生长代谢类激素对肾小球系膜细胞增殖及系膜基质合成的影响

肾小球系膜细胞增生和硬化是多种病理类型肾炎的共同病理特征,大量临床和实验研究证明体内多种内分泌、旁分泌和自分泌的细胞因子或生长因子参与和调节肾小球系膜细胞增生和硬化。我们探讨内分泌代谢因素在肾小球硬化中的作用,应用细胞培养的方法研究了生长代谢类激素对系膜增殖及分泌纤维连结蛋白(FN)的影响作用,旨在探讨系膜增生的细胞生物学机制。 1 材料与方法     

洛伐他汀对大鼠肾系膜细胞增殖和细胞外基质分泌的影响

目的 研究洛伐他汀对肾小球系膜细胞增殖和细胞外基质分泌的影响和机制。方法 用不同浓度的洛伐他汀及甲羟戊酸和拢牛儿基牻牛儿醇焦磷酸盐(GGPP)处理大鼠肾小球系膜细胞,测定溴化脱氧尿嘧啶核苷(Brdu)的整合来评估DNA的合成,ELISA法测定上清液中胶原Ⅳ、Ⅰ和纤维连结蛋白含量,用蛋白质印迹法分析原癌基因蛋白(Ras)活动及丝裂原活化蛋白激酶的活化。结果洛伐他汀抑制血小板源生长因子(PDGF)引起BrdU整合进入系膜细胞和细胞外基质胶原Ⅳ、Ⅰ和纤维连结蛋白的分泌,同时也抑制PDGF引起的Ras活动及丝裂原活化蛋白激酶的活化,甲羟戊酸和GGPP能部分拮抗洛伐他汀的上述抑制作用。结论 洛伐他汀可显著抑制系膜细胞增殖及细胞外基质的分泌,其机制可能通过抑制甲羟戊酸及其代谢产物的合成而阻止Ras活动及丝裂原活化蛋白激酶的活化,影响信号传导有关。     

益肾蠲湿合剂治疗糖尿病肾病疗效观察

目的:观察益肾蠲湿合剂治疗糖尿病肾病的疗效。方法:96例糖尿病肾病患者随机分为治疗组48例,对照组48例。2组在一般治疗处理的基础上,治疗组予益肾蠲湿合剂加减口服,对照组予血管紧张素转换酶抑制剂口服,共治疗3个月,观察2组治疗前、后的临床疗效。结果:治疗组的临床疗效明显优于对照组(P<0.01)。结论:应用益肾蠲湿合剂治疗糖尿病肾病疗效满意。     

冬虫夏草对肾小球系膜细胞增殖的抑制作用

目的:研究冬虫夏草对大鼠肾小球系膜细胞增殖的影响。方法:用四甲基偶氮唑蓝(MTT)法测定冬虫夏草对肾小球系膜细胞增殖的影响。结果:冬虫夏草抑制脂多糖刺激的大鼠系膜细胞增殖(P<0.05)。结论:冬虫夏草对脂多糖诱导的大鼠肾小球系膜细胞增殖有抑制作用。     

糖基化终产物对人肾小球系膜细胞细胞外基质金属蛋白酶促进因子基因表达的影响

目的 探讨糖基化终产物(AGEs)对人肾小球系膜细胞(HMRC)细胞外基质金属蛋白酶诱导因子(EMMPRIN)基因表达的影响.方法 在培养的人肾系膜细胞(HRMC)中分别加入不同浓度AGEs干预24 h和同一浓度AGEs干预HRMC不同的时间点,以无血清培养基(DMEM)和相应浓度的牛血清白蛋白(BSA)为对照.RT-PCR法检测EMMPRIN mRNA的表达水平.结果 与对照组相比,经各浓度及各时间点AGEs干预后, HMRC的EMMPRIN mRNA水平与对照组相比有显著差异(P＜0.01),且随AGEs浓度以及干预时间增加,EMMPRIN mRNA水平也明显逐渐降低(P＜0.05).结论 EMMPRIN可能是AGEs所致糖尿病肾病的因素之一.     

醛固酮对肾小球系膜细胞合成分泌纤维连接蛋白的影响

目的研究醛固酮(aldosteroen,ALD)及其受体拮抗剂螺内酯(spironolactone,SPI)对大鼠肾小球系膜细胞合成分泌纤维连接蛋白(fibronectin,FN)的影响。方法以不同浓度的ALD(10-11、10-9、10-7mol/L)和/或10-7mol/L SPI刺激系膜细胞48 h后,应用酶联免疫吸附试验(ELISA)测定培养上清中FN的浓度,应用半定量反转录-聚合酶链反应(RT-PCR)检测细胞FN mRNA的表达;以10-9mol/L浓度的ALD刺激系膜细胞不同时间(24、48、72 h)后,应用ELISA测定培养上清中FN的浓度,应用半定量RT-PCR方法检测细胞FN mRNA的表达。结果ELISA结果显示,ALD促进大鼠肾小球系膜细胞合成分泌FN,且具有浓度依赖和时间依赖性的特点,SPI能够拮抗ALD的作用。半定量RT-PCR方法结果显示,ALD促进培养的大鼠肾小球系膜细胞FN mR-NA的表达,且具有浓度依赖和时间依赖性的特点,SPI能够拮抗ALD的作用。结论ALD从蛋白和基因水平促进大鼠肾小球系膜细胞合成分泌FN,且具有浓度依赖和时间依赖性的特点,SPI能够拮抗ALD的作用,从而可能有益于延缓肾小球硬化的进展。     

肾小球系膜细胞增殖抑制的研究进展

肾小球系膜细胞异常增殖对肾小球疾病进展有重要作用,探讨其发生机制对肾小球疾病的防治具有重要意义.本文综述了近年来肾小球系膜细胞增殖机制及抑制因子的研究进展.     

细胞外基质磷酸糖蛋白对细胞增殖能力的影响

目的:探讨细胞外基质磷酸糖蛋白对细胞增殖能力的影响。方法:构建含细胞外基质磷酸糖蛋白基因的质粒,稳定转染293T细胞,通过激光共聚焦显微镜与RT-PCR检测转染细胞株hMEPE蛋白表达结果。通过MTT实验检测细胞增殖与迁移能力。通过RT-PCR检测p53基因的表达水平的差异,进一步探讨肿瘤细胞增殖的分子机制。结果:与转染空载体细胞相比,稳定转染hmepe细胞株增殖能力有明显提高,RT-PCR结果显示p53基因在转染目的基因细胞中较转染空载体细胞其表达增高。结论:细胞外基质磷酸糖蛋白可以提高细胞增殖能力。     

普伐他汀对高糖培养肾小球系膜细胞细胞外基质产生和氧化应激的影响

目的探讨普伐他汀对糖尿病肾病的保护机制。方法大鼠肾小球系膜细胞分别用正常浓度葡萄糖(5.5 mmo.lL-1),高浓度葡萄糖(25 mmo.lL-1)及葡萄糖25 mmo.lL-1+普伐他汀100μmo.lL-1培养。用CCK-8试剂盒测定细胞增殖,比色法检测培养液中超氧化物歧化酶(SOD)活性、谷胱甘肽(GSH)和丙二醛(MDA)含量。ELISA法检测培养上清液Ⅳ型胶原(ColⅣ-)、纤连蛋白(FN)、转化生长因子β1(TGFβ-1)和基质金属蛋白酶组织抑制因子(TIMP-1)含量;明胶酶谱法检测培养上清明胶酶A及B的活性。结果与对照组比较,高糖组肾小球系膜细胞增殖增加,基质蛋白ColⅣ-和FN合成增多,明胶酶A及B活性下降,TIMP-1含量和TGFβ-1分泌增加,总SOD活性和Cu,Zn-SOD活性下降,GSH含量下降,MDA含量增加。与高糖组比较,普伐他汀干预后可逆转上述变化。结论普伐他汀可抑制高糖培养的肾小球系膜细胞增殖,减少胞外基质合成,增加胞外基质降解,并缓解高糖诱导的氧化应激。     

醛固酮及其受体拮抗剂对肾小球系膜细胞增殖的影响

目的 研究醛固酮(ALD)及其受体拮抗剂螺内酯(SPI)对大鼠肾小球系膜细胞增殖和细胞周期的影响.方法 应用MTT法检测系膜细胞增殖,用流式细胞术检测细胞周期各时相的百分比.结果 MTT结果显示,ALD抑制肾小球系膜细胞增殖(与对照组比较P＜0.01),并具有一定的剂量依赖和时间依赖性,SPI能拮抗其作用.流式细胞术结果显示,ALD作用于系膜细胞24 h,G1期细胞数增多,S期细胞数减少(与对照组比较,P＜0.01).结论 ALD通过调控细胞周期抑制肾小球系膜细胞增殖.     

普伐他汀联合二甲双胍对大鼠肾小球系膜细胞增殖的影响

目的 探讨普伐他汀联合二甲双胍对肾小球系膜细胞（GMC）增殖的影响.方法 通过体外培养GMC,用高浓度葡萄糖刺激GMC增殖,选择最佳刺激浓度,并在普伐他汀联合二甲双胍不同剂量的条件下,比较GMC在不同剂量、不同时间的增殖情况.结果 葡萄糖最佳刺激浓度为0.6 g/L;普伐他汀联合二甲双胍高剂量组对葡萄糖刺激的GMC增殖抑制作用最明显,与低剂量组比较差异有统计学意义（P〈0.05）,但与中剂量组比较差异无统计学意义（P〉0.05）.结论 普伐他汀联合二甲双胍对GMC增殖具有抑制作用,可治疗糖尿病肾病.     

HMGB1/TLR4介导狼疮性肾炎肾小球细胞增殖和细胞外基质沉积

目的探究高迁移率族蛋白1(high mobility group protein B1,HMGB1)和其受体Toll样受体4(Toll like receptor 4,TLR4)介导狼疮性肾炎(lupus nephritis,LN)肾小球细胞增殖和细胞外基质沉积机制。方法采用免疫组化、免疫细胞化学技术检测LN患者肾穿标本和MRL/lpr小鼠肾小球细胞中TLR4表达水平;重组HMGB1刺激人肾小球系膜细胞(human mesangial cell,HMC),运用Western blot技术检测TLR4和髓样分化因子88(myeloid differentiation factor 88,Myd88)表达水平;LN患者置换血浆刺激HMC,采用CCK8试剂盒检测细胞增殖水平,Western blot技术检测纤连蛋白(fibronectin,FN)表达水平,ELISA技术检测HMC培养上清中FN水平。结果与对照组相比,LN患者及小鼠肾小球细胞中TLR4水平均上调;与对照组相比,重组HMGB1刺激HMC后,TLR4和Myd88水平上调;而抑制HMGB1及TLR4均可改善LN患者置换血浆介导的HMC增殖水平升高及FN合成和分泌增加(均P<0.05)。结论HMGB1可能通过激活其受体TLR4介导LN系膜细胞的增殖活性增强及细胞外基质沉积增加。     

Inhibitory effects and mechanism of 25-OH-PPD on glomerular mesangial cell proliferation induced by high glucose

ObjectiveTo investigate the protective effects and potential mechanism of the compound 25-OH-PPD (PPD) on the glomerular mesangial cells (GMC) under high glucose condition.MethodsThe hypertrophic GMC cells were established by DMEM containing glucose and randomly divided into five groups, including the normal control group (Control), the high glucose model group (HG, 25 mmol L⁻¹), the PPD low dose group (1 μmol L⁻¹, PPD-L), the PPD middle dose group (5 μmol L⁻¹, PPD −M) and the PPD high dose group (10 μmol L⁻¹, UCN-H). The GMC were incubated for 48 h under different treatment factors. Total protein content was determined by Lowry method. The diameter of the single GMC and volume were measured by computer photograph analysis system. The GMC cell viability was analyzed by MTT assay. The level of malondialdehyde (MDA), the content of glutathione (GSH) and superoxide dismutase (SOD) activity were measured by ELISA. [Ca²⁺]_і transient was measured by Till image system and by cell-loading Fura-2/AM. The expression of COX-1 and COX-2 were also determined using ELISA method.ResultsThe viability of GMC and the total protein content were decreased in HG group, different dosage PPD group could increase these indexes (P < 0.05). The level of MDA was increased, the content of GSH and SOD was decreased in HG group, while PPD could reduce the MDA and enhance GSH and SOD (P < 0.05). Following treatment with different dosage (PPD-L, PPD-M or PPD-H), the [Ca²⁺]_і transient was reduced (P < 0.05 or P < 0.01). Moreover, the expression of COX-1 was decreased while COX-2 expression was increased in different dosage PPD groups.ConclusionThe protective effects of PPD on GMC from HG-induced hypertrophy may be associated with the inhibition of [Ca²⁺]_і transient and decreasing expression of COX-1 via the oxidative-stress injure pathway.     

Protective effects of polyphenols against cadmium-induced glomerular mesangial cell myocontracture

The main objective of this work was to determine the ability of polyphenols (procyanidoloic oligomers; PCO) to diminish the contracture of CdCl₂-induced mesangial cells (smooth muscle cell type). Glomeruli were isolated by passing rat renal cortex pulp through calibrated sieves followed by a culture step for outgrowth of cells. PCO lethality was measured by microassay (Neutral Red uptake). This study has revealed an absence of PCO toxicity during exposure for 24 h and for concentrations ranging from 0.031 to 1‰ (w/v) on rat renal mesangial cells. We observed a lack of cytotoxicity response for the PCO mixture dissolved in medium. Quantitative assessments of the planar cell surface area (PCSA) were performed with an accurate automatized image analyser. The use of isolated cultured mesangial cells permits us to evaluate by quantitative morphometric analysis the contracture elicited either with CdCl₂ salts alone or by previous incubation with a non-lethal dose of PCO. When renal mesangial cells were exposed for 10 min to the PCO mixture, the Cd-mediated myocontracturant response of the mesangial cells was totally abolished. These results suggest that polyphenols could be effective against renal damages induced by cadmium. Received: 23 August 1999 / Accepted: 6 September 1999     

Modulation of cell proliferation and hypertrophy by gangliosides in cultured human glomerular mesangial cells

Glomerular mesangial cells (GMCs) in diverse renal diseases undergo cell proliferation and/or hypertrophy, and gangliosides have been reported to play an important role in modulating cell structure and function. This study compared the effects of transforming growth factor-beta1 (TGF-beta1) and the effects of the application of exogenous gangliosides on GMCs and investigated whether the application of exogenous gangliosides regulated cellular proliferation and hypertrophy. Human GMCs were cultured with exogenous gangliosides and TGF-beta1 in a media containing 10% fetal bovine serum and in a media without the fetal bovine serum. Exogenous gangliosides biphasically changed the proliferation of human GMCs (0.1-1.0 mg/mL). A low concentration (0.1 mg/mL) of gangliosides mainly increased the number of human GMCs, whereas cellular proliferation was significantly reduced by raising the concentration of exogenous gangliosides. TGF-beta1 greatly reduced the number of human GMCs in a concentration-dependent manner (1-10 ng/mL). Serum deprivation accelerated the gangliosides- and TGF-beta1-induced inhibition of mesangial cell proliferation to a greater extent. Gangliosides (1.0 mg/ mL) and TGF-beta1 (10 ng/mL) both caused a significant increase in the incorporation of [3H]leucine per cell in the serum-deprived condition, whereas it was completely reversed in serum-supplemented condition. Similar results to the [3H]leucine incorporation were also observed in the changes in cell size measured by flow cytometric analysis. These results show that exogenous gangliosides modulate cell proliferation and hypertrophy in cultured human GMCs, and these cellular responses were regulated differently based on whether the media contained serum or not. Results from the present study raise new possibilities about the potential involvement of gangliosides in the development of mesangial cell proliferation and hypertrophy.     

美洲大蠊浸膏对大鼠HSC和SEC增殖及细胞外基质分泌的影响

目的探究美洲大蠊浸膏(APE)对体外培养大鼠肝星状细胞株(HSC-T6)和肝窦内皮细胞(SEC)增殖及分泌细胞外基质的影响。方法用稀释不同倍数的美洲大蠊浸膏干预大鼠肝星状细胞株(HSCT6)和肝窦内皮细胞(SEC)。M TT法测定HSC-T6和SEC的增殖情况;ELISA测定药物干预后细胞培养上清液中α-SMA、Ⅳ型胶原和层粘连蛋白(LN)的含量。结果美洲大蠊浸膏叮抑制HSC-T6和SEC的增殖,并在一定范围内呈剂量效应关系。美洲大蠊浸膏组细胞上清中α-SMA、Ⅳ型胶原和层粘连蛋白(LN)的含量低于对照组。结论美洲大蠊浸膏可抑制HSC-T6和SEC增殖,并不同程度抑制α-SMA、Ⅳ-C和LN的分泌,有可能成为防治肝纤维化的临床用药。     

氯通道阻滞剂抑制人肾小球系膜细胞增殖

目的研究氯通道阻滞剂对肾小球系膜细胞增殖的作用。方法细胞计数和3H-TdR参入量测定确定细胞增殖,应用流式细胞术检测细胞周期时相。结果同对照组相比,氯通道阻滞剂5-硝基-2-(3-苯丙胺)苯甲酸5-nitro-(3-phenylpropylamino)-benzoicacid,NPPB、尼氟灭酸使人肾小球系膜细胞数和3H-TdR参入量明显减少(P<0·01,n=8),并呈剂量依赖关系,但不增加系膜细胞乳酸脱氢酶释放量(P>0·05,n=8)。NPPB和尼氟灭酸均使细胞周期停滞在G0/G1期(n=3)。结论氯通道阻滞剂NPPB、尼氟灭酸对人肾小球系膜细胞增殖具有抑制作用。     

甲基阿魏酸对肝星状细胞增殖和细胞外基质的影响

目的研究甲基阿魏酸对大鼠肝星状细胞增殖和细胞外基质的影响。方法采用四甲基噻唑蓝法检测MFA对HSC的毒性;采用酶联免疫法法检测HSC上清液羟脯氨酸、金属蛋白酶组织抑制因子和透明质酸。结果 MFA可以抑制HSC的增殖,呈现剂量和时间依赖性;MFA各剂量组均可显著降低HSC细胞上清液HYP、HA、TIMP-1含量,呈现剂量依赖关系。结论 MFA通过抑制HSC的增殖,从而使ECM合成减少,肝纤维化减轻。