高糖对体外培养肾小球及肾小球系膜细胞分泌TNF的影响

采用酶联免疫吸附实验观察两种不同浓度的葡萄糖对体外培养的大鼠肾小球及ＧＭＣ分泌ＴＮＦ的影响，结果表明：分别２４，１６小时后，与对照组相比肾小球与ＧＭＣ分泌ＧＮＦ明显增加，Ｐ〈０．０１。不同浓度的葡萄糖环境对肾小球与ＧＭＳ分泌ＴＮＦ的影响无明显差异，Ｐ〉０．０５。     

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     

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.     

肝素对大鼠肾小球系膜细胞增殖和核因子κB的抑制作用

<正>肾小球系膜细胞增殖在肾小球肾炎的发生发展中起重要的病理作用。有研究表明,核因子κB(nuclear factor-κB,NF-κB)与系膜细胞增殖密切相关[1,2]。近年来有不少体外、体内试验证实肝素能抑制系膜细胞增殖,减轻肾脏病变[3,4],但其是否能通过NF-κB起作用,目前还没有明确报道。本研究通过研究肝素对大鼠肾小球系膜细胞NF-κB的作用及其对系膜细胞增殖的影响,旨在探讨肝素抑制系膜细胞增殖的细胞内机制。     

丙泊酚对高糖诱导的人肾小球系膜细胞氧化应激和炎症反应的影响

目的探讨丙泊酚对高糖诱导的人肾小球系膜细胞损伤的影响。方法该研究于 2021年 3月至 2022年 3月进行。体外培养人肾小球系膜细胞,采用 30 mol/L葡萄糖诱导建立人肾小球系膜细胞细胞损伤模型,用丙泊酚浓度 10、20、40 μmol/L处理细胞,细胞分为对照组、高糖组、高糖 +低、中、高剂量丙泊酚组。酶联免疫吸附测定( ELISA)检测超氧化物歧化酶( SOD)、谷胱甘肽过氧化物酶( GSH-PX)、丙二醛、活性氧、白细胞介素( IL)-10、肿瘤坏死因子 α(TNF-α)IL-1β表达;蛋白质印迹法检测诱导型一氧化氮合酶( iNOS)、细胞间黏附分子 -1(ICAM-1)、单核细胞趋化蛋白 -1(MCP-1)、 IL-10、TNF-α、IL-1β蛋白表达。结果高糖组的丙二醛［( 16.7±1.7)mmol/L比( 3.8±0.4)mmol/L］、活性氧［( 9.6±0.9)μg/L比( 3.5±0.3)μg/L］、 IL-10［( 65.3±6.9)ng/L比(26.9±3.2)ng/L］、 TNF-α［( 105.6±10.9)ng/L比( 42.8±4.8)ng/L］和 IL-1β［( 79.7±8.2)ng/L比( 31.2±3.6)ng/L］明显高于对照组; iNOS、ICAM-1、MCP-1、IL-10、TNF-α、IL-1β蛋白表达明显高于对照组; SOD、GSH-Px明显低于对照组,均 P<0.05。高糖 +低剂量丙泊酚组、高糖 +中剂量丙泊酚组、高糖 +高剂量丙泊酚组的丙二醛、活性氧、 IL-10、TNF-α和 IL-1β明显低于高糖组; iNOS、 ICAM-1、MCP-1、IL-10、TNF-α、IL-1β蛋白表达明显低于高糖组,均 P<0.05;SOD、GSH-Px明显高于高糖组,均 P<0.05。结论丙泊酚能减轻高糖诱导的人肾小球系膜细胞氧化应激和炎症反应,发挥保护肾脏的作用。     

灵芝多糖对肾小球系膜细胞增殖的影响和体外抗氧化活性的研究

采用纤维素酶法提取灵芝多糖,获得两个组分cGLP1和cGLP2,以MTT法测定两组分对高糖诱导人-肾小球系膜细胞(HMC,human Mesangial Cell)增殖的影响,并通过对O2-、·OH两种自由基的清除作用以及总抗氧化能力来测定其抗氧化活性.进而考查酶法提取的灵芝多糖(Ganoderma Lucidump01ysaccharide)对高糖引起肾小球系膜细胞增殖的抑制作用及其体外抗氧化活性.结果显示 cGLP2对高糖所致的HMC增殖有显著的抑制作用.且cCLP2对羟自由基具有明显的清除作用,并具有较强的抑制过氧化脂质反应的能力.表明灵芝多糖酶提组分cGLP2能显著抑制高糖诱导的HMC增殖,这可能与cGLP2抗氧化活性相关.     

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。     

硫辛酸对高糖诱导的肾小球系膜细胞NLRP3炎症小体表达的影响

目的 观察高血糖对培养的人肾小球系膜细胞（HMC）中NLRP3炎症小体表达的影响,并探讨硫辛酸（LA）对上述过程的干预机制,以探寻糖尿病肾病（DN）发病新机制和防治新策略。方法 传代培养人HMC,根据不同的干预因素分为正常对照（NG）组、高糖（HG）组、LA组、HG+LA组。采用CCK-8方法分别检测不同浓度的LA对HMC活性的影响,流式细胞仪检测活性氧（ROS）水平,RT-PCR检测NLRP3炎症小体mRNA的表达,Western-blot检测LA对HMC中NLRP3炎症小体的蛋白表达的影响。结果 CCK-8结果显示,LA的最佳保护浓度为200μmol/l;流式细胞仪检测结果显示,HG组ROS表达水平高于NG组,差异有统计学意义（P<0.05）。RT-PCR及Westernblot检测结果显示,HG组NLRP3炎症小体表达高于NG组,差异有统计学意义（P<0.05）;加入LA后,NLRP3炎症小体的表达均下降,差异有统计学意义（P<0.05）。结论 高糖环境可促进人HMC的增殖,在一定浓度范围内,适当浓度的LA能抑制HMC的增殖;高血糖可通过氧化应激途径增加NLRP...     

维甲酸对高糖培养肾小球系膜细胞增殖及分泌转化生长因子-β的实验研究

<正>糖尿病肾病是糖尿病常见并发症,研究在高糖条件下培养肾小球系膜细胞生物学功能的改变及干预治疗,对于糖尿病肾病的防治具有重要意义。近年来维生素A代谢产物或类似物家族中被称为维甲酸(retinoic acid)的一类化合物在肾脏病的动物模型中显示出良好的肾脏保护作用[1]。维甲酸在     

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.     

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。     

灯盏花素对高糖环境肾系膜细胞c-fos、c-jun蛋白表达的影响

目的　观察高葡萄糖环境中 ,蛋白激酶C(PKC)抑制剂灯盏花素对肾小球系膜细胞 (GMC)c fos、c jun蛋白表达和Ⅳ型胶原 (C Ⅳ )合成的影响 ,探索糖尿病肾病防治的新途径。方法　原代培养大鼠GMC ,分别置于正常葡萄糖 (对照组 )、高葡萄糖 (高糖组 )和高葡萄糖加灯盏花素 (高糖加灯盏花素组 )环境中 ,观察干预 2 4h、4 8h和 1wk后GMCc fos、c jun蛋白表达、C Ⅳ合成和PKC活性的变化。结果　与对照组比较 ,高糖组干预 2 4h后c fos、c jun蛋白表达同时明显增高 ,4 8h后c fos开始下降 ,而c jun 1wk后仍保持高水平 ,高糖组C Ⅳ合成 1wk后增加 ,各观察时点PKC活性均较对照组明显增高 ;而高糖加灯盏花素组各时点c fos、c jun蛋白表达、C Ⅳ合成和PKC活性均低于高糖组。结论　高葡萄糖可促使GMC中c fos、c jun蛋白表达和C Ⅳ合成增加 ,此可能为PKC活化所介导 ,灯盏花素可通过抑制PKC活化而有效阻止高葡萄糖引起的上述变化。     

1,25（OH）2D3对人肾小球系膜细胞增殖及PCNA表达的影响

目的探讨1,25-二羟基维生素D3[1,25（OH）₂D₃]对人肾小球系膜细胞中增殖细胞核抗原（PCNA）表达及细胞增殖的影响。方法体外培养人肾小球系膜细胞,取传代培养至3⁸代细胞随机分为4组:正常对照组（加含5%胎牛血清DMEM培养基）,增殖对照组（EGF组,加10μg/L的EGF）,一般干预组[VD组,加10^-8mol/L 1,25（OH）₂D₃],增殖干预组[EGF+VD组,加10μg/L EGF及10^-8mol/L1,25（OH）₂D₃],均作用48 h。流式细胞术检测各组细胞周期;Western blot检测各组PCNA表达的情况。结果 （1）细胞周期。与正常对照组相比,EGF组G₁期细胞明显减少,S、G2/M期细胞增多,增殖指数（PI）较高;VD组G₁期细胞明显增多,S、G₂/M期细胞减少,PI较低;与EGF组相比,VD组和EGF+VD组G₁期细胞增多,S、G₂/M期细胞减少,PI较低,差异均有统计学意义。（2）系膜细胞中PCNA蛋白的表达。与正常对照组相比,EGF组PCNA的表达较高,VD组PCNA的表达较低;与EGF组相比,VD组和EGF+VD组PCNA的表达较低。结论 1,25（OH）₂D₃通过阻滞细胞周期、抑制PCNA的表达,从而抑制人肾小球系膜细胞的增殖。     

缬沙坦对高糖培养系膜细胞信号转导和转录活化因子1、3表达的影响

目的探讨高糖状态下肾小球系膜细胞中信号转导和转录活化因子1、3的改变以及血管紧张素受体1拮抗剂(AT1Ra)缬沙坦的影响。方法体外培养大鼠肾小球系膜细胞,分别给予高糖和缬沙坦干预,采用W estern印迹检测信号转导和转录活化因子1、3(STAT1、STAT3)及其磷酸化蛋白(p-STAT1、p-STAT3)的表达,酶联免疫吸附实验(ELISA)和放免法测定细胞上清液中TGF-β1、纤维连接蛋白(F ibronectin,FN)和IV型胶原的含量,逆转录-聚合酶链反应(RT-PCR)检测TGF-β1mRNA的表达。结果与低糖对照组相比,高糖组系膜细胞p-STAT1和p-STAT3表达明显上调,TGF-β1、FN和IV型胶原含量增加,TGF-β1mRNA的表达增加。缬沙坦组p-STAT1和p-STAT3的表达明显下调,TGF-β1、FN和IV型胶原的含量减少,同时TGF-β1mRNA的表达降低。结论高糖状态下p-STAT1和p-STAT3表达明显升高,缬沙坦抑制肾小球系膜细胞TGF-β1和细胞外基质的分泌可能部分是通过影响STAT1和STAT3的激活而实现。     

Puerarin reduces increased c-fos, c-jun, and type Ⅳ collagen expression caused by high glucose in glomerular mesangial cells

AIM: Increased expression of c-fos, c-jun and type IV collagen (CoIV) in glomerular mesangial cells (GMC) are important characteristics of diabetic nephropathy. Both c-fos and c-jun regulate the gene expression of extracellular matrix components, and CoIV is the main component of the extracellular matrix. It has been reported that puerarin inhibits aggregation of the extracellular matrix in diabetic rats by an as yet unknown mechanism. The aim of this study is to investigate the effect of puerarin on c-fos, c-jun and CoIV expression in GMC cultured in medium containing 5.6 or 27.8 mmol/L glucose. METHODS: The expressions of c-fos and c-jun were measured at the protein level using flow cytometry. CoIV content was detected using radioimmunoassay. Protein kinase C (PKC) activity was measured using liquid scintillation counting. RESULTS: Puerarin (10(-5) mmol/L) significantly ameliorated the high-glucose effect on c-fos, c-jun and CoIV expression. This effect is accompanied by a reduced PKC activity in these cells. CONCLUSION: Our results suggest that reduced PKC activity and expression of c-fos and c-jun in GMC might participate in the mechanisms underlying the therapeutic effect of puerarin on diabetic nephropathy.     

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.     

黄连素对高糖培养的大鼠肾小球系膜细胞FN及p38MAPK信号通路的影响

目的研究黄连素对高糖培养下大鼠肾小球系膜细胞细胞外基质成分纤维连接蛋白及p38MAPK信号通路的影响,进一步探讨黄连素抗糖尿病肾病的作用机制。方法实验分组:正常对照组、甘露醇组、高糖组、高糖+SB203580组、高糖+黄连素低剂量组、高糖+黄连素高剂量组共6组,观察黄连素对高糖培养下的大鼠肾小球系膜细胞纤维连接蛋白以及p38MAPK信号通路蛋白表达的影响。结果与高糖组相比,黄连素降低系膜细胞纤维连接蛋白的蛋白表达水平,抑制p38MAPK及其下游核转录因子CREB的磷酸化。结论黄连素抗糖尿病肾病的作用可能与其减少细胞外基质成分FN的积聚,抑制p38MAPK信号通路激活密切相关。     

螺内酯对高糖及醛固酮诱导的肾小球系膜细胞CTGF表达的影响

目的通过研究螺内酯(SPI)对高糖(HG)和醛固酮(ALD)刺激的体外培养的大鼠肾小球系膜细胞(MsC)结缔组织生长因子(CTGF)mRNA和蛋白表达的影响,探讨SPI的肾脏保护机制。方法将大鼠MsC分为:A组,正常对照组(5.6 mmol.L-1)、B组,高糖组(30 mmol.L-1)、C组,HG+SPI干预组(10-9,10-8,10-7mol.L-1)、D组,ALD组(5.6 mmol.L-1葡萄糖+10-7mol.L-1ALD)、E组,ALD+SPI干预组(10-9,10-8,10-7mol.L-1)。酶联免疫法检测上清液中CTGF蛋白的含量,RT-RCR法检测MsC CTGFmRNA表达。结果①正常糖浓度的培养基中可测到MsCCTGF mRNA和蛋白表达;②与A组比较,B组和D组MsCCTGF mRNA表达和上清液CTGF水平明显升高,差异有显著性;③与B组比较,C组中经10-8,10-7mol.L-1SPI干预MsC CTGF mRNA表达和上清液CTGF水平明显降低,P<0.05,但10-9mol.L-1SPI干预MsC CTGF mRNA表达和上清液CTGF水平无变化,P>0.05;④与D组比较,E组MsCCTGFmRNA表达和上清液CTGF水平明显降低,P<0.05。结论 SPI可抑制HG和ALD刺激的大鼠MsC CTGFmRNA表达和蛋白合成,该作用可能是其抗纤维化和防治肾损害的机制之一。