胰岛素样生长因子-Ⅰ对人肾小管上皮细胞转分化及胶原合成的作用

目的探讨胰岛素样生长因子-Ⅰ(IGF-Ⅰ)在人肾小管上皮细胞转分化及胶原合成中的作用。方法将体外培养的人肾小管上皮细胞HK2分为2组(1)对照组;(2)IGF-Ⅰ组培养液中加入终浓度分别为25、50、100、200μg/L的IGF-Ⅰ。用倒置显微镜观察细胞形态变化,以RT-PCR检测HK2细胞α-平滑肌肌动蛋白(α-SMA)、胶原Ⅰα和胶原ⅢαmRNA表达的变化。ELISA方法检测培养上清中胶原Ⅰ的浓度。结果IGF-Ⅰ刺激使HK2细胞由椭圆形变为梭形。不同浓度的IGF-Ⅰ作用于HK2细胞48h后,α-SMA、胶原Ⅰα和胶原ⅢαmRNA水平显著升高(P<0.05);ELISA结果显示,100及200μg/LIGF-Ⅰ组中HK2细胞分泌的胶原Ⅰ显著增加(P<0.05)。结论IGF-Ⅰ在体外能够诱导人肾小管上皮细胞转分化,并促进其胶原的合成。     

小分子干扰RNA沉默CTGF对肾小管上皮细胞的作用及对氧化应激的影响

目的研究小分子干扰RNA沉默结缔组织生长因子(CTGF)基因对高糖诱导肾小管上皮细胞的作用及对氧化应激的影响。方法肾小管上皮细胞(HK-2)分组:①正常对照组(A组),培养基含D-葡萄糖1 g/L;②高糖组(B组),培养基含D-葡萄糖4.5 g/L;③高糖+阴性对照组(C组):细胞转染含无关序列的重组质粒后于D-葡萄糖4.5 g/L的高糖培养基中培养;④高糖+干扰组(D组):细胞转染针对CTG F的siRNA表达质粒后于D-葡萄糖4.5 g/L的高糖培养基中培养。Q-PCR和western blot检测CTGF的表达。ELISA检测氧化应激指标F2-aisoprostanes、8-OHdG、NT、MDA水平。结果与A组相比,B和C组CTGF mRNA和蛋白表达水平上调(P0.05);B组和C组CTGF mRNA和蛋白表达水平无明显变化(P0.05);与C组相比,D组CTGF mRNA和蛋白表达水平下调(P0.05)。与A组相比,B和C组F2-aisoprostanes、8-OHdG、NT、MDA水平上调(P0.05);B组和C组F2-aisoprostanes、8-OHdG、NT、MDA水平无明显变化(P0.05);与C组相比,D组F2-aisoprostanes、8-OHdG、NT、MDA水平下调(P0.05)。结论沉默CTGF基因可抑制高糖诱导的肾小管上皮细胞氧化应激。     

不同分子量尿毒血清组分对人肾小管上皮细胞CTGF基因和蛋白表达的影响

目的：探讨不同分子量尿毒血清组分对人肾小管上皮细胞结缔组织生长因子（CTGF）基因和蛋白表达的影响。方法:无菌条件下收集40份尿毒症病人血清和20例正常人血清，应用Centricon Plus 20 Centrifugal Filter Devices将尿毒血清分离成分子量 >10 000 D，5 000-10 000 D，<5 000 D 3个组分。应用Western blotting方法检测CTGF的蛋白表达，RT-PCR方法检测CTGF mRNA表达。结果:2.5%-20%浓度尿毒血清组CTGF基因表达均明显高于正常对照组，以10%尿毒血清组最高；分子量 <5 000 D尿毒血清组CTGF基因表达与正常对照组差异无显著，而分子量 5 000-10 000 D和 >10 000 D尿毒血清组CTGF基因表达均高于正常对照组，以分子量 >10 000 D尿毒血清组最高。不同浓度尿毒血清组CTGF蛋白表达均高于正常对照组，且有随着尿毒血清浓度的升高而增高，在不同分子量尿毒血清组中，以分子量 >10 000 D组增高最为显著。结论：尿毒症毒素通过影响人肾小管上皮细胞致纤维化细胞因子CTGF基因和蛋白表达从而在人肾小管-间质纤维化中起重要作用，而以分子量大于 10 000 D的尿毒症毒素在其中起主要作用。     

结缔组织生长因子诱导人肾小管上皮细胞向间充质细胞转化的实验研究

目的 探讨结缔组织生长因子(CTGF)对肾小管上皮细胞向间充质细胞转化的影响.方法 体外培养的人肾小管上皮细胞(HK-2),按是否用CTGF处理将其分为实验组和对照组.直接免疫荧光法、间接免疫化学检测培养72 h后肾小管上皮细胞E-cadherin、波形蛋白、α-平滑肌肌动蛋白((α-SMA)、细胞外信号调节激酶2(ERK2)的表达;Western blot方法 在72 h检测E-cadherin、波形蛋白和ERK2的表达;Boyden小室在1、3、5 d检测细胞迁移能力的变化.结果 (1)实验组上皮细胞表型标志蛋白E-cadherin随着时间的延长而表达递减,波形蛋白表达渐增,ERK2表达上调;(2)在第1天,实验组与对照组细胞迁移能力差异无统计学意义,第3天实验组迁移至滤膜下面细胞多于对照组,第5天实验组细胞迁移数明显高于对照组[(45.0±1.1):(14.0±1.2),P<0.05].结论 在体外,人肾小管上皮细胞由CTGF刺激表现出向间充质细胞转化的特性,并且ERK2信号转导通路可能参与了对这一过程的调控效应.     

结缔组织生长因子诱导人肾小管上皮细胞向间充质细胞转化的实验研究

Objective To investigate the role of connective tissue growth factor ( CTGF) in epithelial mesenchymal transition of HK-2 cells in vitro.Methods HK-2 cells were randomly divided into two groups; (1) control group including cells cultured in DMEM medium supplemented with 10% fetal bovine serum only; and (2) experimental group including cells cultured in DMEM medium supplemented with 10% fetal bovine serum and recombinant CTGF at a final concentration of 5 p-g/L The cells were collected at 72 h time points.Direct immunoiluorescence staining and immunohistochemistry were used to evaluate the E-cadherin,Vimentin,α-SMA and ERK2 in cells.Western-blotting was used to detect the E-cadherin,Vimentin and ERK2 protein expression.Boyden Chamber was used to detect the migration of tubular endothelium at 1 d,3 d and S d.Results There were less E-cadherin but more Vimentin expressed in cells of the experimental group.The presence of α-SMA was detected at 48 h with peak at 72 h in the cells of the experimental group.On the first day,the cellular migration in the two groups showed no difference.However,after 3 days,the transformed cells migrated surpassed the control group with peak at the 5th day [ (45.0±1.1) : (14.0±1.2),P < 0.05 ) ].Conclusion Connective tissue growth factor induces mesenchymal transformation of HK-2 cells,in which the ERK2 signaling pathway may play an important role.     

结缔组织生长因子诱导人肾小管上皮细胞向间充质细胞转化的实验研究

Objective To investigate the role of connective tissue growth factor ( CTGF) in epithelial mesenchymal transition of HK-2 cells in vitro.Methods HK-2 cells were randomly divided into two groups; (1) control group including cells cultured in DMEM medium supplemented with 10% fetal bovine serum only; and (2) experimental group including cells cultured in DMEM medium supplemented with 10% fetal bovine serum and recombinant CTGF at a final concentration of 5 p-g/L The cells were collected at 72 h time points.Direct immunoiluorescence staining and immunohistochemistry were used to evaluate the E-cadherin,Vimentin,α-SMA and ERK2 in cells.Western-blotting was used to detect the E-cadherin,Vimentin and ERK2 protein expression.Boyden Chamber was used to detect the migration of tubular endothelium at 1 d,3 d and S d.Results There were less E-cadherin but more Vimentin expressed in cells of the experimental group.The presence of α-SMA was detected at 48 h with peak at 72 h in the cells of the experimental group.On the first day,the cellular migration in the two groups showed no difference.However,after 3 days,the transformed cells migrated surpassed the control group with peak at the 5th day [ (45.0±1.1) : (14.0±1.2),P < 0.05 ) ].Conclusion Connective tissue growth factor induces mesenchymal transformation of HK-2 cells,in which the ERK2 signaling pathway may play an important role.     

结缔组织生长因子诱导人肾小管上皮细胞向间充质细胞转化的实验研究

Objective To investigate the role of connective tissue growth factor ( CTGF) in epithelial mesenchymal transition of HK-2 cells in vitro.Methods HK-2 cells were randomly divided into two groups; (1) control group including cells cultured in DMEM medium supplemented with 10% fetal bovine serum only; and (2) experimental group including cells cultured in DMEM medium supplemented with 10% fetal bovine serum and recombinant CTGF at a final concentration of 5 p-g/L The cells were collected at 72 h time points.Direct immunoiluorescence staining and immunohistochemistry were used to evaluate the E-cadherin,Vimentin,α-SMA and ERK2 in cells.Western-blotting was used to detect the E-cadherin,Vimentin and ERK2 protein expression.Boyden Chamber was used to detect the migration of tubular endothelium at 1 d,3 d and S d.Results There were less E-cadherin but more Vimentin expressed in cells of the experimental group.The presence of α-SMA was detected at 48 h with peak at 72 h in the cells of the experimental group.On the first day,the cellular migration in the two groups showed no difference.However,after 3 days,the transformed cells migrated surpassed the control group with peak at the 5th day [ (45.0±1.1) : (14.0±1.2),P < 0.05 ) ].Conclusion Connective tissue growth factor induces mesenchymal transformation of HK-2 cells,in which the ERK2 signaling pathway may play an important role.     

结缔组织生长因子诱导人肾小管上皮细胞向间充质细胞转化的实验研究

Objective To investigate the role of connective tissue growth factor ( CTGF) in epithelial mesenchymal transition of HK-2 cells in vitro.Methods HK-2 cells were randomly divided into two groups; (1) control group including cells cultured in DMEM medium supplemented with 10% fetal bovine serum only; and (2) experimental group including cells cultured in DMEM medium supplemented with 10% fetal bovine serum and recombinant CTGF at a final concentration of 5 p-g/L The cells were collected at 72 h time points.Direct immunoiluorescence staining and immunohistochemistry were used to evaluate the E-cadherin,Vimentin,α-SMA and ERK2 in cells.Western-blotting was used to detect the E-cadherin,Vimentin and ERK2 protein expression.Boyden Chamber was used to detect the migration of tubular endothelium at 1 d,3 d and S d.Results There were less E-cadherin but more Vimentin expressed in cells of the experimental group.The presence of α-SMA was detected at 48 h with peak at 72 h in the cells of the experimental group.On the first day,the cellular migration in the two groups showed no difference.However,after 3 days,the transformed cells migrated surpassed the control group with peak at the 5th day [ (45.0±1.1) : (14.0±1.2),P < 0.05 ) ].Conclusion Connective tissue growth factor induces mesenchymal transformation of HK-2 cells,in which the ERK2 signaling pathway may play an important role.     

结缔组织生长因子诱导人肾小管上皮细胞向间充质细胞转化的实验研究

Objective To investigate the role of connective tissue growth factor ( CTGF) in epithelial mesenchymal transition of HK-2 cells in vitro.Methods HK-2 cells were randomly divided into two groups; (1) control group including cells cultured in DMEM medium supplemented with 10% fetal bovine serum only; and (2) experimental group including cells cultured in DMEM medium supplemented with 10% fetal bovine serum and recombinant CTGF at a final concentration of 5 p-g/L The cells were collected at 72 h time points.Direct immunoiluorescence staining and immunohistochemistry were used to evaluate the E-cadherin,Vimentin,α-SMA and ERK2 in cells.Western-blotting was used to detect the E-cadherin,Vimentin and ERK2 protein expression.Boyden Chamber was used to detect the migration of tubular endothelium at 1 d,3 d and S d.Results There were less E-cadherin but more Vimentin expressed in cells of the experimental group.The presence of α-SMA was detected at 48 h with peak at 72 h in the cells of the experimental group.On the first day,the cellular migration in the two groups showed no difference.However,after 3 days,the transformed cells migrated surpassed the control group with peak at the 5th day [ (45.0±1.1) : (14.0±1.2),P < 0.05 ) ].Conclusion Connective tissue growth factor induces mesenchymal transformation of HK-2 cells,in which the ERK2 signaling pathway may play an important role.     

结缔组织生长因子诱导人肾小管上皮细胞向间充质细胞转化的实验研究

Objective To investigate the role of connective tissue growth factor ( CTGF) in epithelial mesenchymal transition of HK-2 cells in vitro.Methods HK-2 cells were randomly divided into two groups; (1) control group including cells cultured in DMEM medium supplemented with 10% fetal bovine serum only; and (2) experimental group including cells cultured in DMEM medium supplemented with 10% fetal bovine serum and recombinant CTGF at a final concentration of 5 p-g/L The cells were collected at 72 h time points.Direct immunoiluorescence staining and immunohistochemistry were used to evaluate the E-cadherin,Vimentin,α-SMA and ERK2 in cells.Western-blotting was used to detect the E-cadherin,Vimentin and ERK2 protein expression.Boyden Chamber was used to detect the migration of tubular endothelium at 1 d,3 d and S d.Results There were less E-cadherin but more Vimentin expressed in cells of the experimental group.The presence of α-SMA was detected at 48 h with peak at 72 h in the cells of the experimental group.On the first day,the cellular migration in the two groups showed no difference.However,after 3 days,the transformed cells migrated surpassed the control group with peak at the 5th day [ (45.0±1.1) : (14.0±1.2),P < 0.05 ) ].Conclusion Connective tissue growth factor induces mesenchymal transformation of HK-2 cells,in which the ERK2 signaling pathway may play an important role.     

结缔组织生长因子在肾间质纤维化中的表达及其调控

CTGF, a member of the CCN family of immediate early genes, is a recently discovered profibrotic growth factor, which is involved in many pathophysiologic procedures. CTGF acts as a downstreame ffector of TGF-β acting on interstitial ceils to enhance the progression of fibrotic renal diseases. It has been shown that CTGF gene expression can be induced or blocked by some kinds of cytokine and drugs. It is an interesting candidate target for future intervention strategies of renal interstitial fibrosis.     

结缔组织生长因子在单侧输尿管梗阻大鼠肾脏中的表达及其意义

目的： 观察结缔组织生长因子（CTGF）在单侧输尿管梗阻（UUO）大鼠模型中的动态表达，探讨CTGF在肾小管间质纤维化中的作用机制。方法： 将48 只Wistar大鼠随机分为UUO组和假手术（SO）组，采用左输尿管结扎术复制UUO模型，于术后1、3、7、14 d分别处死2组大鼠取左肾。采用Masson染色评定肾小管间质损伤程度；逆转录-聚合酶链式反应(RT-PCR) 方法检测肾组织转化生长因子-β₁（TGF-β₁）、CTGF、Ⅰ型胶原（ColⅠ）和纤溶酶原激活物抑制物-1（PAI-1）mRNA表达；免疫组织化学方法测定TGF-β₁、CTGF、ColⅠ、PAI-1表达；Western blotting方法检测CTGF蛋白表达量的变化。结果: UUO术后1d，梗阻肾TGF-β₁ mRNA表达开始升高，第3-14d时升高更显著（P<0.01），CTGF、ColⅠ、PAI-1 mRNA水平随之逐渐升高。免疫组织化学染色发现，UUO大鼠肾小管和间质区域CTGF表达随病程进展逐渐增强；相关分析显示，UUO术后第7d，CTGF表达量与肾小管间质损伤指数、肾小管间质TGF-β₁、ColⅠ、PAI-1表达强度均呈正相关，相关系数（r）分别为0.62、0.85、0.78和0.76（均P<0.01）。Western blotting结果显示，CTGF蛋白水平在术后第3d开始上升，随病程进展更显著。结论: CTGF可通过促进细胞外基质产生和抑制细胞外基质降解双重途径诱导肾小管间质纤维化的发生和进展。     

Genistein ameliorates parathyroid hormone-induced epithelial-to-mesenchymal transition and inhibits expression of connective tissue growth factor in human renal proximal tubular cells

Introduction

Genistein, a soybean and soy-based product, has been reported to inhibit the growth of a wide range of cancer cells, but there is no evidence concerning its treatment of chronic kidney disease. The aim was to investigate whether genistein has potential to inhibit parathyroid hormone (PTH)-induced renal interstitial fibrosis.

Material and methods

Using human renal tubular epithelial HK-2 cells, α-smooth muscle actin (α-SMA) was assessed by using immunofluorescence detection. α-Smooth muscle actin, E-cadherin and connective tissue growth factor (CTGF) were measured by Western blot analysis. The promoter activity of the CTGF gene was examined by the luciferase reporter assay.

Results

When cells were treated with PTH (0.1 nM) for 48 h, α-SMA protein expression was induced significantly, the protein expression of E-cadherin decreased substantially, and the promoter activity of the CTGF gene as well as its mRNA and protein expression levels increased (p < 0.01). Interestingly, genistein effectively inhibited PTH-induced α-SMA expression, restored E-cadherin expression, decreased mRNA and protein expression of CTGF, and suppressed the promoter activity of CTGF in a dose-dependent manner.

Conclusions

Genistein has the ability to block the biomarker for renal transdifferentiation and epithelial-to-mesenchymal transition, α-SMA, following PTH treatment and inhibit CTGF expression in human renal tubular epithelial cells; these might be important modes of actions that contribute to genistein anti-fibrogenic effects and may have great implications for its potential in clinical treatment of renal interstitial fibrosis.     

结缔组织生长因子在转化生长因子β诱导的肾小管上皮细胞转分化中的作用

目的：探讨结缔组织生长因子（CTGF）在转化生长因子-β1（TGF-β1）诱导的肾小管上皮细胞表型转化中的可能作用。方法： 将NRK52E肾小管上皮细胞分组处理，光镜、扫描电镜、透射电镜观察细胞形态的改变，细胞免疫组化检测ɑ-平滑肌肌动蛋白（α-SMA）和细胞角蛋白-18的表达，RT-PCR和Western blot检测Ⅰ型胶原的表达。 结果：TGF-β1 10 μg/L作用3 d，NRK52E小管上皮细胞失去正常的椭圆形，变得肥大，胞体拉长，扫描电镜下，见成纤维细胞状，失去上皮细胞特有的顶端-基底极性和表面的微绒毛结构，透射电镜下胞浆中见到微丝和致密体结构，骨架标志上肾小管上皮细胞较具特征性的细胞角蛋白-18表达减少,肌成纤维细胞标志性的α-SMA表达增多，Ⅰ型胶原分泌增多；加入TGF-β1中和抗体和CTGF反义寡核苷酸可以大部分阻断TGF-β的作用，而正义的CTGF寡核苷酸不能阻断TGF-β的作用。 结论： NRK52E细胞中，CTGF作为TGF-β的下游效应因子，介导了TGF-β诱导的肾小管上皮细胞转分化。     

结缔组织生长因子促纤维化作用及其表达调节的研究进展

Connective tissue growth factor (CTGF) has recently received much attention as a possible key determinant of progressive fibrosis. It promotes tissue fibrosis through different pathways, such as cell proliferation, extracellular matrix accumulation and cell transdifferentiation. A number of regulators of CTGF expression have been identified, including transformiing growth factor β, vascular endothelial growth factor, tumor necrosis factor α, etc. The mechanism of profibrotic effect by CI‘GF was reviewed.     

结缔组织生长因子在单侧输尿管梗阻大鼠肾组织中的表达

目的：检测大鼠单侧输尿管梗阻（UUO）模型不同时期，肾组织中结缔组织生长因子（CTGF）、转化生长因子β1（TGF-β1）和α-平滑肌肌动蛋白（α-SMA）的表达，观察比较在间质纤维化不同阶段，3者的动态变化及关系。 方法： 采用雄性SD大鼠36只，分为假手术组和模型组，模型组行左侧输尿管结扎术，再分3、7、14、21和28 d共6组，每组6只，于各时点处死大鼠，取肾组织，常规HE、Masson染色，按小管间质损害的特征进行半定量评分。免疫组化检测CTGF、TGF-β1和α-SMA表达。 结果： 随梗阻时间的延长，小管间质纤维化加重，28 d间质已基本被纤维化组织所代替。随间质纤维化程度的加重，CTGF和α-SMA表达逐渐增加，两者与小管间质损害积分呈正相关，CTGF与α-SMA的表达之间也呈正相关。TGF-β1表达在7-14 d达高峰后，逐渐减少，但仍高于对照组。 结论： UUO致CTGF表达增加可能与TGF-β升高有关，CTGF可能通过促进间质中肌成纤维细胞的形成而参与肾间质纤维化。     

心肌细胞结缔组织生长因子的表达及TGF-β1对其表达的影响

目的： 研究纯化的心肌细胞中结缔组织生长因子（CTGF）基因及蛋白的表达，并探讨转化生长因子β1（TGF-β₁）对其表达的影响。方法: 分离培养乳鼠心肌细胞，48 h后用流式分选方法（FACS）分选纯化心肌细胞，纯化的心肌细胞继续培养48 h。分为对照组（空白纯化心肌细胞组）和TGF-β₁刺激组，用RT-PCR方法测定两组细胞中TGF-β₁、CTGF、纤维连接蛋白（FN） mRNA的含量， 用Western blotting法检测两组细胞胞浆蛋白中CTGF、FN含量。结果: 心肌细胞中有较低水平的CTGF表达，在予以 TGF-β₁刺激后细胞内CTGF mRNA与蛋白含量分别提高 2.49 倍(P<0.01) 和3.63 倍（P<0.01），FN mRNA与蛋白含量分别提高 1倍(P<0.01)和4.18倍（P<0.01），而TGF-β1mRNA水平减少34%（P<0.01）。结论: 纯化的心肌细胞有CTGF的基础表达，并且TGF-β1可以明显上调其表达，提示心肌细胞主动参与了心肌纤维化过程。     

低氧刺激结缔组织生长因子表达与肾间质纤维化

目的：观察低氧对结缔组织生长因子（CTGF）表达的影响，探讨低氧致肾间质纤维化的机制。方法： 单侧输尿管结扎（UUO）9 d大鼠动物模型，用RT-PCR方法检测肾组织中低氧标记分子-低氧诱导因子（HIF-1α）的mRNA水平，免疫组化方法观察假手术组及模型组肾组织中HIF-1α和CTGF的表达及部位，Western蛋白印迹技术检测肾组织CTGF的蛋白水平。体外实验，正常大鼠肾间质成纤维细胞（NRK-49F）分别置于低氧（1%O2）和正常氧条件下培养6 h，应用RT-PCR和Western蛋白印迹检测CTGF mRNA和蛋白表达水平。结果： 对照组肾组织未能检测到HIF-1α mRNA和HIF-1α蛋白表达；模型组肾组织有高水平HIF-1α mRNA，并出现HIF-1α蛋白表达，主要分布在小管-间质细胞；CTGF蛋白与HIF-1α蛋白表达部位及程度一致；低氧（1% O2）培养刺激NRK-49F细胞表达CTGF mRNA和蛋白。结论： 低氧刺激的CTGF的表达增加与肾间质纤维化的发生有关。     

百令对慢性肾衰竭模型大鼠肾脏保护和肾结缔组织生长因子表达的影响

目的 观察百令对5/6肾切除大鼠的肾脏保护作用及对肾结缔组织生长因子(CTGF)表达的影响,探讨其延缓肾衰竭进展及抗纤维化的相关机制. 方法 50只SD大鼠随机取8只为假手术组,其余行5/6肾切除术.根据术后3周血肌酐(Scr)值分为模型组、天然虫草组(2.0 g·kg-1·d-1)、百令治疗组(2.0 g·kg-1·d-1)和百令高剂量组(3.0 g·kg-1·d-1).术后4周给药.治疗1个月后检测Scr、尿素氮(BUN)浓度;光镜下观察肾脏病理改变,免疫组化方法检测肾组织CTGF、α平滑肌肌动蛋白(α-SMA)的表达水平,采用图像分析系统进行定量分析. 结果 治疗后模型组大鼠Scr、BUN明显高于假手术组(P<0.01),肾小球与肾小管间质均有明显病理改变,CTGF、α-SMA的表达明显上调;而药物治疗组的Scr、BUN明显低于模型组(P<0.05),肾脏病理损伤减轻,CTGF、α-SMA的表达降低(P<0.05). 结论 百令能改善5/6肾切除大鼠的肾功能,减轻肾脏病理损害,其机制可能与下调肾组织CTGF的表达有关.