高浓度尿酸对人近端肾小管上皮细胞氧化应激的影响

目的观察高浓度尿酸对人近端肾小管上皮细胞氧化应激的影响,探讨其可能的作用途径。方法体外培养人近端肾小管上皮HK-2细胞,以720μmol/L的尿酸干预0、12、24、48 h后收集细胞;DCHF-DA染色检测细胞内活性氧（ROS）含量,分光光度计检测细胞上清液中的丙二醛（MDA）、总超氧化物岐化酶（T-SOD）,MitoSOXTM染色观察活细胞线粒体中ROS的生成情况。结果尿酸干预12 h时细胞内总ROS含量较未干预时无明显变化,细胞上清中MDA含量、T-SOD活性亦较未干预时无明显差异（P均〉0.05）;干预24 h时总ROS含量有所升高,MDA含量上升,T-SOD活性降低,48 h时上述变化更为明显（P均〈0.05）;线粒体内ROS也在尿酸干预24 h时合成上调,48 h时升高更为显著（P〈0.05）。结论高浓度尿酸可诱导HK-2细胞氧化应激反应,线粒体ROS合成增多可能是其作用基础。     

尿酸对肾小管上皮细胞sICAM-1表达的影响及其机制探讨

目的探讨高尿酸损害肾小管上皮细胞的可能机制。方法将培养后的人肾小管上皮细胞株HK-2细胞(浓度为5×105/mL)分为6组,分别接种于不同细胞培养板。对照组加入10%FBS普通PRMI 1640培养液,尿酸2 mg/dL组、尿酸7 mg/dL组、尿酸12 mg/dL组、尿酸17 mg/dL组、尿酸22 mg/dL组加入相应剂量的尿酸。分别于干预12、24、48 h采用ELISA法检测各组HK-2细胞液中可溶性细胞间黏附分子-1(sICAM-1)表达,免疫组化法检测NF-κB表达。结果与对照组比较,尿酸干预12、24、48 h时,尿酸17 mg/dL组、尿酸22 mg/dL组sICAM-1的表达随时间明显增多(P<0.05),尿酸17 mg/dL组、尿酸22 mg/dL组NF-κB表达随时间明显增多(P<0.05)。sICAM-1与NF-κB的表达呈正相关(r=0.998,P<0.05)。结论高浓度尿酸可以引起人肾小管上皮细胞sICAM-1的表达增加,其机制可能为激活NF-κB途径引起肾小管上皮细胞sICAM-1表达增加。     

肾小管上皮细胞的免疫活性作用

研究表明，皮肤、消化道、关节、甲状腺、胰腺、肝脏和肾脏等处的实质细胞与免疫细胞发生相互作用以启动或调控局部的免疫反应。在肾脏疾病中，肾小管上皮细胞通过其抗原呈递功能参与肾脏疾病的发生、发展。研究显示，肾小管上皮细胞具备免疫细胞的潜质，在某些肾脏病理情况下表现出免疫细胞的活性，从而主动参与小管间质局部的免疫反应、炎症以及间质纤维化过程。本文将对肾小管上皮细胞免疫活性的激活、机制和结局作一综述。     

醛固酮对人肾小管上皮细胞表达纤溶酶原激活物抑制物-1和纤维连接蛋白的影响

目的 探讨醛固酮(ALD)对人肾小管上皮细胞表达纤溶酶原激活物抑制物-1(PAI-1)和纤维连接蛋白(FN)的影响.方法 人肾小管上皮细胞(HK-2)培养于含5％ FCS的DMEM/F12培养液中,以不同浓度的ALD(10-11、10 -10、10-9、10-8 mol/L)刺激HK-2细胞48 h后,应用半定量RT-PCR法检测细胞中PAI-1和FN mRNA的表达,ELISA法测定培养上清液中FN的含量.结果 ALD呈剂量依赖性地上调HK-2细胞中PAI-1和FN的基因表达,并增加培养上清液中FN的含量(P＜0.05).结论 ALD通过促进HK-2细胞PAI-1的基因表达,并促进其合成分泌FN,导致肾间质纤维化的进展.     

大鼠近端肾小管上皮细胞的原代培养和鉴定

目的 建立较好的大鼠近端肾小管上皮细胞原代培养模型.方法 无菌取Wistar大鼠肾脏,取皮质剪碎,经Ⅰ型胶原酶消化和45%Percoll连续密度梯度离心进行纯化,用含10%胎牛血清的DMEM/F12培养基原代培养并传代,观察细胞形态并以免疫细胞化学染色及酶化学染色鉴定.结果 培养4～5 d细胞融合成单层,呈典型的鹅卵石样,细胞角蛋白18及碱性磷酸酶染色均呈阳性,细胞可传2～3代.结论 此法可在短期获得数量较多、重复性好的近端肾小管上皮细胞,为体外研究肾小管细胞的病变提供了实验平台.     

左旋肉碱对氧化应激损伤肾小管上皮细胞的保护作用

目的观察左旋肉碱对过氧化氢（H2O2）应激损伤人肾小管上皮细胞的保护作用,并探讨其可能机制。方法用H2O2作用于人肾小管上皮细胞系HK-2,建立肾小管上皮细胞氧化应激损伤模型;MTT法检测左旋肉碱预处理后HK-2细胞的活力;用酶化学法测定HK-2细胞超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH—Px）、过氧化氢酶（CAT）活性及总抗氧化能力（T—AOC）、丙二醛（MDA）;荧光显微镜观察及流式细胞仪测定细胞内活性氧（ROS）及HK-2细胞凋亡率。结果左旋肉碱预处理12h能抑制H2O2损伤所导致的HK-2细胞活力降低,增加细胞中SOD、GSH—Px和CAT含量,提高细胞T-AOC,降低细胞中MDA和ROS水平,抑制HK-2细胞凋亡。结论左旋肉碱对氧化应激所致的肾小管上皮细胞损伤具有保护作用,其机制可能与增强细胞抗氧化能力、减少自由基生成、抑制脂质过氧化反应及细胞凋亡有关。     

马兜铃酸对肾小管上皮细胞氧化应激效应的作用

目的探讨马兜铃酸（AA）对人肾小管上皮细胞（HK-2）氧化应激的作用。方法体外培养HK-2细胞,AA组在培养体系中加入AA 20 mg/m l刺激培养48 h;另设对照组。采用MTT法观察HK-2细胞活性,透射电镜观察细胞的超微结构,流式细胞术检测细胞凋亡率,比色法检测丙二醛（MDA）、谷胱甘肽过氧化物酶（GSH-PX）活力,黄嘌呤氧化酶法检测超氧化物歧化酶（SOD）活力。结果与对照组比较,AA组HK-2细胞活性显著抑制,细胞凋亡比例明显增加,SOD、GSH-PX活力明显下降,MDA含量明显增加（P均〈0.05）。结论 AA诱导HK-2细胞凋亡的机制与其导致的氧化应激损伤有关,氧化应激作用可能为马兜铃酸肾病的发病机制之一。     

膜性肾病尿蛋白对人肾小管上皮细胞凋亡的影响

目的探讨膜性肾病尿蛋白对人肾小管上皮细胞凋亡存在剂量依赖关系。方法提取膜性肾病患者尿蛋白,进行蛋白成分分析、细菌内毒素试验、蛋白溶液的肌酐水平等质量鉴定后,试验组分别按1.0、2.0、4.0、8.0 mg/ml蛋白浓度刺激人肾小管上皮细胞48 h,空白组无蛋白,Ho-echst33258染色法观察人肾小管上皮细胞凋亡情况。结果膜性肾病尿蛋白由56.4%白蛋白、34.6%转铁蛋白、6.9%IgG组成。肌酐水平、内毒素均低于人体正常范围的下限。Hoechst33258染色法分析发现试验组与空白组之间,较大浓度尿蛋白组与较小浓度尿蛋白组之间人肾小管上皮细胞凋亡指数有显著差异(P<0.05)。结论肾小管上皮细胞凋亡程度与膜性肾病尿蛋白存在浓度依赖关系。     

大黄酸对糖尿病大鼠肾小管上皮细胞表型转化的影响

目的:探讨大黄酸防治糖尿病肾病的机制.方法:60只雄性Wistar大鼠随机分为时照组(n=20只)和糖尿病模型组(n=40只),模型组大鼠给予腹腔内一次注射STZ 60 mg/kg诱导糖尿病模型,再随机分为糖尿病组和大黄酸组.大黄酸组给予大黄酸150 mg/(kg·d)灌胃.16周后测定大鼠24 h尿蛋白,光镜观察肾小管间质损害及纤维化,免疫组织化学法检测肾小管上皮细胞内肌成纤维细胞标志蛋白α-SMA和间质细胞标志蛋白Vimentin的表达,并对结果进行半定量分析.结果:糖尿病组大鼠24 h尿蛋白排泄增加(P＜0.01),肾小管上皮细胞α-SMA和Vimentin阳性表达面积分别为(0.2763±0.0529)和(0.1388±0.0336);大黄酸组大鼠24 h尿蛋白排泄较糖尿病组减少,肾小管间质损伤和间质纤维化程度减轻,其肾小管上皮细胞α-SMA、Vimentin阳性表达面积分别为(0.1449±0.0447)和(0.822±0.0176),表达较糖尿病组显著下调(P＜0.01).结论:大黄酸能显著下调糖尿病大鼠肾小管上皮细胞α-SMA、Vimentin表达,阻抑肾小管上皮细胞的表型转化.     

质粒表达型小分子干扰RNA对人肾小管上皮细胞单核细胞趋化蛋白-1抑制作用的研究

目的研究质粒表达型小分子干扰RNA(siRNA)对人肾小管上皮细胞(HKC)单核细胞趋化蛋白-1(MCP-1)的抑制作用。方法2002年2～7月对同济大学附属东方医院针对人MCP-1 mRNA67、116、142位点设计并合成的3对MCP-1siRNA序列,构建MCP-1特异性siRNA真核表达载体pRNAT-MCP-1-Ⅰ、pRNAT-MCP-1-Ⅱ、pRNAT-MCP-1-Ⅲ,以脂质体法转染至HKC,转染24h后分别采用实时定量反转录-聚合酶链反应(RT-PCR)及Western blot技术检测HKC内MCP-1 mRNA、蛋白表达。结果siRNA表达载体的转染效率为60%,与正常对照组相比,3组不同质粒表达型siRNAMCP-1 mRNA和蛋白表达均显著降低(P<0.01)。结论siRNA能高效抑制HKC的MCP-1基因表达,提示siRNA在防治肾间质纤维化中具有潜在作用。     

Regulation of lysyl oxidase in vascular cells: lysyl oxidase as a new player in cardiovascular diseases

Lysyl oxidase (LOX) plays a crucial role in the maintenance of extracellular matrix stability and could participate in vascular remodelling associated with cardiovascular diseases. Evidence from in vitro and in vivo studies shows that LOX downregulation is associated with the endothelial dysfunction characteristic of earlier stages of the atherosclerotic process. Conversely, upregulation of this enzyme in vascular cells could induce neointimal thickening in atherosclerosis and restenosis. In fact, LOX is chemotactic for vascular smooth muscle cells and monocytes, is modulated by proliferative stimulus in these cells, and could control other cellular processes such as gene expression and cell transformation. Furthermore, it is conceivable that LOX downregulation could underlie plaque instability and contribute to the destructive remodelling that takes place during aneurysm development. Overall, LOX could play a key role in vascular homeostasis and, hence, it emerges as a new player in cardiovascular diseases. This review addresses the experimental evidence related to the role of LOX in vascular disorders and the potential benefits of controlling its expression and function.     

Control of fibronectin synthesis by rat granulosa cells in culture

The secreted and cellular [35S]methionine-radiolabeled proteins of cultured rat granulosa cells were separated by electrophoresis on sodium dodecylsulfate (SDS) polyacrylamide gradient slab gels. From 24 to 72 h of culture FSH increased the intensity of labeling of most of the secreted proteins. A 220,000-dalton protein, however, increased in intensity only in control cultures and became the major secreted protein after 72 h, comprising 20% of the total radiolabeled proteins. This protein was identified as fibronectin by immunoprecipitation. There was no increase in the secreted or cellular fibronectin in FSH- or testosterone- and insulin-treated cultures. These studies indicate that a component of extracellular matrix is a major secretory product of unstimulated immature granulosa cells. As hormones induce the differentiated functions of granulosa cells in culture, the secretion of fibronectin is inhibited.     

大鼠降结肠上皮γ-氨基丁酸及谷氨酸脱羧酶的表达特征

目的:检测γ-氨基丁酸(gamma-aminobutyricacid,GABA)和谷氨酸脱羧酶(glutamicaciddecarboxylase,GAD)在大鼠降结肠上皮的表达及分布特征,并探讨GABA与上皮细胞分化增殖的关系.方法:用免疫荧光及激光共聚焦显微扫描技术,检测GABA、GAD65及GAD67在大鼠降结肠上皮中的表达,并以麦芽凝聚素组织化学染色与免疫荧光结合的双重染色显示GABA和GAD65表达细胞的分布特征.同时,用RT-PCR和原位分子杂交方法检测GADmRNA的表达.此外,用3H-胸腺嘧啶放射自显影法显示降结肠上皮的增殖带.结果:RT-PCR显示降结肠黏膜中GAD65及GAD67mRNA均阳性,原位杂交显示阳性杂交信号主要分布在上皮细胞的隐窝和腔面,且GAD65信号较GAD67强.GABA及GAD65免疫反应阳性细胞主要分布在降结肠的腔面和隐窝的上1/3上皮细胞的胞质,而GAD67阳性细胞仅分布腔面,此外,GABA及GAD65阳性染色也见于黏膜固有层.双重染色显示杯状细胞中GABA及GAD65均阴性.3H-胸腺嘧啶标记阳性细胞主要在隐窝的中下段.结论:GABA及GAD65分布在大鼠降结肠上皮的成熟带及功能带,GABA系统可能参与上皮细胞的分化与增殖的调节.     

血管紧张素Ⅱ对肾小管上皮细胞Klotho基因表达的影响

目的:观察血管紧张素Ⅱ(AngⅡ)刺激正常大鼠肾小管上皮细胞(NRK-52E)后Klotho、p53、p21mRNA及蛋白的表达和细胞凋亡情况,探讨高血压肾损害肾小管上皮细胞凋亡的相关机制。方法:AngⅡ按浓度梯度0(对照组)、10-10、10-9、10-8、10-7、10-6mol/L分组培养NRK-52E24h,确定适宜干预浓度后,按时间梯度0h、6h、12h、18h、24h分组培养确定最佳干预时间。分别用RT-PCR和Western印迹法检测Klotho、p53、p21mRNA及蛋白的表达;分光光度法检测Caspase-3的活性;AnnexinV-FITC双染法及流式细胞仪检测细胞凋亡率。结果:随着AngⅡ干预NRK-52E浓度增高(10-10mol/L～10-6mol/L)及作用时间延长(6h～24h),Caspase-3活性逐渐增高,凋亡率逐渐增加(P<0.01)。与对照组比较,AngⅡ组Klotho表达明显下调,p53和p21表达上调,Caspase-3活性增高,凋亡率增加(P<0.01)。结论:AngⅡ可通过抑制Klotho表达,增加p53和p21的表达,活化Caspase-3,从而诱导肾小管上皮细胞发生凋亡,这可能是其在高血压肾损害肾小管细胞凋亡中的作用机制之一。     

Abnormal deposition of collagen around hepatocytes in Wilson's disease is associated with hepatocyte specific expression of lysyl oxidase and lysyl oxidase like protein-2

BACKGROUND/AIMS: Lysyl-oxidases catalyze the oxidation of lysine residues in collagen and elastin thereby promoting their polymerization. We have studied here the expression of four lysyl-oxidases in normal and diseased human liver. METHODS: The expression of the different lysyl-oxidases in paraffin embedded liver sections was studied using in-situ hybridization and immunohistochemistry. The enzymatic activity of lysyl-oxidase like protein-2 (Loxl2 or LOR-1) using a previously described lysyl-oxidase assay. RESULTS: We have found that the four lysyl-oxidases which we examined are not significantly expressed in the normal liver. By contrast, Wilson's disease and primary biliary cirrhosis (PBC) patients express lysyl-oxidase (Lox) and lysyl-oxidase like protein-2 (Loxl2 or LOR-1) in hepatocytes, and the expression is accompanied by collagen deposition around the hepatocytes. Lysyl-oxidases are also expressed in additional fibrotic liver diseases such as hepatitis B and C but in these diseases the expression is confined to the fibrotic lesions and collagen does not accumulate around hepatocytes. We have found that Loxl2 is able to oxidize lysine residues of collagen, and behaves in that respect similarly to Lox. The copper chelator D-penicillamine inhibits Loxl2 induced oxidation of collagen but the Lox inhibitor beta-aminopropionitrile did not inhibit the oxidation using a BAPN concentration at which Lox activity was completely inhibited. Loxl2 also catalyzed the oxidation of cell surface proteins on HepG2 hepatoblastoma cells and inhibited their proliferation. CONCLUSIONS: Upregulation of Lox and Loxl2 in hepatocytes of Wilson's disease and PBC patients may contribute to liver damage by various mechanisms. The upregulation of Lox and Loxl2 in Wilson's disease could perhaps be utilized for diagnostic purposes since their expression is up-regulated in hepatocytes even before the onset of fibrosis.     

促红细胞生成素对马兜铃酸致肾小管上皮细胞凋亡的影响

目的：探讨促红细胞生成素（EPO）对马兜铃酸（AA）所刺激的肾小管上皮细胞结构损伤的影响。方法：以不同浓度AA（10、20、40μg／ml）刺激LLC—PK1细胞株，同时培养体系中加入不同浓度的EPO（5、10、20U／m1），另设对照组。各组细胞培养24h后，透射电镜观察细胞的超微结构，TUNEL法原位检测细胞凋亡情况，流式细胞仪检测凋亡细胞的比例。结果：电镜显示：与AA10μg／ml组相比，AA10μg／ml＋EPO20U／ml组有少数细胞线粒体肿胀或呈早期凋亡改变（异染色质），大部分细胞结构基本正常；AA10μg／ml＋EPO10U／ml组与前者相似，有少数细胞出现凋亡。AA20μg／ml和AA40μg／ml刺激的细胞经不同浓度EPO干预后，细胞的损伤无明显变化。TUNEL结果：经AA10μg／ml、20μg／ml刺激后，核染色阳性的细胞百分比与对照组比较明显增加（P〈0．05）。与AA10μg／ml组比较，EPO10U／ml和EPO20U／ml可明显降低阳性细胞的百分比（P〈0．05）。流式细胞仪显示：经AA10μg／ml、20μg／ml刺激24h后，细胞凋亡的比例明显增加（P〈0．05），AA20μg／ml组凋亡和坏死的细胞比例高于AA10μg／ml组。与AA10μg／ml组比较，EPO10U／ml和EPO20U／ml干预后细胞凋亡比例有所下降，以EPO20U／ml作用显著（P〈0．05）。AA20μg／ml组细胞凋亡的比例与EPO干预后的各组比较无显著差异。结论：EPO通过抑制细胞的凋亡从而对AA所刺激的肾小管上皮细胞结构的损伤产生保护作用，但对细胞的坏死改变无明显影响。     

High levels of homocysteine inhibit lysyl oxidase (LOX) and downregulate LOX expression in vascular endothelial cells

BACKGROUND: Hyperhomocysteinemia, an independent risk factor for cardiovascular disease and atherothrombosis, alters endothelial function through a mechanism not fully understood. Downregulation of lysyl oxidase (LOX), an enzyme involved in extracellular matrix maturation, impairs the endothelial barrier function and could be involved in homocysteine (HC)-induced endothelial dysfunction. OBJECTIVE: The aim of this study was to analyze the effect of HC on LOX regulation in vascular endothelial cells. RESULTS: HC at pathophysiological concentrations (35 microM) inhibited LOX activity in porcine aortic endothelial cells. Homocysteine thiolactone and related molecules containing sulfhydryl groups (cysteine), but not methionine or homocystine (non-containing thiol-group) inhibited LOX. In addition, the blockade of HC-sulfhydryl group by N-ethylmaleimide abrogated HC-induced LOX downregulation. This process was triggered by oxidative stress since superoxide dismutase and vitamin C reverted LOX inhibition caused by HC. On the contrary, the effect was not mediated through the induction of endoplasmic reticulum stress. Finally, higher doses of HC (200 microM), common in severe hyperhomocysteinemia, decreased LOX mRNA levels ( approximately 2-fold) and LOX promoter activity in transient transfection experiments. CONCLUSIONS: These findings suggest that LOX inhibition contributes to the endothelial dysfunction associated with hyperhomocysteinemia. This effect was dependent on a mechanism involving both an inhibition of LOX activity and a reduction of LOX expression.