肿瘤坏死因子受体在肾小球系膜细胞中的基因表达及蛋白合成

目的：探讨肿瘤坏死因子α（ＴＮＦα）对肾小球系膜细胞（ＭＣ）作用的机理。方法：逆转录多聚酶链反应（ＲＴＰＣＲ）、原位杂交和免疫组织化学方法检测培养大鼠和人ＭＣ肿瘤坏死因子Ⅰ型受体（ＴＮＦＲ１）的基因表达及蛋白合成；原位杂交方法观察５例系膜增生性肾小球肾炎肾穿刺组织的ＴＮＦＲ１基因表达。结果：培养的大鼠和人ＭＣ有ＴＮＦＲ１ｍＲＮＡ的表达并有ＴＮＦＲ１蛋白的合成，系膜增生性肾小球肾炎增生的ＭＣ内有ＴＮＦＲ１表达。结论：ＴＮＦα通过与ＭＣ表面特异性受体相结合而发挥作用。     

丙型肝炎病毒感染后肾小球肾炎

目的探讨丙型肝炎病毒（ＨＣＶ）感染与肾小球肾炎病变的关系。方法应用免疫组化技术以丙型肝炎病毒抗体（抗ＨＣＶ）ＮＳ３和抗ＨＣＶＮＳ５单克隆抗体对２１例丙型肝炎患者尸检石蜡包埋肾组织中的丙型肝炎抗原（ＨＣＡｇ）进行检测。结果ＨＣＡｇ的检出率为６１９％（１３／２１），其中膜增殖性肾炎（ＭＰＧＮ）１１例，系膜增殖性肾炎（ＭｓＰＧＮ）１例，１例未见明显病变。ＨＣＡｇ阳性颗粒定位于肾小球系膜区、系膜细胞及肾小管上皮细胞胞浆内。２１例中有１７例血清乙型肝炎病毒（ＨＢＶ）标志呈阳性，同单纯ＨＢＶ感染组相比，本组患者出现肾小球肾炎病变更为多见（χ２＝８５，Ｐ＜００１）。结论ＭＰＧＮ型是ＨＣＶ感染后肾小球肾炎病变的主要类型。     

低密度脂蛋白对人肾小球系膜细胞增殖的影响

用酶法测定了正常人、肾病综合征（ＮＳ）未服用糖皮质激素和服用糖皮质激素治疗患者的血清脂蛋白组分，用体外肾小球细胞培养的方法观察了正常人及ＮＳ患者的低密度脂蛋白（ＬＤＬ）对正常成年人肾小球系膜细胞（ＨＭＣ）增殖的影响。结果表明：（１）ＮＳ患者的ＬＤＬ构成成分发生改变，其对ＨＭＣ的增殖影响与正常人不同，小剂量时促进ＨＭＣ增殖作用较正常人增强，较大剂量时使ＨＭＣ增殖高峰的下降提前出现。（２）糖皮质激素如无治疗ＮＳ的疗效时，不影响ＮＳ患者ＬＤＬ的构成成分和对ＨＭＣ的作用。系膜细胞病变是肾小球疾病的重要病理部位，我们认为脂质代谢异常可能加速肾脏疾病的进展。     

肿瘤坏死因子受体在肾小球系膜细胞中的基因表达及蛋…

探讨肿瘤坏死因子α对肾小球系膜细胞作用和机理。方法逆转录－多聚酶链反应、原位杂交和免疫组织化学方法检测培养大鼠和ＭＣ肿瘤坏死因子Ⅰ型受体的基因表达及蛋白合成；原位杂交方法观察５例系膜增生性肾小球肾炎肾穿刺组织的ＴＮＦ－Ｒ１基因表达。结论：ＴＮＦ－α通过与ＭＣ表面特异性受体相结合而发挥作用。     

脂多糖、植物血凝素对人肾小球系膜细胞分泌IL-18的影响

系膜细胞(ｍｅｓａｎｇｉａｌｃｅｌｌ,ＭＣ)数量的异常增加或减少是肾小球疾病的常见特征。在肾小球肾炎发病过程中,不管是肾小球受到损伤还是肾小球细胞过度增生的修复阶段,以及慢性肾小球细胞衰竭及肾小球硬化,都存在细胞凋亡。而Ｆａｓ介导的细胞凋亡可能参与肾小球损伤过程中肾小球系膜细胞的死亡[1]。白细胞介素18(ＩＬ18)是新近发现的一种细胞因子,可直接增强ＦａｓＬ介导的Ｔｈ1细胞毒效应[2]。为了解ＭＣ是否表达ＩＬ18,以及ＩＬ18在肾小球系膜细胞凋亡中的作用,我们对脂多糖(ＬＰＳ)、植物血凝素(ＰＨＡ)诱导人肾小球ＭＣ分泌ＩＬ…     

TGF-β1和ECM与糖尿病肾病关系的实验研究

目的 观察高糖条件下大鼠肾小球系膜细胞（ＧＭＣ）分泌细胞外基质（ＥＣＭ）成分中纤粘连蛋白（ＦＮ）、层粘连蛋白（ＬＡＭ）、Ⅱ及Ⅳ型胶原和转化生长因子β（ＴＧＦ－β１）的变化,探讨细胞因子和细胞外基质在糖尿病肾病发病中的作用。方法 采用体外大鼠肾小球系膜细胞培养,用ＥＬＩＳＡ方法测定培养上清液中的ＥＣＭ和ＴＧＦ－β１。结果（１）高糖条件下,ＧＭＣ合成和分泌ＴＧＦ－β１增加。（２）高糖对体外培养的系膜细     

糖皮质激素受体对肾小球系膜细胞表达细胞间黏附分子-1的影响

近年来免疫病理学研究已经证实细胞间黏附分子１（ＩＣＡＭ１）作为细胞黏附分子中免疫球蛋白超家族成员之一,通过与炎性细胞表面同族型配体之间相互作用形成黏附,在免疫监督、炎症反应、吞噬过程、动脉粥样硬化等过程中起着重要的作用［１,２］。目前证实,肾小球系膜细胞（ＭＣ）表面低表达ＩＣＡＭ１,而细胞因子白细胞介素１β（ＩＬ１β）、肿瘤坏死因子α（ＴＮＦα）、干扰素γ（ＩＦＮγ）等刺激并上调系膜细胞表达ＩＣＡＭ１。本研究目的是进一步探讨糖皮质激素（ＧＣ）,如氟美松（Ｄｅｘ）对ＴＮＦα诱…     

霉酚酸酯对大鼠抗Thy-1系膜增殖性肾小球肾炎模型的作用

系膜增殖性肾小球肾炎（ＭｓＰＧＮ）是肾小球肾炎最常见的病理类型，以系膜细胞（ＭＣ）增殖和细胞外基质（ＥＣＭ）增多为主要病理特征，因此抑制ＭＣ增殖及ＥＣＭ的聚积是延缓肾小球肾炎进展的关键环节之一。个别体外研究提示，霉酚酸酯（ＭＭＦ）具有抑制ＭＣ增殖的作...     

系膜增殖性肾炎白细胞介素6表达与细胞外基质产生的研究

系膜细胞增生、细胞外基质（ＥＣＭ）聚积和扩张是系膜增殖性肾小球肾炎（ＭｓＰＧＮ）发生发展的重要病理基础［１］。研究表明,系膜细胞可分泌具有生物活性的白细胞介素６（ＩＬ６）,ＩＬ６又可促进系膜细胞生长［２］。人类ＭｓＰＧＮ中ＩＬ６的表达及其与ＥＣ...     

低密度脂蛋白对人肾小球系膜细胞产生炎性介质的影响

目的：探讨脂蛋白在肾小球损伤中的作用，观察人低密度脂蛋白（ＬＤＬ）对体外培养的人肾小球系膜细胞（ＧＭＣ）产生活性氧（ＲＯＳ）、血小板活化因子（ＰＡＦ）、肿瘤坏死因子（ＴＮＦ）和乳酸脱氢酶（ＬＤＨ）的影响，并与经ＬＤＬ刺激后ＧＭＣ的增殖水平相比较。方法：将ＧＭＣ加或不加ＬＤＬ刺激，分别收集３０ｍｉｎ、１８ｈ或４８ｈ后的上清，测定其Ｈ２Ｏ２、Ｏ２＾、ＴＮＦ、ＰＡＦ和ＬＤＨ的水平，以四唑蓝（ＭＴＴ）摄入     

粘着斑激酶反义寡核苷酸与胃癌细胞生长及凋亡的关系

根据FAKcDNA序列设计合成与FAKmRNA碱基序列互补的FAK反义寡核苷酸,导入BGC－823胃癌细胞,通过MTT比色法、细胞免疫化学染色法、逆转录－聚合酶链反应（RT－PCR）、细胞周期及电镜等方法,检测FAK反义寡核苷酸对人胃癌细胞的影响。结果示：①胃癌细胞生长明显受到抑制,抑制效应呈浓度和时间依赖性。②细胞内P125蛋白和FAKmRNA表达均明显下降。③经FAK反义寡核苷酸处理后BGC－823胃癌细胞发生凋亡,FCM普上可见明显的凋亡峰,电镜观察呈现凋亡早期形态改变,认为FAK反义寡核甘酸导入BGC－823胃癌细胞后,使BGC－823胃癌细胞FAKmPNA及P125蛋白表达水平下降,抑制PGC－823胃癌细胞增列,同时诱导BGC－823胃癌细胞发生凋亡。     

周期素激酶抑制剂p27在肿瘤坏死因子-α诱导系膜细胞增生中的作用

目的 研究周期素激酶抑制剂p27在肿瘤坏死因子－α（TNF－α）诱导系膜细胞（MC）增生中的作用。方法 采用蛋白印迹（Western杂交）方法测定MC裂解液p27蛋白水平。[^3H]胸腺嘧啶核苷（[^3H]TdR）测定MC增生的情况,观察p27反义寡核苷酸（ODN）转染对TNF-α刺激MC中的p27水平及其对增生程度的影响。结果 TNF－α（200000U/L）可使无血清培养24h的MC中的p27水平降低（P＜0.01）,同时使[^3H]TdR掺入增加（P＜0.01）;p27反义ODN转染可降低TNF－α刺激24h的MC中的p27水平（P＜0.01）,同时可使[^3H]TdR掺入增加更为明显（P＜0.05）。结论 p27水平降低可能在TNF－α诱导MC增生中起重要作用。     

肾炎宁对LPS诱导人肾小球系膜细胞增殖及细胞外信号调节蛋白激酶的影响

目的 探讨肾炎宁对人肾小球系膜细胞(HMC)细胞增殖及细胞外信号调节蛋白激酶(extracellular signal-regulated protein kinase,ERK)的影响.方法 应用细胞培养技术,进行HMC培养,采取血清药理学方法,制备肾炎宁药物血清,利用脂多糖(LPS)为刺激因子,应用四唑盐(MTT)比色法和酶联免疫吸附试验(ELISA)检测细胞的增殖和磷酸化ERK1/2的表达.结果 肾炎宁可显著抑制HMC增殖,抑制率在12、24和48 h分别为16.0%、15.4%、18.2%,LPS组磷酸化ERK1/2的含量与空白组比较明显升高(P<0.05);中药组与空白组比较,磷酸化ERK1/2含量明显降低(P<0.05);LPS+肾炎宁组与LPS组比较,磷酸化ERK1/2表达明显降低 (P<0.05).结论 肾炎宁具有抑制正常培养条件及LPS刺激条件下磷酸化ERK1/2,从而发挥对肾小球硬化的治疗作用.     

Inhibition by transforming growth factor-beta1 of the cellular action of arginine vasopressin in cultured rat glomerular mesangial cells.

The present study was undertaken to determine whether transforming growth factor (TGF)-beta1 modulates the cellular actions of arginine vasopressin (AVP) in cultured rat glomerular mesangial cells. AVP increased cytosolic free calcium ([Ca2+]i), and TGF-beta1 dose-dependently reduced the AVP-mobilized [Ca2+]i. Such an inhibition by exogenous TGF-beta1 was abolished by liposomal transfection of antisense oligodeoxynucleotide for the TGF-beta type II receptor. AVP activated mitogen-activated protein (MAP) kinase, which was significantly reduced by 1 ng/ml TGF-beta1. AVP increased [3H]thymidine incorporation into mesangial cells in a dose-dependent manner, and 1 ng/ml TGF-beta1 significantly reduced the AVP-stimulated [3H]thymidine incorporation. However, 10 microM antisense oligodeoxynucleotide for the TGF-beta type II receptor seemed to attenuate the inhibition by TGF-beta1. 1 X 10(-7) M AVP significantly increased inositol 1,4,5-trisphosphate (IP3) production by 1.8-fold, but this production was totally blunted by 1 ng/ml TGF-beta1. TGF-beta1 did not affect [3H]AVP receptor binding. 1 X 10(-6) M AVP concentration stimulated TGF-beta1 production in mesangial cells by 4-fold. These results indicate that TGF-beta1 inhibits the cellular signaling of AVP at steps beyond the AVP receptors and prior to the phospholipase C activation, and that TGF-beta1 may participate in a negative feedback regulation on the cellular action of AVP in glomerular mesangial cells.     

乳腺癌细胞中HMGA2的表达及其反义寡核苷酸对乳腺癌细胞黏附侵袭的影响

目的观察HMGA2在乳腺癌细胞中的表达以及其反义寡核苷酸(ASODN)对乳腺癌细胞黏附和侵袭能力的影响。方法采用流式细胞术检测检测乳腺癌细胞株中HMGA2蛋白的表达;将HMGA2 ASODN及其对照序列(空白对照和错配序列)通过脂质体转染MCF-7细胞,参照V lodavsky实验方法和采用Boyden小室法检测细胞的黏附能力和侵袭能力。结果 HMGA2 ASODN能特异性下调MCF-7细胞HMGA2蛋白表达,而且能抑制其细胞黏附和侵袭能力。结论 HMGA2 ASODN抑制乳腺癌细胞黏附和侵袭能力具有可行性。     

Release of endothelins and platelet-activating factor by a rat pleural mesothelial cell line.

Thrombin is a multifunctional serine protease. It is generated in inflammatory processes and induces the proliferation and chemotaxis of a variety of cells including mesothelial cells (MTCs). MTCs are epithelial cells derived from the mesoderm, as are the vascular endothelial cells. Since thrombin acts on endothelial cells to produce platelet-activating factor (PAF) and endothelin (ET)-1, it was hypothesized that MTCs also produce PAF and ET via the action of thrombin. Rat pleural MTC (RMTC, 4/4 R.M.-4) monolayers were cultural in tissue culture dishes for various periods. The supernatants were fractionated by means of high-performance liquid chromatography to determine the ET isoforms and PAF species present. Immunoreactive ET was measured using an enzyme-linked immunosorbent assay, and PAF was measured by means of a bioassay using a platelet aggregometer. ET-1, ET-2 and ET-3 were detected in RMTC-conditioned medium, and the predominant isoforms were ET-1 and ET-2. RMTCs mainly released C16:0 PAF into the supernatant. Immunoreactive ET and PAF were released via the action of thrombin. Synthetic PAF significantly induced secretion of ET, but the PAF receptor antagonists, WEB2086 and E6123, failed to modulate thrombin-induced ET release. These results indicate that thrombin acts on pleural rat mesothelial cells to release ET and PAF, which may play a role in the development of pleurisy.     

Modulation of apoptosis, tumorigenesity and metastatic potential with antisense H-ras oligodeoxynucleotides in a high metastatic tumor model of hepatoma: LCI-D20

BACKGROUND/AIMS: To investigate the effect of antisense H-ras DNA on tumorigenesity, apoptosis and metastasis of a high metastatic tumor model of human hepatocellular carcinoma in nude mice LCI-D20. METHODOLOGY: LCI-D20 cells in primary culture were treated with 10 microns/L antisense oligodeoxynucleotide (ODN) drugs in vitro. 1.5 x 10(6) LCI-D20 cells with or without pretreatment were inoculated into each elevated subcutaneous (s.c.) flap in 14 nude mice, 6 animals for antisense H-ras oligodeoxynucleotide treated cells, 4 for H-ras non-specific antisense oligodeoxynucleotide treated cells, and the rest 4 for cells without pretreatment. RESULTS: In in vitro cell culture study, 5-day continuous suppression of H-ras expression by antisense H-ras oligodeoxynucleotide resulted in significant inhibition of the proliferation of LCI-D20 cells (t = 31.529, P < 0.01). In situ end-labeling detection showed that apoptotic cell death was significantly increased in cells with 5-day treatment of antisense H-ras oligodeoxynucleotide (34.0 +/- 4.5%) in comparing with cells without treatment (2.5 +/- 1.2%, t = 13.434, P < 0.01) or treated with non-specific antisense oligodeoxynucleotide (4.8 +/- 1.4%, t = 12.453, P < 0.01) at the corresponding time. In the in vivo experiment, at week 6, no palpable tumor could be found in 50% (3/6) of animals receiving cells with pretreatment of antisense H-ras oligodeoxynucleotide, while 100% (4/4, 4/4) of animals in the 2 control groups developed palpable tumors. Tumor growth in antisense H-ras treated animals was significantly retarded in comparison with that of the untreated (t = 3.509, P < 0.01) or non-specific antisense oligodeoxynucleotide treated animals (t = 3.452, P < 0.01). 75% to 100% of animals in the 2 control groups developed lung metastases, while in antisense H-ras treated animals lung metastasis foci could not be found by random serial section and microscopy (u = 2.536, P < 0.01; u = 3.162, P < 0.01, respectively). CONCLUSIONS: Specific inhibition of H-ras expression by antisense H-ras oligodeoxynucleotides could not only induce apoptotic cell death, inhibit the growth rate of LCI-D20 cells in vitro and in vivo, but also alter in vivo tumorigenesity and metastatic potential of LCI-D20 cells.     

PI3K/Akt/mTOR信号通路对大鼠肾小球系膜细胞增殖的调控作用探讨

目的:探讨磷脂酰肌醇-3(PI3K)/蛋白激酶(Akt)/哺乳动物雷帕霉素靶蛋白(m TOR)信号通路对大鼠肾小球系膜细胞(MC)增殖的调控作用。方法:体外培养大鼠MC,随机分为正常对照组、表皮生长因子(EGF)(10ng/m L)组、PI3K抑制剂LY294002组(2μg/m L)、EGF联合LY294002组。采用四甲基偶氮唑蓝(MTT)法检测4组MC的增殖,流式细胞术检测MC周期,免疫荧光法检测系膜细胞m TOR的表达。结果:与正常对照组相比,EGF组能显著促进大鼠MC增殖,G0/G1期细胞减少,S期及G2/M期细胞增加,且m TOR表达增加;LY294002组大鼠MC增殖受抑制,G0/G1期细胞增加,S期及G2/M期细胞减少,且m TOR表达降低;与EGF组比较,EGF联合LY294002干预组MC增殖受抑制,G0/G1期细胞增多,S期及G2/M期细胞减少,且m TOR表达降低。结论:m TOR信号通路参与了大鼠肾小球MC增殖的调控,抑制该信号通路的活化可显著抑制MC的增殖和MC周期的进程。     

Oxidized LDL, glomerular mesangial cells and collagen

Lipid deposits, foam cell collection and accumulation of mesangial matrix components are recognized as early events in the development of focal segmental glomerulosclerosis (FSGS). Studies have suggested that oxidative stress is increased in uremic patients. Oxidized low-density lipoprotein (Ox-LDL) has been identified in the lesions of FSGS. Dietary antioxidants reduced not only the staining intensity of Ox-LDL but also the severity of renal injury in rats with experimental FSGS possibly by making lipoproteins resistant to oxidation. In vitro studies showed that LDL during its incubation with human mesangial cells (HMC) was peroxidatively modified and stimulated alpha1(I), alpha1(III), and alpha1(IV) collagen mRNA expression. Vitamin E, an antioxidant, and antibody against Ox-LDL caused a marked reduction in collagen mRNA stimulated by LDL. These findings suggest that LDL deposited and oxidized in the glomeruli may be implicated in the development of glomerulosclerosis by facilitating excessive mesangial matrix generation.