological change of NRK-52E as the development of an elongated and fusiforill appearance, and up-regulated protein and mRNA expressions ofα-SMA, CTGF and down-regulated E-cadherin expression. Transformation of tubular epithelial cells was inhibited in TGF-β1 plus rrALR group as compared with TGF-β1 treated group. The protein and mRNA expression of E-cadherin was increased and levels ofα-SMA and CTGF were decreased in cells exposed TGF-β1 plus rrALR medium.
Conclusion: 1.There was no transdifferentiating effect when NRK52E cells cultured with rALR alone. 2. rrALR may inhibit transdifferentiation of NRK52E cells into myofibroblast-like cells and CTGF protein and mRNA expression induced by TGF-β1.
方法:经PCR鉴定的重组质粒pPIC9K-rALR酶切线性化后转入宿主菌GS115中,在甲醇诱导下进行表达。表达产物rALR经超滤方法纯化后,用15% SDS-PAGE和Western blot印迹鉴定。采用3H-TdR掺入法,观察rALR在体外对人肝癌细胞株QGY的促增殖作用以鉴定其活性。
结果:目的蛋白rALR以外分泌方式在宿主菌GS115中获得高效表达,其分子量约为15KD,与预计理论值相符合,超滤纯化得到的目的蛋白经SDS-PAGE和Western bolt鉴定为单一条带,在体外rALR确能刺激QGY增殖,而且这种刺激作用存在着量效关系。
结论:rALR在毕赤酵母菌中被高效表达和成功纯化,在体外rALR确能以剂量依赖关系刺激人肝癌细胞株QGY增殖。
目的:探讨重组大鼠肝再生因子(rrALR)对转化生长因子β1(TGF-β1)诱导的肾小管上皮细胞转分化的影响。
方法:观察细胞形态学变化,检测α平滑肌肌动蛋白(α-SMA)、E-钙粘蛋白(E-cadherin)和结缔组织生长因子(CTGF)蛋白及mRNA的表达水平。
结果:NRK-52E细胞在加入TGF-β1刺激培养后,细胞形态由方形或多角形变为梭形成纤维细胞;α-SMA和CTGF的mRNA及蛋白表达增加,E-cadherin mRNA及蛋白表达明显降低。而TGF-β1和rrALR共培养组,多数细胞保持正常上皮细胞形态,NRK52E细胞转分化程度减轻,α-SMA和CTGF蛋白及mRNA表达增高程度高于正常对照组,但明显低于TGF-β1刺激组(P
结论:rrALR能抑制TGF-β1诱导的NRK52E2细胞转分化,减少细胞CTGF表达。
Objective: To express rALR in GS115 with constructed pichia pastoris expression plasmid of rat augmenter of liver regeneration (rALR) and purify it by ultrafiltration and identify its bioactivity in vitro, for studying its biological functions.
Methods: The recombinant plasmid pPIC9K-rALR was transformed into GS115 by electroporation. The rALR was expressed in GS115 under the induction of methanol and purified with ultrafiltration. The purified product was identified by 15% SDS-PAGE and Western blot. The effects of rALR on the proliferations of QGY were evaluated by 3H-TdR methods in vitro.
Results: The rALR was efficiently expressed as a secretive protein in GS115. Its molecular weight(1.5×104 dalton) was in correspondance with theoretic values. Single band of rALR was observed in SDS-PAGE electrophoresis and in western blot after ultrafiltration. The rALR promoted the proliferation of QGY by a dose-dependent way in vitro.
Conclusion: The rALR was efficiently expressed in GS115 and successfully purified by ultrafiltration. The proliferation of QGY was stimulated by rALR in vitro with a dose-dependent way.
Objective: To investigate the effect of recombination rat augmenter of liver regeneration(rrALR) on TGF-β1-induced renal tubular epithelial cells transdifferentiation(TEMT). The cells were cultured for 48 hours. Morphological change of cells
Methods: The cells of normal rat kidney tubular epithelial cell line(NRK-52E) were divided into four groups, normal control cells, rrALR treated cells, TGF-β1 treated cells, TGF-β1 plus rrALR treated cells. was observed and expressions ofα-SMA, CTGF, E-cadherin protein and mRNA were analysed.
Results: The results showed that TGF-β1 induced cell morphological change of NRK-52E as the development of an elongated and fusiforill appearance, and up-regulated protein and mRNA expressions ofα-SMA, CTGF and down-regulated E-cadherin expression. Transformation of tubular epithelial cells was inhibited in TGF-β1 plus rrALR group as compared with TGF-β1 treated group. The protein and mRNA expression of E-cadherin was increased and levels ofα-SMA and CTGF were decreased in cells exposed TGF-β1 plus rrALR medium.
Conclusion: 1.There was no transdifferentiating effect when NRK52E cells cultured with rALR alone. 2. rrALR may inhibit transdifferentiation of NRK52E cells into myofibroblast-like cells and CTGF protein and mRNA expression induced by TGF-β1.
引文
[1] Hagiya MD,Francavilla A,Polimeno L,et al.Cloning and sequence analysis of the rat augmenter of liver regeneration (ALR) gene:Expression of biologically active recombinant ALR and demonstration of tissue distribution [J].Proc Natl Acad Sci USA. 1994;91:8142–8146
[2] Francavilla A,Hagiya M,Porter KA,et al.Augmenter of liver regeneration:its place in the universe of hepatic growth factors [J].Hepatology, 1994, 20(3):747-757
[3] Yang X, Wang A, Zhou P,et al.Protective effect of recombinant human augmenter of liver regeneration on CCl4-induced hepatitis in mice [J].Chin Med J (Engl). 1998; 111(7): 625-9
[4] Polimeno L,Margiotta M,Marangi L,et al. Molecular mechanisms of augmenter of liver regeneration as immunoregulator: its effect on interferon-gamma expression in rat liver [J]. Dig Liver Dis. 2000;32(3):217-25
[5] 王爱民,杨晓明,郭瑞峰,等. 重组人肝再生增强因子对大鼠实验性肝纤维化的保护作用[J]。中华医学杂志,1998,78(9): 707-708
[6] Hagiya M,Francavilla A,Polimeno L,et al.Cloning and sequence analysis of the rat augmenter of liver regeneration (ALR) gene:Expression of biologically active recombinant ALR and demonstration of tissue distribution[J]. Proc. Natl. Acad. Sci. USA. 1994, 91:8142-8146
[7] 张玲,宋晓英,刘杞. 肝再生增强因子在抗-Thy1.1 肾炎大鼠肾组织中生物活性作用研究 [J]。中华肾脏病杂志,2004,2004,20(2)
[8] 张玲,廖晓辉,刘杞等。肝再生增强因子在急性肾衰大鼠模型肾组织中的表达及意义 [J]。重庆医学,2003,32(6):647-649
[9] Cereghino JL,Cregg JM. Heterologous protein expression in the methylotrophic yeast Pichia pastoris[J]. FEMS-microbiol-rev 2000 Jan, 24(1): 45-66
[10] A Manual of methods for expression of recombninant proteins in pichia pastoris.Invitrogen Corporation.
[11] Thill GP,Dowis GR,Stillman C,et al. Positive and negative effects of multi-copy integrated expression vectors on protein expression in Pichia pastoris[J]. Mol Gen Genet.1994; 243(5):489-99.
[12] 文晓彦,郑法雷,苏震等。肝细胞生长因子对人肾小管上皮细胞转分化抑制作用的初步研究 [J]。中国中西医结合肾病杂志,2002,3(7)380-382
[13]Tang L(唐玲),Sun H,et al.Inhibitory effect of blocking expression of human augmenter of liver regeneration(hALR) on proliferation of hepatocellular carcinoma cell line HepG. Ai Zheng(癌症) 2006;25(6):671-676.( Chinese)
[14]廖晓辉,张玲,刘杞。重组大鼠肝再生增强因子对肾小管上皮细胞增殖以及凋亡的影响[J]。中华肾脏病杂志,2005,21(12):747-751
[15]Jinde K,Nikolic-Paterson DJ,et al.Tubular phenotypic change in progressive tubulointerstitial fibrosis in human glomerulonephritis [J].Am J Kidney Dis 2001;38:761-769
[16]Kalluri R,Neilson Eg.Epithelial-mesenchymal transition and its implications for fibrosis[J].J Clin Invest,2003,112:1776-1784
[17] Hay ED,Zuk A.Transformations between epithelium and mesenchyme: normal, pathological,and experimentally induced[J]. Am J Kidney Dis,1995, 26(4):678-690 [18.]Yoshida S,Watanabe T,et al. Inflammatory myofibroblastic tumor of the renal pelvis. Hinyokika Kiyo 2006;52(1): 3l-33.
[19]Sadlier DM,Connolly SB,et al.Sequential extracellular matrix-focused and baited-global cluster analysis of serial transcriptomic profiles identifies candidate modulators of renal tubulointerstitial fibrosis in murine adriamycin-induced nephropathy. J Biol Chem. 2004;279(28):29670-29680.
[20]Qi W,Chen X,et al. The differential regulation of Smad7 in kidney tubule cells by connective tissue growth factor and transforming growth factor-beta1. Nephrology (Carlton) 2007;12(3):267-274.
[21]Yokoi H,Mukoyama M,et al. Reduction in connective tissue growth factor by antisense treatment ameliorates renal tubulointerstitial fibrosis. J Am Soc Nephrol 2004;15(6):1430-1440.
[1] Strutz F, Zeisberg M, J Am Soc Nephrol. 2006,17(11):2992-8.
[2] Iwano M ,Neilson EG , Curropin Nephml Hypertens. 2004,13(3):279—84.
[3] Chevalier RL,Curr Opin Pediatr . 2006,18(2):153—160.
[4] Bhowmick NA et a1, Science. 2004 Feb 6;303(5659):848-51.
[5] Zeisberg M , Kalluri R, J Mol Med. 2004,82(3):175—181.
[6] Selbi Wd et a1, J Am Soc Nephrol. 2004, 15(5):1199-211.
[7] Allison MM.j med libr assoc. 2006,94(2 Suppl):E74-9.
[8] Forino M et a1,1nt J Exp Pathol. 2006,87(3):197—208.
[9] Roberts AB et a1, Cytokine Growth Factor Rev.2006,17(1— 2):l9—27.
[10] Rhyu DY et a1, J Am Soc Nephrol.2005,16(3):667—675.
[11] Hirschberg R et a1, Curr Opin Nephrol Hypertens. 2005,14(1): 43-52.
[12] Wang SE et a1, Mol Cell Biol. 2005,25(11):4703-15.
[13] Kopp,Jeffrey B, Kidney Internation.2002,61(1):351-352.
[14] Oostingh GJ et a1, Xenotransplantation. 2003,10(6):545-51.
[15] Mathew S et a1,Eur J Clin Invest. 2006,36 Suppl 2:43-50.
[2] Francavilla A,Hagiya M,Porter KA,et al.Augmenter of liver regeneration:its place in the universe of hepatic growth factors [J].Hepatology, 1994, 20(3):747-757
[3] Yang X, Wang A, Zhou P,et al.Protective effect of recombinant human augmenter of liver regeneration on CCl4-induced hepatitis in mice [J].Chin Med J (Engl). 1998; 111(7): 625-9
[4] Polimeno L,Margiotta M,Marangi L,et al. Molecular mechanisms of augmenter of liver regeneration as immunoregulator: its effect on interferon-gamma expression in rat liver [J]. Dig Liver Dis. 2000;32(3):217-25
[5] 王爱民,杨晓明,郭瑞峰,等. 重组人肝再生增强因子对大鼠实验性肝纤维化的保护作用[J]。中华医学杂志,1998,78(9): 707-708
[6] Hagiya M,Francavilla A,Polimeno L,et al.Cloning and sequence analysis of the rat augmenter of liver regeneration (ALR) gene:Expression of biologically active recombinant ALR and demonstration of tissue distribution[J]. Proc. Natl. Acad. Sci. USA. 1994, 91:8142-8146
[7] 张玲,宋晓英,刘杞. 肝再生增强因子在抗-Thy1.1 肾炎大鼠肾组织中生物活性作用研究 [J]。中华肾脏病杂志,2004,2004,20(2)
[8] 张玲,廖晓辉,刘杞等。肝再生增强因子在急性肾衰大鼠模型肾组织中的表达及意义 [J]。重庆医学,2003,32(6):647-649
[9] Cereghino JL,Cregg JM. Heterologous protein expression in the methylotrophic yeast Pichia pastoris[J]. FEMS-microbiol-rev 2000 Jan, 24(1): 45-66
[10] A Manual of methods for expression of recombninant proteins in pichia pastoris.Invitrogen Corporation.
[11] Thill GP,Dowis GR,Stillman C,et al. Positive and negative effects of multi-copy integrated expression vectors on protein expression in Pichia pastoris[J]. Mol Gen Genet.1994; 243(5):489-99.
[12] 文晓彦,郑法雷,苏震等。肝细胞生长因子对人肾小管上皮细胞转分化抑制作用的初步研究 [J]。中国中西医结合肾病杂志,2002,3(7)380-382
[13]Tang L(唐玲),Sun H,et al.Inhibitory effect of blocking expression of human augmenter of liver regeneration(hALR) on proliferation of hepatocellular carcinoma cell line HepG. Ai Zheng(癌症) 2006;25(6):671-676.( Chinese)
[14]廖晓辉,张玲,刘杞。重组大鼠肝再生增强因子对肾小管上皮细胞增殖以及凋亡的影响[J]。中华肾脏病杂志,2005,21(12):747-751
[15]Jinde K,Nikolic-Paterson DJ,et al.Tubular phenotypic change in progressive tubulointerstitial fibrosis in human glomerulonephritis [J].Am J Kidney Dis 2001;38:761-769
[16]Kalluri R,Neilson Eg.Epithelial-mesenchymal transition and its implications for fibrosis[J].J Clin Invest,2003,112:1776-1784
[17] Hay ED,Zuk A.Transformations between epithelium and mesenchyme: normal, pathological,and experimentally induced[J]. Am J Kidney Dis,1995, 26(4):678-690 [18.]Yoshida S,Watanabe T,et al. Inflammatory myofibroblastic tumor of the renal pelvis. Hinyokika Kiyo 2006;52(1): 3l-33.
[19]Sadlier DM,Connolly SB,et al.Sequential extracellular matrix-focused and baited-global cluster analysis of serial transcriptomic profiles identifies candidate modulators of renal tubulointerstitial fibrosis in murine adriamycin-induced nephropathy. J Biol Chem. 2004;279(28):29670-29680.
[20]Qi W,Chen X,et al. The differential regulation of Smad7 in kidney tubule cells by connective tissue growth factor and transforming growth factor-beta1. Nephrology (Carlton) 2007;12(3):267-274.
[21]Yokoi H,Mukoyama M,et al. Reduction in connective tissue growth factor by antisense treatment ameliorates renal tubulointerstitial fibrosis. J Am Soc Nephrol 2004;15(6):1430-1440.
[1] Strutz F, Zeisberg M, J Am Soc Nephrol. 2006,17(11):2992-8.
[2] Iwano M ,Neilson EG , Curropin Nephml Hypertens. 2004,13(3):279—84.
[3] Chevalier RL,Curr Opin Pediatr . 2006,18(2):153—160.
[4] Bhowmick NA et a1, Science. 2004 Feb 6;303(5659):848-51.
[5] Zeisberg M , Kalluri R, J Mol Med. 2004,82(3):175—181.
[6] Selbi Wd et a1, J Am Soc Nephrol. 2004, 15(5):1199-211.
[7] Allison MM.j med libr assoc. 2006,94(2 Suppl):E74-9.
[8] Forino M et a1,1nt J Exp Pathol. 2006,87(3):197—208.
[9] Roberts AB et a1, Cytokine Growth Factor Rev.2006,17(1— 2):l9—27.
[10] Rhyu DY et a1, J Am Soc Nephrol.2005,16(3):667—675.
[11] Hirschberg R et a1, Curr Opin Nephrol Hypertens. 2005,14(1): 43-52.
[12] Wang SE et a1, Mol Cell Biol. 2005,25(11):4703-15.
[13] Kopp,Jeffrey B, Kidney Internation.2002,61(1):351-352.
[14] Oostingh GJ et a1, Xenotransplantation. 2003,10(6):545-51.
[15] Mathew S et a1,Eur J Clin Invest. 2006,36 Suppl 2:43-50.