EFFECTS OF PDGF-BB ON INTRACELLULAR CALCIUM CONCENTRATION AND PROLIFERATION IN CULTURED GLOMERULAR MESANGIAL CELLS

Objective To investigate the relationship between the alteration of intracellular calcium concentration and proliferation in cultured glomerular mesangial cells. Methods Rat mesangial cells were cultured. lntracellular calcium concentrations were measured by confocal Laser Scanning Microscopy and Fura-3 fluorescence dyeing techniques. Cell growth was measured by MTT assay. Results PDGF-BB increased intracellular calcium concentrations in a dose-dependent manner, and at the same time promote the proliferation of mesangial cells. After preincubation with calcium channel blocker nifedipine or angiotensin converting enzyme inhibitor captopril, both the increase of intracellular calcium concentrations and cell proliferations induced by PDGF-BB were inhibited. Tripteriglum Wilfordii Glycosides （TMG） significantly inhibited the mesangial cell proliferations, but it had no significant effect on intracellular calcium concentrations. Conclusion There was a positive relationship between the elevation of intracellular calcium concentration and cell proliferation in glomerular mesangial cells, but the increase of intracellular calcium concentrations wasn＇t the only way for proliferation.     

THE LOCALIZATION OF ADRENOMEDULLIN IN RAT KIDNEY TISSUE AND ITS INHIBITORY EFFECT ON THE GROWTH OF CULTURED RAT MESANGIAL CELLSA

Objective:To observe the localization of adrenomedullin(AM) in rat kidney tissue and its inhibitory effect on the growth of cultured rat mesangial cells (MsC).Methods:A monoclonal antibody against AM developed by our laboratory was used to detect the localization of AM protein in rat kidney tissue by avidin-biotin complex immunohistochemistry.The expressions of AM and its receptor CRLR mRNA on cultured glomerular epithelial cells (GEC)and MsC were investigated by Northern blot assay,and the possible effect of AM secreted by GEC on MsC proliferation was observed using [^3H] thymidine incorporation as an index.Results:A specific monoclonal antibody against AM was successfull developed.AM was immunohistochemically localized mainly in glomeruli (GEC and endothelial cells),some cortical proximal tubules,medullary collecting duct cells,interstitial cells,vascular smooth muscle cells and endothelial cells.Northern blot assay showed the AM mRNA was expressed only on cultured GEC,but not on MsC,however,AM receptor CRLR mRNA was only expressed on MsC.GEC conditioned medium containing AM can inhibit MsC growth and AM receptor blocker CGRP8-37 may partially decreased this inhibitory effect.Conclusion:AM produced by GEC inhibits the proliferation of MsC,which suggests that AM as an important regulator is involved in glomerular normal physiological functions and pathologic processes.     

ENDOGENOUS EXPRESSION AND HLA STABILIZATION ASSAY OF PLASMODIUM FALCIPARUM CTL EPITOPEMINIGENE IN HUMAN HLA－A2. 1 AND HLA－BS1 CELLS

Objective. To evaluate the Plasmodium falciparum CTL epitope vaccines in HLA class I allele specific human cell lines that have high frequency among Chinese population. Methods. Synthesized oligonucleotides encoding for P.f. CTL epitope genes, constructed eukaryotic expression plasmids, transfected the minigenes into HLA class I allele specific human cell lines and identified endogenous expressing of the minigenes by RT-PCR and HLA stabilization assay. Results. Two mini-genes encoding Plasmedium falciparum CTL epitopes were designed and cloned, respectively, into an eukaryotic expressing vector to form TR26 which was restricted to HLA-B51,SH6 which was restrictedto HLA-A2.1, and TS, which had the two aforementioned mini-genes fused in tandem. All of these CTL epitope genes were transfected and endogenously expressed in respective cell lines containing appropriate HLA molecules.The obviously increased expressions of HLA class I molecules were detected in the transfected cell lines. It was demonstrated that the two discrete Plasmedium falciparum epitope genes were effectively processed and presented, and the close proximity of the two epitope genes in one chain as in mini-gene TS did not interfere with the process-ing and presenting of each epitope gene in corresponding cell line. Conclusion. A successful expression and presentation of multiple CTL epitope mini-gene in MHC class I allele specific human cell lines were demonstrated by an in vitro assay, which could be corresponding to the vaccina-tion of CTL vaccines in people with different MHC I molecules. This work also suggested the possibility of constructing a multiple CI~ epitope plasmodium falciparum DNA vaccine that could cover most of Chinese population.     

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

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转化生长因子及大黄酸对肾小球系膜细胞葡萄糖转运蛋白功能的影响

目的对人肾小球系膜细胞葡萄糖转运蛋白-1（GLUT1）进行鉴定。研究GLUT1的作用特点,TGF-β1对其影响以及大黄酸的干预作用。方法分别用RT-PCR,免疫荧光染色,流式细胞仪和［3H］-2脱氧葡萄糖摄入率,对系膜细胞GLUT1mRNA表达,蛋白质分布和功能进行鉴定。观察不同浓度TGF-β1在加或不加大黄酸的情况下对系膜细胞葡萄糖摄人以及GLUT1mRNA表达的影响。结果人类肾小球系膜细胞上存在功能性的GLUT1。TGF-β1能刺激系膜细胞GLUT1mRNA的表达,并增加系膜细胞葡萄糖的摄入率[TGF-β1（836±35）％,对照（154±12）％、P＜0.01]。大黄酸对正常糖浓度培养条件下系膜细胞糖摄入无影响,但能明显抑制TGF-β1增加系膜细胞糖摄入的作用［TGF-β1为（198±28）％、大黄酸为（576±12）％,P＜0.01]。结论首次报道人系膜细胞上存在GLUT1,TGF-β1能从转录水平上调控GLUT1的功能,使系膜细胞葡萄糖摄入增加,但该作用能够明显地被大黄酸所抑制。     

转化生长因子大黄酸对肾小球系膜细胞葡萄糖转运蛋白功能的影响

目的 对人肾小球系膜细胞葡萄糖转运蛋白－１（ＧＬＵＴ１）进行鉴定。研究ＧＬＵＴ１的作用特点,ＴＧＦ－β１对其影响以及大黄酸的干预作用。方法 分别用ＲＴ－ＰＣＲ,免疫荧光染色,流式细胞仪和［＾３Ｈ］－２脱氧葡萄糖摄入率,对系膜细胞ＧＬＵＴ１ｍＲＮＡ表达,蛋白质分布和功能进行鉴定。观察不同浓度ＴＧＦ－β１在加或不加大黄酸的情况下对系膜细胞葡萄糖摄入以及ＧＬＵＴ１ｍＲＮＡ表达的影响。结果 人类肾小球系膜     

高糖对肾小球系膜细胞Smurf2表达的影响

目的观察不同浓度高糖刺激后大鼠肾小球系膜细胞胞内Smad泛素调节因子2(Smurf2)的表达,探讨泛素化降解在糖尿病肾病中的作用。方法将体外培养的大鼠肾系膜细胞分别设正常对照组(葡萄糖浓度5.6 mmol/L)、20 mmol/L高糖组、30 mmol/L高糖组、甘露醇组。分别用real ti me quantitative PCR法和细胞免疫荧光染色法及激光共聚焦显微镜检测各组细胞Smurf2的mRNA和蛋白的表达。结果(1)正常对照组系膜细胞Smurf2的mRNA和蛋白表达较弱。(2)高糖组Smurf2的mRNA和蛋白表达较正常对照组增强(P<0.05),呈浓度依赖性。结论高糖可诱导肾系膜细胞Smurf2表达增强。提示泛素-蛋白酶体途径可能参与了糖尿病肾病的病理进程。     

甲羟戊酸对人类肾小球系膜细胞增殖的影响

目的探讨甲羟戊酸(MVA)对人类肾小球系膜细胞(HMC)增殖的作用和机制。方法用酶联免疫检测仪检测不同浓度MVA作用下HMC在490 nm波长处的光吸收(A490 nm)值以测定不同浓度和时间点MVA作用下HMC的增殖水平。结果MVA在10-100 nmol/L范围内对HMC增殖有促进作用,且作用呈浓度依赖性,100 nmol/L MVA作用下A490 nm值从正常组0.408±0.019上升到0.509±0.011。100 nmol/L MVA对HMC的增殖作用在12-48 h内呈时间依赖性。12,24,48 hA490 nm值分别为0.201±0.040,0.326±0.019,0.509±0.011。结论MVA是HMC增殖的促进剂。     

辛伐他汀对高糖培养肾小球系膜细胞增殖和细胞周期的影响及机制

目的：研究辛伐他汀（SIM ）对高糖培养肾小球系膜细胞（GMC）增殖和细胞周期的影响,并观察其炎症因子和细胞外基质的变化。方法将大鼠 GMCs 分为4组：低糖组[LG 组,葡萄糖（Glu）5．6 mmol／L ],高糖组（HG 组,Glu 30 mmol／L ）, HG ＋ SIM （10μg／L）组和 HG ＋ SIM （25μg／L）组。采用4-甲基偶氮四唑蓝（M TT ）法检测各组细胞的增殖能力,同时比较各组细胞的 G0／G1,G2＋ M 期和 S 期细胞比例。比较各组细胞上清液中的转化生长因子β1（TGF-β1）、纤维黏连蛋白（FN）和Ⅳ型胶原（COL4）水平。结果 HG 组不同时刻的吸光度（A）高于 HG ＋ SIM （10μg／L）组、HG ＋ SIM （25μg／L）和 LG 组,差异有统计学意义（P＜0．05）。 LG 组的 G0／G1期细胞比例为（35．67±3．38）％,S 期细胞比例为（41．24±5．64）％;HG 组的 G0／G1期细胞比例为（25．88±4．02）％,S 期细胞比例为（28．65±1．88）％;HG ＋ SIM （10μg／L）组的 G0／G1期细胞比例为（28．12±2．01）％,S 期细胞比例为（35．55±2．09）％;HG ＋ SIM （25μg／L）组的 G0／G1期细胞比例为（31．85±3．52）％,S 期细胞比例为（39．82±2．23）％,差异有统计学意义（P＜0．05）。 HG 组上清液 TGF-β1、FN 和 COL4水平高于 HG ＋ SIM （10μg／L）组、HG ＋ SIM （25μg／L）和 LG组,差异有统计学意义（P＜0．05）。结论 SIM 可以通过降低 TGF-β1水平来抑制 GMCs 的增殖并调整细胞周期,抑制 GMCs 肥大。     

Interleukin 1 beta gene expression by mouse glomerular mesangial cells]

Interleukin 1 beta (IL-1 beta) plasmid DNA was isolated and purified with alkaline lysis method and equilibrium centrifugation in cesium chloride. IL-1 beta plasmid RNA was cut by restriction endonucleases EcoRI and XbaI, and after that it was 1 loaded in 0.75% low-melting-temperature agarose and underwent electrophoresis. 1.2 Kb DNA fragments were extracted from gel and for further purification it was used as IL-1 beta cDNA probe. RNA from mouse glomerular mesangial cells, tubular epithelial cells and P388D, cells was cut with EcoRI separately and then hybridized in dot blots with alpha 32P labeled IL-1 cDNA probe. The dot blot autoradiograph showed expression of the IL-1 beta gene in mesangial cell and P388D1 cell but showed no expression in epithelium. These results not only identified the specificity of IL-1 beta probe but also suggested that the local production of IL-1 beta may be important in the pathogenesis of glomerulonephritis.     

洛伐他汀对人类肾小球系膜细胞增殖及细胞周期的影响

目的探讨洛伐他汀(LOV)对人类肾小球系膜细胞(HMC)增殖及细胞周期的影响。方法人类系膜细胞常规培养。实验分为3组:正常对照组(10%胎牛血清)、洛伐他汀组(10μmol/LLOV)、甲羟戊酸(MVA)干预组(10μmol/LLOV 100μmol/LMVA)。每组分别在干预因素作用24,48h时用MTT法检测HMC在490nm波长处的光吸收(A490nm)值,观察LOV对HMC增殖的影响。用流式细胞仪测定HMC的细胞周期G1(DNA合成前期)与S期(DNA合成期)的比率,观察LOV对HMC周期进展的影响。结果MTT法结果显示24,48hLOV抑制HMC增殖,A490nm值分别为0.108±0.010和0.216±0.009,与正常组比较P<0.05。100μmol/LMVA可逆转其作用。细胞周期结果显示24,48hLOV抑制HMC细胞周期进展,S/G1比例分别为0.150±0.009和0.301±0.010,与正常组比较P<0.05。100μmol/LMVA可逆转其作用。结论LOV抑制HMC增殖,阻止细胞周期进展,MVA可逆转其抑制作用。