高糖对大鼠肾系膜细胞泛素及泛素化蛋白表达的影响

目的 探讨高糖对大鼠肾系膜细胞泛素及泛素化蛋白表达的影响。 方法 将大鼠HBZY-1肾系膜细胞进行体外培养,高糖作为刺激因子,分别设正常对照组(5.6 mmol/L葡萄糖)、高糖组（10、20、30 mmol/L葡萄糖）、甘露醇组。刺激12、24、48 h后,分别用实时定量PCR法和Western印迹法检测各组系膜细胞泛素mRNA和泛素化蛋白的表达。 结果 泛素化蛋白多以大分子蛋白为主。与正常对照组比较,30 mmol/L葡萄糖刺激24 h和48 h后泛素化蛋白的表达分别增加36％和52％（均P ＜ 0.01）;20 mmol/L和30 mmol/L葡萄糖作用48 h泛素化蛋白的表达分别增加31％和52％（均P ＜ 0.01）;高糖作用24、48 h,泛素mRNA的表达显著增加（均P ＜ 0.05）。 结论 高糖呈时间浓度依赖性地促进大鼠肾系膜细胞泛素mRNA及泛素化蛋白的表达,增加泛素蛋白酶体途径活性。     

葡萄糖转运蛋白4及其下游信号分子在高糖刺激下肾小球系膜细胞中的作用

目的 探讨高糖和胰岛素对肾小球系膜细胞（GMC）葡萄糖转运蛋白4（GLUT4）和Cbl相关蛋白（CAP）的mRNA表达及细胞骨架纤维状肌动蛋白F-actin 的影响,探讨糖尿病肾病发生发展中GLUT4 及其下游分子F-actin和CAP的重要作用。 方法 将细胞分为8组：正常对照组、生理浓度胰岛素（10－9 mol/L）组、低浓度胰岛素（10－8 mol/L）组、高浓度胰岛素（10－6 mol/L）组、高糖（30 mmol/L）组、甘露醇组（25 mmol/L甘露醇＋5 mmol/L葡萄糖）、高糖加高浓度胰岛素组、高糖加生理浓度胰岛素组。采用RT-PCR法和免疫组化法,观察不同情况下GMC中GLUT4蛋白和mRNA以及CAP mRNA 的表达及其变化。Rhodamine-phalloidin染色和激光共聚焦显微镜观察F-actin形态及荧光强度。 结果 正常对照组GMC中GLUT4蛋白和mRNA以及CAP mRNA有一定表达,而生理浓度胰岛素组与正常对照组差异均无统计学意义。高糖组GLUT4蛋白（P < 0.01)和mRNA（P < 0.05）以及CAP mRNA（P < 0.01)表达均显著减少,F-actin解聚增加（P < 0.01）;而甘露醇组以上各指标与对照组差异均无统计学意义。低浓度胰岛素组和高浓度胰岛素组GLUT4 mRNA表达分别为生理浓度胰岛素组的2.06倍和2.66倍,GLUT4蛋白表达分别为对照组的1.93倍和2.83倍,CAP mRNA表达分别为对照组的1.91倍和2.15倍,F-actin荧光强度分别为对照组的1.296倍及1.224倍,均呈一定的浓度依赖性。高糖加高浓度胰岛素组GLUT4 mRNA表达为高糖组的2.15倍（P < 0.05）,GLUT4蛋白表达为高糖组的2.08倍（P < 0.01）,CAP mRNA表达为高糖组的2.14倍（P < 0.01）,F-actin荧光强度为高糖组的1.838倍（P < 0.01）。GLUT4 mRNA与CAP mRNA呈正相关（r = 0.905,P = 0.002）;GLUT4与F-actin呈正相关（r = 0.929,P = 0.001）。 结论 （1）正常GMC中GLUT4 mRNA与蛋白、CAP mRNA有一定表达。（2）高糖可抑制GLUT4的蛋白和mRNA以及CAP mRNA表达,促进F-actin解聚。（3）胰岛素能部分拮抗高糖导致系膜细胞中GLUT4的蛋白和mRNA以及CAP mRNA表达的下调作用。（4）GLUT4、CAP和F-actin是糖尿病肾病发生发展的重要影响因子之一。     

Effect of high glucose on the Notch signal pathway in mesangial cells and intervention of the cordyceps sinensis

Objective To investigate the expression of Notch signaling molecules, transforming growth factor-β (TGF-β) and fibronectin (FN) in mesangial cell induced by high glucose, and the underlying mechanism of cordyceps sinensis. Methods Rat glomerular mesangial cells were divided into following groups: normal control group (5.5 mmol/L glucose), hypertonic control group (5.5 mmol/L glucose+19.5 mmol/L mannitol), high glucose group (25.0 mmol/L glucose), DAPT inhibitor group (25.0 mmol/L glucose+1 μmol/L DAPT), cordyceps sinensis intervention group (25.0 mmol/L glucose+10 mg/L cordyceps sinensis). Cell proliferation was detected by MTT. The protein and mRNA expression of Notch signaling molecules (Notch1, Jagged1 and Hes1), TGF-β and FN was detected by Western blotting and real time PCR. Results Compared with normal control group, high glucose induced mesangial cell proliferation, as well as the mRNA and protein expression of Notch1, Jagged1, Hes1, TGF-β1 and FN was up-regulated in high glucose group (all P＜0.05). Compared with that in high-glucose group, DAPT and cordyceps sinensis inhibited high glucose-induced mesangial cell proliferation and down-regulated the mRNA and protein expression of Notch pathway, TGF-β1 and FN (all P＜0.05). Conclusion By inhibiting the abnormal activation of Notch signaling pathway and TGF-β signaling pathway, cordyceps sinensis may alleviate high glucose-induced mesangial cell proliferation and reduce extracellular matrix accumulation, thus protecting kidney.     

蛋白激酶C激活在高糖诱导肾系膜细胞中的作用

目的：探讨高糖对系膜细胞蛋白激酶C（PKC）活性的影响及PKC在系膜细胞增殖、细胞外基质积聚中的作用。方法：采用大鼠系膜细胞进行体外培养,高糖作为激动剂,佛波酯（PMA）作为PKC抑制剂,甘露醇作为渗透压对照,用液闪仪测定PKC活性,^3H-TdR渗入法检测细胞增殖,ELISA法测定培养上清中纤维连接蛋白（FN）含量。结果：高糖可增加系膜细胞颗粒部分PKC活性、抑制细胞增殖、促进FN分泌,且与渗透压无关。抑制PKC后,可阻止高糖诱导的FN分泌。结论：高糖可激活系膜细胞PKC,促进细胞外基质积聚和糖尿病肾症的发生。     

Effects of silibinin on expression of integrin linked kinase,transforming growth factor β1 and α-smooth muscle actin in rat peritoneal mesothelial cells induced by high glucose

Objective To observe the effect of silibinin on the expression of integrin linked kinase (ILK), transforming growth factor β1 (TGF-β1) and α-smooth muscle actin (α-SMA) in rat peritoneal mesothelial cells (RPMCs) induced by high glucose. Methods RPMCs were isolated, cultured and passaged by trypsin, then identified. The second generation of cultured RPMCs were used in the experiment. RPMCs were divided into normal control group, high glucose(1.5%, 2.5%, 4.25%) for 24 hours, high glucose (2.5%) for 12, 24, 48, 72 hours, high glucose (2.5%) for 24 hours after silibinin (5, 10, 20 mg/L) preincubate for 2 hours. ILK and α-SMA mRNA were detected by real-time PCR. ILK protein was detected by Western blotting. TGF-β1 protein in supernatants was detected by ELISA. Results Compared with the control group, the expresssion of ILK, TGF-β1 and α-SAM was significantly increased in groups stimulated by high glucose (all P＜0.05). Silibinin could significantly decrease the expression of ILK, TGF-β1 and α-SMA induced by high glucose (all P＜0.05). Conclusions High glucose can up-regulate the expression of ILK, TGF-β1 and α-SMA. Silibinin can reverse these changes.     

磷脂酶D对高糖培养的肾小球系膜细胞骨架的影响

目的 研究高糖环境下肾小球系膜细胞（GMC）内磷脂酶D（PLD）的活性改变及其对细胞骨架的影响。方法 对高糖（30mmol/L)刺激48h的大鼠GMC，用酶联比色法测定磷脂酰胆碱专一性磷脂酶D（PC-PLD）活性，底物磷酸化法检测蛋白激酶C（PKC）的活性。用免疫荧光标记和共聚焦显微镜显示并测量F-actin的表达。结果 高糖刺激48h后，GMC内PC-PLD和PKC活性明显增高，而F-actin荧光表达减少，排列紊乱，给予PC-PLD抑制剂后，高糖培养的GMCPKC活性明显下降。F-actin的荧光表达和排列都得到显著改善。结论 高糖时PLD活性增高可以通过PKC途径影响GMC骨架的组装状态。改变系膜细胞收缩功能。     

缬沙坦对高糖上调系膜细胞细胞外调节蛋白激酶表达的影响

目的观察在高糖刺激下，系膜细胞细胞外调节蛋白激酶（ERKI／2）的活性变化以及缬沙坦对其影响，探讨缬沙坦保护肾脏作用的可能机制。方法原代培养大鼠肾脏系膜细胞，随机分为4组：低糖组（NG，d-葡萄糖5．5mmol／L）、高糖组（HG，d-葡萄糖30mmol／L）、甘露醇组（MG，d-葡萄糖5．5mmol／L＋甘露醇24．5mmol／L）和缬沙坦组（HG＋Val，d-葡萄糖30mmol／L＋缬沙坦10μmol／L）。用免疫细胞化学法及Western印迹法对系膜细胞中磷酸化ERK1／2（p-ERK1／2）的表达进行定位及半定量分析；RT—PCR法检测细胞中TGF-β1 mRNA的表达；放射免疫法测定各组细胞上清中Ⅳ型胶原的含量。结果高糖组系膜细胞中P-ERK1／2蛋白的表达较低糖组明显增高，并由胞质向胞核内转移，呈时间依赖方式（P〈0．01）；TGF-β1 mRNA及细胞上清液中Ⅳ型胶原水平均高于低糖组（P〈0．01）。而缬沙坦组上述指标均较同时相点高糖组显著降低，差异有统计学意义（P〈0．01）。甘露醇组与低糖组各指标间差异均无统计学意义。结论高糖可显著激活系膜细胞ERK信号通路，缬沙坦可抑制高糖的激活作用。     

高糖培养下人肾小球系膜细胞蛋白表达谱变化的研究

目的 利用比较蛋白质组学双向电泳技术研究体系,观察高糖培养下人肾小球系膜细胞（HMC）蛋白表达谱的变化。 方法 人肾小球系膜细胞分为高糖培养组（30 mmol/L）和正常糖组（5 mmol/L）,培养48 h后收集细胞,提取总蛋白,以DIGE饱和荧光标记,双向荧光差异凝胶电泳。应用Typhoon多功能成像系统扫描凝胶,DeCyder 2-D差异分析软件进行图像分析,寻找差异表达蛋白质。采用基质辅助激光解析电离飞行时间质谱（MALDI-TOF-MS）和蛋白质数据库检索鉴定差异蛋白。 结果 通过DeCyder 2-D差异分析软件,发现正常糖组和高糖培养组之间差异大于1.5倍的蛋白质斑点147个,对其中96个差异蛋白质斑点进行了肽质指纹图分析,鉴定出37种蛋白质。其中磷脂酰乙醇胺结合蛋白 1（PEBP-1）、粒溶素、ATP合成酶H+转运线粒体F0复合体亚基F2仅在高糖组表达。高糖刺激后表达上调的蛋白质有24个,包括嗜酸细胞阳离子蛋白、RGS膜相互作用蛋白16（MIR16）、肽酰-脯氨酰-顺反式异构酶、disks large homolog DLG2、早发性乳腺癌2（BRCA2）、儿茶酚-邻-甲基转移酶等;表达下调的蛋白有5个,包括O-GlcNAc transferase-interacting protein 106 000 isoform 1、proteasome beta 6 subunit precursor、NEFA-interacting nuclear protein NIP30等。 结论 高糖培养下HMC内147种蛋白质的表达发生变化。这些蛋白质广泛参与高糖对HMC的细胞骨架、糖代谢、细胞分裂、基因转录、信号转导、磷酸化、细胞增殖、凋亡等的调节。深入分析这些差异表达蛋白的功能与调控,有望为阐明糖尿病肾病的发病机制提供重要的实验依据。     

高糖刺激肾小球系膜细胞葡萄糖转运蛋白4及p21的表达及其意义

目的探讨早期糖尿病肾病(DN)肾小球系膜细胞(GMC)中葡萄糖转运蛋白(GLUT)4、p21mRNA表达变化及其与GMC肥大的关系。方法大鼠1097系膜细胞株分为高糖组、甘露醇组、不同浓度胰岛素组、高糖加不同浓度胰岛素组、正常对照组。用RT-PCR法检测各组GLUT4mRNA、p21mRNA的表达。流式细胞仪测各组GMC体积大小。结果正常对照组GMC有一定GLUT4mRNA、p21mRNA表达。高糖组GLUT4mRNA表达明显下降,p21mRNA表达明显增加。胰岛素刺激GMCGLUT4mRNA表达存在浓度依赖关系。p21mRNA表达越高,细胞前向角度散射光(FSC)越强,GMC体积越大。结论高糖刺激导致GMC肥大,GMC的p21mRNA表达上调和GLUT4mRNA表达下调与DN早期GMC肥大-肾小球肥大有关。     

Abated microRNA-21 attenuates high glucose-induced autophagy inhibition in rat mesangial cells by PTEN/Akt/mTOR pathway

Objective To investigate the effects of abated microRNA-21 (miRNA-21) on phosphatase and tensin homologue on chromosome ten protein (PTEN) and PI3K/Akt/mTOR pathway, as well as their further influence on the autophagy in high glucose (HG, 25.0 mmol/L) induced rat glomerular mesangial cells. Methods MiRNA-21 inhibitor and negative control were transfected by liposome 2000 into rat glomerular mesangial cells (HBZY-1). The cells were divided into normal glucose (5.5 mmol/L) group, normal glucose+negative control group, normal glucose+miRNA-21 inhibitor group, HG group, HG+negative control group and HG+miRNA-21 inhibitor group. Cell proliferation and hypertrophy were assayed by MTT and the ratio of total protein to cell number respectively. The miRNA-21 expression was detected using real time PCR. The expressions of PTEN/Akt/mTOR signaling signatures, autophagy-associated protein (p62 and LC3 Ⅱ) and collagen Ⅰ was detected by Western blotting and real time PCR. Autophagosomes were observed using electron microscopy. Results Compared with those in normal glucose group, in HG group cells had hypertrophy and proliferation, up-regulated miRNA-21 expression, and down-regulated PTEN protein and mRNA expressions (all P＜0.01). Also there were and up-regulated p-Akt, p-mTOR, p62 and collagen Ⅰ expression, and lower LC3 Ⅱ expression and autophagosomes (all P＜0.01). Further, compared with those in HG group, cells hypertrophy and proliferation in HG+miRNA-21 inhibitor group were reduced, expressions of p-Akt, p-mTOR, p62 and collagen Ⅰ were down-regulated, while expressions of PTEN and LC3 Ⅱ and autophagosomes were up-regulated (all P＜0.01). Conclusions MiRNA-21 inhibitor up-regulates PTEN expression, which inhibits the activation of Akt/mTOR signaling pathway, ameliorates cell hypertrophy, proliferation and enhances autophagy to reduce extracellular matrix accumulation.     

衣霉素对高糖刺激肾系膜细胞增殖的影响及其机制研究

目的 观察N-糖链抑制剂衣霉素(TM)对肾小球系膜细胞(GMC)β1整合素以及其下游重要信号分子粘着斑激酶(FAK)和细胞周期正调控蛋白cyclinD1表达的影响。了解衣霉素在抑制高糖刺激GMC增殖中的重要作用。方法 体外培养的大鼠系膜细胞株分为：正常对照组，高糖组，甘露醇组，高糖加不同浓度衣霉素(TM)组。用流式细胞技术检测B1整合素的表达；免疫组化方法测定FAK和细胞周期素D1(cyclin D1)的表达，四唑盐比色(MTT)法测定细胞增殖。结果 正常系膜细胞中B1整合素、FAK和cyclin D1均有一定表达，高糖作用24h可以使3者表达明显升高，细胞增殖能力增强，且与渗透压无关。衣霉素(TM)可呈浓度依赖性的抑制高糖的这种作用。结论 N-糖链抑制剂TM通过阻断细胞表面糖蛋白的糖基化过程，形成脱糖蛋白，从而抑制β1整合素的表达，并进一步影响细胞FAK和cyclin D1表达，使高糖诱导的细胞增殖受抑制。     

高糖对大鼠肾小球内皮细胞肾素-血管紧张素系统的影响及机制

目的 探讨高糖对大鼠肾小球内皮细胞肾素-血管紧张素系统（RAS）的影响及机制。 方法 5 mmol/L、30 mmol/L葡萄糖（加或不加卡托普利、糜蛋白酶抑制剂）分别作用于细胞12、24、48、72 h后,用ELISA法检测上清与细胞内的血管紧张素Ⅱ（AngⅡ）水平;实时荧光定量PCR法检测血管紧张素原、肾素mRNA的表达;Western印迹法检测细胞AngⅡ1型受体（AT1R）、AngⅡ2 型受体（AT2R）、血管紧张素原及肾素的表达;激光共聚焦间接免疫荧光法检测AT1R、AT2R、肾素在细胞内的分布及改变。 结果 与对照组相比,高糖刺激细胞12、72 h时,上清AngⅡ水平均升高（P < 0.05）;而在细胞内AngⅡ水平只在72 h时有升高 （P < 0.05）;在高糖刺激24、48 h时,细胞内外AngⅡ水平均未发生明显变化。加入卡托普利、糜蛋白酶抑制剂预处理,然后高糖刺激细胞72 h,上清和胞内AngⅡ水平较不加抑制剂时显著下降（P < 0.05）。高糖可使血管紧张素原mRNA表达上调、肾素 mRNA表达下调（均P < 0.05）。同时,高糖可使血管紧张素原蛋白表达上调、AT1R蛋白表达下调,而AT2R、肾素蛋白表达量无明显变化,但激光共聚焦间接免疫荧光观察到AT2R发生由细胞核到细胞质的转移。 结论 高糖可激活大鼠肾小球内皮细胞内的RAS,这种作用至少与血管紧张素转换酶（ACE）和非ACE途径有关。高糖可通过增加底物水平引起肾小球内皮细胞AngⅡ的改变,同时AngⅡ受体在高糖刺激下也发生相应的改变。     

The effects and underlying mechanism of CD36 in high glucose - induced rat glomerular mesangial cells apoptosis

Objective To investigate the effects and underlying mechanism of the scavenger receptor CD36 in high glucose - induced rat glomerular mesangial cells apoptosis. Methods The mesangial cells of rats were divided into 4 groups: control group (5.6 mmol/L glucose), mannitol group (24.2 mmol/L mannitol+5.6 mmol/L glucose), high glucose group (30 mmol/L glucose), CD36 mono- antibody group (30 mmol/L glucose+CD36 mono-antibody). The intracellular ROS level was detected by confocal microscopy with fluorescent probe CM - H2DCFDA. MDA, GSH - PX, 8 - OHDGA in cell supernatant were detected. Apoptosis was determined by flow cytometry followed by Annexin V-FITC/PI double stains. The expression of CD36, Bax and Bcl-2 were detected by RT-PCR and Western blotting. Results The expression of CD36 was detected in glomerular mesangial cells. The highest level was found in high glucose group in 24 hours. There was no significant difference found between control group and mannitol group with respect to intracellular ROS generation, MDA, 8-OHDG, GSH-PX level, apoptosis rate, expression of CD36, Bax and Bcl-2 (all P＞0.05). There was no significant difference in the expression of CD36 between CD36 mono - antibody group and high glucose group (P＞0.05）. Compared to control group, the intracellular ROS generation, MDA and 8-OHDG levels, apoptosis rate, the expression of CD36 and Bax were significantly increased, the GSH-PX level and the expression of Bcl-2 were significantly lower in high glucose group (all P＜0.05). Compared to the high glucose group, the intracellular ROS generation, MDA and 8-OHDG levels, apoptosis rate, the expression of Bax were suppressed but the GSH-PX level and the expression of Bcl-2 increased in CD36 mono-antibody group (all P＜0.05). The intracellular ROS level was positively correlated with apoptosis rate, protein expression of CD36 and Bax gene, was negatively correlated with Bcl - 2 protein expression. Conclusions CD36 was involved in the high glucose induced apoptosis of mesangial cells which was potentially mediated by an increased level of oxidative stress.     

整合素连接激酶对瘢痕成纤维细胞VEGF的调控作用

目的 探讨整合素连接激酶(intergrin-linked kinase,ILK)在人瘢痕成纤维细胞中的表达及对成纤维细胞VEGF的调控作用.方法 8例人增生性瘢痕标本采用组织块法分离培养瘢痕成纤维细胞,选取第5～6代细胞备用.实验分为3组①对照组:不做任何处理,只用含10%FCS的DMEM培养液同步培养;②空质粒组:用空质粒转染人瘢痕成纤维细胞;③ILK cDNA表达质粒转染组:用ILK cDNA表达质粒转染人瘢痕成纤维细胞.首先通过免疫细胞化学染色法检测ILKcDNA转染前后成纤维细胞ILK、VEGF的表达变化;应用实时荧光定量PCR(RT-PCR)及Western blot检测成纤维细胞ILK、VEGF的mRNA和蛋白水平变化;最后采用酶联免疫吸附试验(ELISA法)检测3组成纤维细胞上清液中的VEGF蛋白含量.结果 免疫细胞化学染色结果显示瘢痕成纤维细胞胞浆中ILK表达阳性,VEGF表达不明显,经ILK cDNA转染细胞后ILK与VEGF表达均增强;RT-PCR显示ILK cDNA转染组VEGF mRNA表达(0.338±0.060)均高于对照组(0.022±0.001)和空质粒组(0.028±0.005,P＜0.05);Western blot结果显示ILK cDNA转染组VEGF蛋白表达(0.819±0.019)显著高于对照组(0.607±0.033)和空质粒组(0.591±0.024,P＜0.05);ELISA法检测ILK cDNA转染组的VEGF蛋白含量高于其他两组(P＜0.05).结论 ILK可以上调VEGF mRNA及蛋白水平,并且促进成纤维细胞分泌VEGF,ILK可能通过提高瘢痕成纤维细胞合成分泌VEGF而促进增生性瘢痕血管生成.

Abstract：

Objective To explore the expression of intergrin-linked kinase (ILK) and its effect on VEGF expression in fibroblasts from human hypertrophic scar. Methods Fibroblasts were isolated from hypertrophic scar of 8 patients and cultured in vitro. Then the cells were divided into three groups: ① Cells were cultured only in DMEM containing 10% FCS in the control group; ② Cells were transfected with empty plasmid in the empty plasmid group; ③ Cells were transfected with plasmid experessing ILKcDNA in the ILK cDNA plasmid transfection group. First, the expression of ILK and VECF was observed by immunocytochemistry before and after ILK cDNA transfection. Second, ILK and VEGF mRNA expression was investigated by real-time PCR ( RT-PCR). Third, the protein expression of ILK and VEGF was detected by Western blot. Finally, the protein level of VEGF in supernatant of fibroblasts was measured by ELISA. Results Before ILK cDNA transfection, the expression of ILK was positive and the VEGF expression was weak in cytoplasm of fibroblasts . After ILK cDNA transfection, both the expression of ILK and VEGF was enhanced. The level of VEGF mRNA was significantly higher in ILK cDNA transfection group (0.338 ±0.060) than that in control group (0.022 ±0.001) and empty plasmid group (0.028 ± 0. 005 , P ±0. 05 ). The level of VEGF protein was significantly higher in ILK cDNA transfection group (0. 819 ±0. 019) than that in control group (0. 607 ±0. 033) and empty plasmid group (0. 591 ±0. 024, P ＜0. 05). Secretion of VEGF increased remarkably in ILK cDNA transfection group comparing with the other two groups (P＜0. 05). Conclusions ILK could up-regulate the VEGF mRNA and protein level in human scar fibroblasts. It may play an important role in the angiogenesis in hypertrophic scar.     

高糖通过蛋白激酶C改变肾小球系膜细胞的间隙连接与细胞表型

目的观察高糖环境下，蛋白激酶C（PKC）活性的变化对肾小球系膜细胞间隙连接与细胞表型的影响。方法将体外培养的大鼠肾小球系膜细胞分为低糖组、高糖组、高糖＋PKC抑制剂十字孢碱（SP）组，测定细胞间隙连接蛋白-43（connexin 43）、α-平滑肌肌动蛋白（α—SMA）的表达。结果①与低糖组相比，高糖组细胞PKC活性、mSMA mRNA表达增高，connexin 43 mRNA表达下降，差异有统计学意义（P〈0．05）；②与高糖组相比，高糖＋SP组细胞PKC活性、α—SMA mRNA表达下降，connexin 43 mRNA表达增高，差异有统计学意义（P〈0．05）。结论高糖通过PKC改变肾小球系膜细胞的间隙连接与细胞表型。     

内质网应激在高糖诱导人肾脏系膜细胞表型转化中的作用

目的 探讨内质网应激在高糖诱导人肾脏系膜细胞表型转化的作用。 方法 将体外培养的人肾脏系膜细胞分为对照组、高糖组（HG组）、高糖+4-苯基丁酸（4-PBA）干预组（4-PBA组）。MTT法测定细胞增殖率,流式细胞术观察细胞周期变化,免疫组织化学染色激光共聚焦显微镜观察细胞α平滑肌肌动蛋白（α-SMA)表达情况,实时荧光定量PCR和Western印迹分别检测mRNA和蛋白表达。 结果（1）高糖组系膜细胞增殖率和进入S期比例显著高于对照组（P < 0.05）,同时,高糖能刺激α-SMA表达增加（P < 0.05）;4-PBA干预可明显抑制高糖刺激的系膜细胞增殖和进入S期及α-SMA表达。（2）相对于对照组,高糖能刺激系膜细胞葡萄糖调节蛋白78（Grp78） mRNA和蛋白表达增加（P < 0.05）;4-PBA干预可显著下调这种效应（P < 0.05）。（3）高糖可诱导系膜细胞转化生长因子（TGF-β1）、纤连蛋白（FN） mRNA和蛋白表达（P < 0.05）;4-PBA干预则明显下调其表达（P < 0.05）。 结论 内质网应激在高糖诱导人肾脏系膜细胞表型转化效应中发挥了重要作用。     

雷帕霉素对肾小球系膜细胞增殖及凋亡的影响

目的 观察不同浓度雷帕霉素对大鼠肾小球系膜细胞增殖及凋亡的影响,探讨其可能的作用机制。 方法 使用不同浓度雷帕霉素（1 μg/L、2 μg/L、4 μg/L、8 μg/L、16 μg/L）处理大鼠肾小球系膜细胞,并设正常对照组。MTT法测不同浓度雷帕霉素干预24 h、48 h、72 h后对细胞增殖的影响,并描记生长曲线。干预72 h后,采用RT-PCR和Western印迹法分别检测各浓度雷帕霉素组细胞周期负调蛋白p27和p53的mRNA和蛋白表达变化;用流式细胞计量术检测各浓度雷帕霉素组细胞周期及凋亡率。 结果 小剂量雷帕霉素（1 μg/L）对系膜细胞增殖即具有明显的抑制作用,停滞于G1期的细胞数明显增加,但对系膜细胞凋亡无明显影响。较大剂量雷帕霉素(8~16 μg/L)能够明显增加肾小球系膜细胞凋亡。雷帕霉素能够增加肾小球系膜细胞p27、p53 mRNA和蛋白表达,且呈剂量依赖性。 结论 雷帕霉素可能通过增加p27、p53表达抑制肾小球系膜细胞增殖和促进系膜细胞凋亡。     

系膜细胞己糖激酶的表达及蛋白激酶C依赖的调控

目的 讨糖尿病相关因素－蛋白激酶Ｃ（ＰＫＣ）对己糖激酶的特异性调节以及与细胞对葡萄糖净利用的相关性，并探讨ＨＫｓ在糖尿病肾病中的作用。方法 ＳＶ４０转基因鼠肾小球系膜细胞株（ＳＶ４０ＭＥＳ１３）作为细胞模型，分别使用６－磷酸葡萄糖脱氢酶耦联比色法和醋酸纤维膜区带电泳、原位底物染色法检测ＨＫｓ总活性及同工酶活性，用Ｔｒａｎｄｅｒ氧化法检测细胞对葡萄糖的净利用。结果 ＳＶ４０ＭＥＳ１３细胞株和原代培养     

白细胞介素1β通过p38信号通路上调大鼠肾小球系膜细胞骨调素mRNA的表达

目的 探讨p38信号通路(1938MAPK)在白细胞介素1(IL-1)β介导的大鼠肾小球系膜细胞表达骨调素(OPN)中的作用。方法 应用Western印迹检测p38MAPK在IL-1β诱导的肾小球系膜细胞炎症反应中的活化程度。应用逆转录-聚合酶链式反应(RT-PCR)法观察p38MAPK特异性阻断剂SB203580对IL-1β诱导的系膜细胞促炎症介质OPNmRNA的影响。结果 IL-1β以时间和剂量依赖方式刺激系膜细胞引起p38MAPK的活化，并明显上调系膜细胞OPNmRNA的表达。p38MAPK特异性抑制剂SB203580以剂量依赖性方式显著抑制IL-1β诱导的OPNmRNA的表达。结论 p38MAPK在IL-16介导的肾小球系膜细胞上调表达黏附分子OPN中起重要作用。