低密度脂蛋白对肾小珠系膜细胞,TGF—β和FN mRNA表达的影响

目的：观察低密度脂蛋白（ＬＤＬ）对体外培养的大鼠肾小球系膜细胞（ＭＳＣ）生长及转化生长因子β（ＴＧＦ－β）和纤维连接蛋白（ＦＮ）基因表达的影响。方法：体外培养的ＭＳＣ培养液中加入ＬＤＬ共同孵育,采用＾３Ｈ－ＴｄＲ渗入法检测ＭＳＣ增殖情况,应用Ｎｏｒｔｈｅｒｎ ｂｌｏｔ检测ＴＧＦ－β ｍＲＮＡ、ＦＮ ｍＲＮＡ的表达,应用斑点杂交法检测抗ＴＧＦ－抗体对ＦＮ ｍＲＮＡ表达的影响。结果（１）ＬＤＬ刺激ＭＳ     

大鼠肾脏系膜细胞转染人TGF—β1基因可增强MMP—2表达

目的 研究转化生长因子－β１（ＴＧＦ－β１）对培养的大鼠肾小球系膜细胞（ＭｓＣ）基质金属蛋白酶－２（ＭＭＰ－２）表达的影响。方法 采用Ｌｉｐｏｆｅｃｔｉｎ将人ＴＧＦ－β１基因转染培养的大鼠ＭｓＣ,经Ｎｏｒｔｈｅｒｎｂｌｏｔ鉴定其转染成功否;又分别应用Ｎｏｒｔｈｅｒｎｂｌｏｔ．Ｗｅｓｔｅｒｎ ｂｌｏｔ,酶谱分析法检测阳性表达人ＴＧＦ－β１的ＭｓＣＭＭＰ－２ｍＲＮＡ蛋白及酶活性变化。结果成功获得稳定过     

环孢素A联合雷公藤单体T_4对人肾小球系膜细胞Ⅳ型胶原合成及Ⅳ型胶原α1、转化生长因子β1mRNA表达的影响

目的探讨环孢素Ａ（ＣsＡ）、雷公藤单体Ｔ４单独或联合使用对肾小球系膜基质合成的影响及可能机制。方法用ＥＬＩＳＡ法测定人系膜细胞培养上清Ⅳ型胶原含量,用ＲＴ－ＰＣＲ法测定人肾小球系膜细胞Ⅳ型胶原α１ｍＲαＮＡ（Ｃｏｌｌα１（Ⅳ）ｍＲＮＡ）、转化生长因子β１（ＴＧＦβ１）ｍＲＮＡ的表达。结果ＣｓＡ可双向调节人系膜细胞Ⅳ型胶原的合成,ＣｓＡ１．０ｕｇ／ｍｌ为促进作用,ＣｓＡ０．０１～０．１ｕｇ／ｍｌ为抑制效应。Ｔ４剂量依赖性促进Ⅳ型胶原产生。两药联合应用具有协同效应,小剂量ＣｓＡ和Ｔ４联合在获得较强免疫抑制作用的同时促胶原生成作用较弱。ＣｓＡ１．０ｕｇ／ｍｌ和Ｔ４１０ｎｇ／ｍｌ均可上调人系膜细胞Ｃｏｌｌβ１（Ⅳ）、ＴＧＦβ１ｍＲＮＡ表达,两者变化趋势基本一致。结论ＣｓＡ和Ｔ４均参与调节基质合成,两药小剂量联合促胶原生成作用减轻,这一作用可能由ＴＧＦβ１介导发生于转录水平。     

大鼠肾脏系膜细胞转染人TGF-β1基因可增强MMP-2表达

目的　研究转化生长因子β１（ＴＧＦβ１）对培养的大鼠肾小球系膜细胞（ＭｓＣ）基质金属蛋白酶２（ＭＭＰ２）表达的影响。方法　采用Ｌｉｐｏｆｅｃｔｉｎ将人ＴＧＦβ１基因转染培养的大鼠ＭｓＣ,经Ｎｏｒｔｈｅｒｎｂｌｏｔ鉴定其转染成功否;又分别应用Ｎｏｒｔｈｅｒｎｂｌｏｔ．Ｗｅｓｔｅｒｎｂｌｏｔ．酶谱分析法检测阳性表达人ＴＧＦβ１的ＭｓＣＭＭＰ２ｍＲＮＡ、蛋白及酶活性变化。结果　成功获得稳定过表达人ＴＧＦβ１的大鼠ＭｓＣ克隆（ＭＴＧ１）,该细胞克隆的ＭＭＰ２ｍＲＮＡ．蛋白表达及其酶活性均获增强。结论　ＴＧＦβ１可增强ＭｓＣＭＭＰ２ｍＲＮＡ、蛋白表达,并提高其酶活性,这为进一步阐明ＴＧＦβ１在肾小球硬化发生机制中的作用提供了重要的实验手段和依据。     

环孢素A联合雷公藤单体T4对人肾小球系膜细胞Ⅳ型胶原合？…

目的 探讨环孢素Ａ（ＣｓＡ）、雷公藤单体Ｔ４单独鄞艇对肾小球系膜基质合成的影响及可能机制。方法用ＥＬＩＳＡ法测定人系膜细胞培养上清Ⅳ型胶原含量用ＲＴ－ＰＣＲ法测定人肾小球系膜细胞Ⅳ型胶原α１ｍＲＮＡ、转化生长因子β１ＴＧＦβ１）ｍＲＮＡ的表达。结果 ＣｓＡ可双向扁队系膜细胞Ⅳ型胶原的合成,ＣｓＡ１．０μｇ／ｍＬ为促进作用,ＣｓＡ０．０１￣０．１μｇ／ｍｌ为抑制效应。Ｔ４剂量依赖性促进Ⅳ型胶原产生。     

α—生育酚抑制高糖诱导大鼠系膜细胞AP—1活性及IGF …

目的 研究α－生育酚对高糖诱导肾小球系膜细胞转录激活蛋白－１（ＡＰ－１）和转化生长因子－β１（ＧＧＦ－β１）的影响,探讨抗氧化剂治疗糖尿病肾病的机制。方法 采用凝胶迁移率实验（Ｇｅｌ ｓｈｉｆｔ ａｓｓａｙ）和超迁移率实验（Ｇｅｌ ｓｕｐｅｒｓｈｉｆｔ ａｓｓａｙ）检测不同浓度及不同时间下葡萄糖对系膜细胞ＡＰ－１活性的影响、ＡＰ－１二聚体中Ｊｕｎ和Ｆｏｓ成分的变化、Ｗｅｓｔｅｒｎ杂交检测ＴＧＦ－β     

细胞因子对人肾小球系膜细胞表达细胞粘附分子的调控

研究白细胞介素－１β（ＩＬ－１β）及肿瘤坏死因子α（ＴＮＦα），对体外培养的人肾小球系膜细胞表达细胞间粘附分子－１（ＩＣＡＭ－１）和血管细胞粘附分子－１（ＶＣＡＭ－１）的调节作用。方法用ｒｈＩＬ－１β（２５ｎｇ／ｍｌ）或ｒｈＴＮＦα（１００ｎｇ／ｍｌ）与系膜细胞共同孵育４、８、１６及３２小时，然后用Ｎｏｒｔｈｅｒｎ杂交检测该细胞ＩＣＡＭ－１和ＶＣＡＭ－１的ｍＲＮＡ表达，并用细胞ＥＬＩＳＡ检测它们的蛋白质表达。结果未予任何刺激的对照组，系膜细胞仅低水平表达ＩＣＡＭ－１、ＶＣＡＭ－１ｍＲＮＡ和蛋白质。ｒｈＩＬ－１β或ｒｈＴＮＦα刺激后，系膜细胞上这两种细胞粘附分子ｍＲＮＡ的表达，均迅速上调（仅ＴＮＦα刺激ＩＣＡＭ－１ｍＲＮＡ表达高峰在８小时，其余均在４小时），蛋白质表达也显著增加（Ｐ＜０．００１））。结论细胞因子ＩＬ－１β及ＴＮＦα可能通过刺激肾小球系膜细胞表达ＩＣＡＭ－１和ＶＣＡＭ－１而参与肾小球肾炎发病。     

EXPRESSION OF TGF－β_1，PDGF AND IGF－1 mRNA IN LUNG OF BLEOMYCIN－A_5－INDUCED PULMONARY FIBROSIS IN RATS

ＥＸＰＲＥＳＳＩＯＮＯＦＴＧＦ－β１，ＰＤＧＦＡＮＤＩＧＦ－１ｍＲＮＡＩＮＬＵＮＧＯＦＢＬＥＯＭＹＣＩＮ－Ａ５－ＩＮＤＵＣＥＤＰＵＬＭＯＮＡＲＹＦＩＢＲＯＳＩＳＩＮＲＡＴＳＬｉＨａｉｃｈａｏ李海潮，ＨｅＢｉｎｇ何冰，ＱｕｅＣｈｅｎｇｌｉ阙呈立ａｎｄＷ...     

五种生长因子mRNA在兔角膜中的表达

目的 了解ＥＧＦ、ＴＧＦ－α、ｂＦＧＦ、ＴＧＦ－β１和ＰＤＧＦ在兔角膜上皮和基质中的表达情况,探讨其在角膜伤口愈合中的作用。方法 应用原位核酸分子杂交方法检测兔角膜上皮和基质中ＥＧＦ、ＴＧＦ－α、ｂＦＧＦ、ＴＧＦ－β１和ＰＤＧＦ ｍＲＮＡＲ的表达。结果 ＥＧＦ、ＴＧＦ－α和ＰＤＧＦ ｍＲＮＡ仅在正常角膜上此细胞层表达,基质中未见表达;ｂＦＧＦ和ＴＧＦ－β１ ｍＲＮＡ在正常角膜伤口和基质中均有表达。     

透析对糖尿病肾衰竭病人外周血单个核细胞TGF—β1mRNA？…

①目的 探讨透析对糖尿病肾衰竭病人转化生长因子（ＴＧＦ－β）表达的影响。②方法 采用逆转录聚合酶链反应（ＲＴ－ＰＣＲ）半定量技术，对糖尿病肾衰竭不同治疗组病人外周血单个核细胞（ＰＢＭＣ）ＴＧＦ－β１ｍＲＮＡ的表达进行检测。③结果 糖尿病肾衰竭透析病人ＴＧＦ－β１ｍＲＮＡ的表达明显高于对照组（ｔ＝１２．５３０，Ｐ〈０．０１），且血液透析病人ＴＧＦ－β１ｍＲＮＡ的表达较腹膜透析病人高（ｔ＝２．１３４，     

Effect of rhein on glucose transporter-1 expression and its function in glomerular mesangial cells

Ｇｌｕｃｏｓｅｉｓｔｈｅｍａｉｎｆｕｅｌｆｏｒｍｏｓｔｍａｍｍａｌｉａｎｃｅｌｌｓ,ａｎｄｃａｎｂｅｔｒａｎｓｐｏｒｔｅｄｉｎｔｏｃｅｌｌｓｂｙｇｌｕｃｏｓｅｔｒａｎｓｐｏｒｔｅｒｓＧｌｕｃｏｓｅｔｒａｎｓｐｏｒｔｅｒ1(ＧＬＵＴ1)ｈａｓｂｅｅｎｃｏｎｓｉｄｅｒｅｄｏｎｅｏｆｔｈｅｆａｃｉｌｉｔａｔｉｖｅｇｌｕｃｏｓｅｔｒａｎｓｐｏｒｔｅｒｓｒｅｓｐｏｎｓｉｂｌｅｆｏｒｃｏｎｓｔｉｔｕｔｉｖｅｇｌｕｃｏｓｅｔｒａｎｓｐｏｒｔ1　Ａｓｔｈｅｐｒｅｄｏｍｉｎａｎｔｆａｃｉｌｉｔａｔｉｖｅｇｌｕｃｏｓｅｔ…     

IDENTIFICATION OF GLUCOSE TRANSPORTER-1 AND ITS FUNCTIONAL ASSAY IN MOUSE GLOMERULAR MESANGIAL CELLS CULTURED IN VITRO

INTRODUCTION Glomerular mesangial cells play a key role in the development of diabetic glomerular lesions. There is a close correlation between expansion of the mesangium of the glomerulus and the clinical manifestations of diabetic nephropathy(1). Although it has become evident that the accumulation of extracellular matrix (ECM) occurs in the diabetic glomerulus and that mesangial cells in culture demonstrate enhanced production of ECM in response to elevated glucose levels (2), the me…     

IDENTIFICATION OF GLUCOSE TRANSPORTER－1 AND ITS FUNCTIONAL ASSAY IN MOUSE GLOMERULAR MESANGIAL CELLS CULTURED IN VITRO

Objective. To evaluate the role of glucose transporter-1 (GLUT1) in the glucose uptake of glomerular mesanglal cells. Methods. Cultured C57/SJL mouse mesangial cells were used in the study. The expression of GLUT1 mRNA was detected by RT-PCR. The expression of GLUT1 protein was detected by immunofluorescence and flow cytometry. The uptake of glucose and its kinetics were determined by 2-deoxy-[3H] -D-glucose uptake. Results. Both GLUT1 mRNA and protein were found in mouse glomerular mesangial cells. 2-deoxy-D-glucose uptake and kinetics assay showed that this glucose transporter had high affinity for glucose and the glucose uptake specificity was further confirmed by phloretin. Conclusion. Functional GLUT1 did present in mouse mesangial cells cultured in vitro and it might be the predominant transporter mediated the uptake of glucose into mesangial cells.     

Regulation of the expression and function of glucose transporter-1 by TGF-β1 and high glucose in mesangial cells

Objective To study the effects of high glucose and transforming growth factor-β1 (TGF-β1) on the expression and function of glucose transporter-1 (GLUT1) in mouse mesangial cells.Methods Cultured mouse mesangial cells were used.The expression of GLUT1 mRNA was detected by Northern Blot; glucose uptake and its kinetics were determined with a 2-Deoxy-［(3)H］-D-glucose uptake assay.Results Mesangial cells exposed to enriched glucose medium (20 mmol/L) for 72 hours demonstrated a decrease in both GLUT1 mRNA and V(max) for uptake of the glucose analog, 2-deoxy-D-glucose　(2DOG), as compared to mesangial cells cultured in physiologic glucose concentrations(5.5 mmol/L).In contrast, hypertonic mannitol had no effect on GLUT1 mRNA levels.TGF-β1 treatment for 10 hours stimulated 2DOG uptake, both in 5.5 mmol/L and 20 mmol/L glucose medium, by approximately 4.28-fold in a dose-dependent manner (2 ng/ml maximum).Kinetic analysis of 2DOG uptake revealed an increase in V(max) and a decrease in K(m) in the presence of TGF-β1. TGF-β1 also up-regulated the expression of GLUT1 mRNA in mesangial cells.The addition of anti-TGF-β neutralizing antibody (30 μg/ml) in mesangial cells cultured in enriched glucose medium (20 mmol/L) led to a 40% decrease in 2DOG uptake.Conclusions The expression of GLUT1 can be suppressed by exposure of mesangial cells to high glucose medium, which may serve as a protective mechanism against possible adverse effects of excessive glucose flux into cells.TGF-β1 stimulates glucose uptake by enhancing the expression and function of GLUT1 in mesangial cells.This effect is independent of the glucose milieu in the cultured medium.     

2型糖尿病肾病患者肾小球系膜细胞表型及功能改变

目的研究2型糖尿病肾病系膜细胞表型和功能的改变,进一步探讨糖尿病肾病的发病机理.方法经肾活检从2型糖尿病肾病患者获取肾组织,体外培养系膜细胞.采用流式细胞术、3H-胸腺嘧啶掺入及细胞倍增时间观察细胞表型改变.系膜细胞α-平滑肌肌动蛋白(α-SMA)、层粘连蛋白、纤维连接蛋白的表达用免疫荧光染色及流式细胞仪检测.用2-脱氧-3H-葡萄糖(2-DG)测定细胞葡萄糖摄入.Northern杂交和流式细胞仪检测葡萄糖转运蛋白1(GLUT1)的表达.细胞谷氨酰胺6-磷酸果糖转氨酶(GFAT)的活性采用比色法测定.结果 2型糖尿病肾病来源的系膜细胞较正常对照表现出细胞体积增大、RNA/DNA比值增加并伴细胞增殖加快,细胞骨架蛋白α-SMA和细胞外基质合成增加.糖尿病肾病系膜细胞的葡萄糖摄入率高于对照(1 592 cpm·105 cell-1 与 1 275 cpm·105 cell-1,P<0.05),同时伴GLUT1mRNA及蛋白质表达增加.此外,糖尿病肾病系膜细胞的GFAT活性明显增高.结论 2型糖尿病肾病系膜细胞具有明显的表型与功能改变.此外,糖尿病肾病患者系膜细胞还表现出细胞糖摄入及己糖胺通路活性增加.上述表型及功能改变可能是糖尿病肾病系膜细胞病变形成的基础.     

Effects of glucose and insulin on the H9c2 （2-1） cell proliferation may be mediated through regulating glucose transporter 4 expression

Background The change of glucose transporter 4 （GLUT4） expression could influence glucose uptake in the myocardial cells and then effect myocardial metabolism, which maybe one of the factor for the diabetes cardiovascular disease. This study aimed to explore the influence of glucose and insulin at different concentrations on H9c2 （2-1） cell proliferation and its GLUT4 expression in vitro, and evaluate the correlation between myocardial cells proliferation and GLUT4 expression. This might be helpful for understanding the relationship between glucose metabolism and cardiovascular disease. Methods According to glucose concentrations in culture medium, cultured H9c2 rat myocardial cells were divided into five groups： control group （NC, glucose concentration 5.0 mmol/L）, low glucose group （LG, glucose concentration 0.1 mmol/L）, high glucose group 1 （HG1, glucose concentration 10 mmol/L）, high glucose group 2 （HG2, glucose concentration 15 mmol/L）, high glucose group 3 （HG3, glucose concentration 20 mmol/L）. Then according to different insulin concentrations in culture medium, each group was further divided into two subgroups： normal insulin subgroup （INSc, insulin concentration 3.8 mU/L）, high insulin subgroup （INSh, insulin concentration 7.6 mU/L）. H9c2 （2-1） cells were cultured for 1, 2, 3 days, the proliferation of cells were assayed by cell counting Kit-8 assay, the expressions of GLUT4 mRNA and protein were detected with RT-PCR and Western Blotting technique, and the relation between myocardial cells proliferation and GLUT4 expression was evaluated.     

凝血酶诱导人胚胎肾系膜细胞增殖和PAI-1表达的研究

目的 :探讨凝血酶对人胚胎肾系膜细胞增殖和纤溶酶原激活物抑制物 1 (PAI 1 )表达的影响。方法 :采用噻唑蓝 (MTT)掺入法、Northern杂交和纤维蛋白平板法分别检测不同浓度凝血酶刺激下人胚胎肾小球系膜细胞增殖、PAI 1mRNA表达和PAI 1蛋白质活性的变化 ,并同时用人工水蛭素进行阻断实验。结果 :凝血酶呈浓度依赖性的方式促进人胚胎肾小球系膜细胞增殖、PAI 1mRNA表达和PAI 1蛋白质活性 ,水蛭素能特异地阻断凝血酶的作用。结论 :凝血酶可通过促进系膜细胞增殖、PAI 1的表达和提高其活性而导致肾脏损伤     

犬低血流心肌缺血诱导GLUT1基因表达增加

目的：通过检测低血流心肌缺血后心肌细胞葡萄糖转运子1(GLUT1)基因的表达,探讨心肌细胞对葡 萄糖摄取增加的代谢机制。方法：建立犬低血流心肌缺血模型,放射 性核素标记方法检测心肌葡萄糖摄取量和GLUT1数量,采用Northern印迹法分析缺血心肌GLU T1 mRNA表达,采用免疫印迹法分析心肌GLUT1多肽表达。结果：与正 常心脏比较,低血流心肌缺血后,缺血心肌GLUT1 mRNA和GLUT1多肽表达明显增加,分别为 正常心肌的3.6倍和1.6倍(P<0.01)。无论在正常或缺血心脏,GLUT1表达均无部位差异 。结论：心肌缺血能诱导缺血心肌局部GLUT1表达增加,致使心肌细胞 在低血流心肌缺血过程中葡萄糖摄取和代谢增强。GLUT1表达增强可能是一种重要的心肌缺 血后代偿性保护机制。     

PPARα激动剂对高糖刺激的肾系膜细胞PEDF和VEGF mRNA表达的影响

目的:研究色素上皮细胞衍生因子(PEDF)和血管内皮生长因子(VEGF)在糖尿病肾病中的作用以及PPARα激动剂与糖尿病肾病的关系。方法:将培养的大鼠HBZY-1肾小球系膜细胞株分为6组,高糖作为刺激因子,PPARα激动剂WY14643作为干预因素。分别设正常组,高糖组,高糖加WY14643治疗组(包括不同的WY14643浓度)以及高糖加WY14643溶剂对照组。用实时定量PCR法测定各组系膜细胞PEDF以及VEGF mRNA表达。结果:(1)与正常组相比较,高糖组肾系膜细胞PEDF mRNA的表达明显降低,而WY14643可逆转高糖导致的肾系膜细胞PEDF mRNA表达的降低。(2)高糖组肾系膜细胞VEGF mRNA的表达较正常组明显增加,而WY14643可减少高糖导致的肾系膜细胞VEGF mRNA表达的增加。结论:高糖可抑制大鼠肾系膜细胞PEDF并增加VEGF的表达,而PPARα激动剂可在一定程度上逆转高糖导致的这种改变。