Effect of taurine on GFAP and TauT expressions in rat retinal Müller cells in high glucose culture

Objective： To detect the expression of glial fibrillary acid protein （GFAP） and taurine transporter （TauT） in the retinal Müller cells in high glucose culture with taurine and to explore the influence of glucose on the taurine transporting, and the possible protective effects of taurine on MUller cells in early diabetic retinopathy. Methods： The Müller cells from the rat retina were cultured in high glucose, and GFAP and Taut expressions were detected in the cells treated with different doses of taurine by immuocytochemical fluorescein staining and Western blotting. Results： High glucose enhanced the expression of GFAP and decreased the expression of TauT in Müller cells. Taurine decreased the up-regulation of GFAP in the cells which was induced by high glucose; 0. 1-10 mmol/L taurine increased the expression of TauT in Müller cells. Conclusion： Taurine can inhibit the changes in Müller cell resulted from high glucose.     

Effect of Dexamethasone and Aquaporin-1 Antisense Oligonucleotides on the Aquaporin-1 Expression in Cultured Human Trabecular Meshwork Cells

The changes in the expression of aquaporin-1 （AQP1） mRNA and protein in cultured human trabecular meshwork （HTM） cells treated with dexamethasone and transfected with antisense oligonucleotides （AS-ODN） were studied, and the implication of AQP1 regulation in corticosteroid-glaucoma and the possibility of AS-ODN inhibiting the AQP1 expression were evaluated. The cultured HTM cells in vitro were treated with different concentrations of dexamethasone and transfected with oligonucleotides for 5 days respectively. Then, total RNA and protein of HTM cells were extracted. The changes of AQP1 mRNA and protein were demonstrated qualitatively and quantitatively by RT-PCR and Western blot. Band intensities were detected by imaging analysis. There was a parallel relationship between the results of RT-PCR and those of Western blot. The expression levels of AQP1 mRNA and protein in dexamethasone-treated groups were increased initially and decreased later as dexamethasone concentration was stepped up. In the 0.04 μg/mL and 0.4 μg/mL groups, the levels of AQP1 were higher than in control group （0 μg/mL）. In the 4 μg/ mL and 40 μg/mL groups, the AQP1 expression levels were lower than in control group. AS-ODN could down-regulate the expression of AQP1 mRNA and protein in a dose-dependent manner. At 5 μg/mL, down-regulation efficiency reached the maximum. There was no statistically significant difference in the expression of AQP1 mRNA and protein between all sense oligonucleotides groups and control group. It was suggested that dexamethasone may induce the changes of the AQP1 expression in HTM cells to be involved in the occurrence of corticosteroid-glaucoma. AS-ODN can down-regulate the AQP1 expression in HTM cells to some extent.     

Regulation of Hypoxic Response Elements on the Expression of Vascular Endothelial Growth Factor Gene Transfected to Rat Skeletal Myoblasts under Hypoxic Environment

The regulation of hypoxic response elements on the expression of vascular endothelial growth factor （VEGF） gene transfected to primary cultured rat skeletal myoblasts under hypoxic environment was investigated. pEGFP-C3-9HRE-CMV-VEGF vector was constructed with molecular biology technique and transfected to primary cultured rat skeletal myoblasts by lipofectamine in vitro. Gene expression of transfected myoblasts was detected by RT-PCR, Western blot and fluorescence microscope under different oxygen concentrations and different hypoxia time. The results showed that in hypoxia group, the VEGF gene bands were seen and with the decrease of oxygen concentrations and prolongation of hypoxia time, the expression of VEGF mRNA was obviously increased. Under hypoxic environment, the expression of VEGF protein in the transfected myoblasts was significantly increased. EGFP was expressed only under hypoxic environment but not under normoxic environment. It was concluded that hypoxia promoter could be constructed with HRE and regulate the expression of VEGF gene under hypoxic and normoxic environment, which could enhance the re- liability of gene therapy.     

Effect of epigenetic modification of maspin on extravillous trophoblastic function

This study investigated the effect of epigenetic modification of maspin on extravillous tro-phoblastic function. The mRNA expression of maspin in placentae from normotensive and preeclamptic pregnant women was detected by RT-PCR. TEV-1 cells, a human first-trimester extravillous trophoblast cell line, were cultured and treated with CoCl2 (300 μmol/L) to induce chemical hypoxia and with 5-aza (500 nmol/L) to induce demethylation. The mRNA expression of maspin in TEV-1 cells subjected to different treatments was determined by RT-PCR, and the proliferative and migratory abilities of TEV-1 cells were assessed by cell counting kit-8 (CCK-8) and Transwell assays. Our results showed that the maspin mRNA expression level in placentae from preeclamptic women was much higher than that from normotensive women. CoCl2 or 5-aza could up-regulate the mRNA expression of maspin and significantly suppress the proliferation and migration of TEV-1 cells. It was concluded that the epigenetic modification in promoter region of maspin contributes to incomplete trophoblast invasion, which offers a novel approach for predicting and treating placental dysfunction.     

Inhibitory Effect of HCPT on Expression of HIF-1α and Downstream Genes in Hypoxic Human Cervical SiHa Cancer Cells

The hypoxic model to simulate hypoxic microenvironment in solid tumors was established and the effect of hydrocamptothecin （HCPT） on the hypoxia-induced over-expression of HIF-1α and VEGF genes was explored. Human cervical cancer SiHa cells were cultured in vitro under hypoxic conditions （37℃, 5% CO2, 1%O2） and treated with different concentrations of HCPT for 24 h. The mRNA and protein expression levels of HIF-1α, VEGF and Glutl in SiHa cells were detected by semi-quantitative RT-PCR and Western blot respectively. Normoxic control groups were exposed to normoxic conditions for 24 h. Under normoxic conditions, HCPT had no obvious effects on the HIF-1α and VEGF gene expression. Hypoxia induced the up-regulation of HIF-1α protein and downstream VEGF gene, and HCPT showed a dose-dependently inhibitory effect on the hypoxia-induced over-expression of HIF-1α protein and VEGF gene expression in SiHa cells, whereas HCPT had no significant effect on the HIF-1α mRNA expression. No difference in HCPT cytotoxic- ity was observed between hypoxic groups and normoxic control groups. It was suggested that HCPT could inhibite the expression of HIF-1α protein and downstream VEGF gene in hypoxic SiHa cells in a dose-dependent manner, and the inhibitory effect was not related with HCPT cytotoxicity.     

Effect of taurine on GFAP and TauT expressions in rat retinal Müller cells in high glucose culture

Objective： To detect the expression of glial fibrillary acid protein （GFAP） and taurine transporter （TauT） in the retinal Müller cells in high glucose culture with taurine and to explore the influence of glucose on the taurine transporting, and the possible protective effects of taurine on MUller cells in early diabetic retinopathy. Methods： The Müller cells from the rat retina were cultured in high glucose, and GFAP and Taut expressions were detected in the cells treated with different doses of taurine by immuocytochemical fluorescein staining and Western blotting. Results： High glucose enhanced the expression of GFAP and decreased the expression of TauT in Müller cells. Taurine decreased the up-regulation of GFAP in the cells which was induced by high glucose; 0. 1-10 mmol/L taurine increased the expression of TauT in Müller cells. Conclusion： Taurine can inhibit the changes in Müller cell resulted from high glucose.     

Local tissue hypoxia and formation of nasal polyps

Objective To explore the response of nasal mucosa epithelial cells to hypoxia in terms of formation of nasal polyps (NP). Methods Epithelial cells of NP and inferior turbinate (IT) were cultured serum-free under normal oxygen and hypoxic circumstances with stimulation of IL-1β and TNFα. The vascular endothelial growth factor (VEGF)mRNA and VEGF protein levels of the cultured cells were detected using in situ hybridization and ELISA, respectively. Results The expression of VEGF mRNA was significantly higher in epithelial cells of NP than in IT exposed to pro-inflammatory cytokines or hypoxia ( P <0.01). VEGF levels were higher in NP epithelial cells than those of IT ( P <0.01) under hypoxia. Conclusion VEGF-induced by hypoxia is very important for the early stages of forming polyps.     

缺氧对Müller细胞水通道蛋白4及血管内皮生长因子的影响

目的:观察缺氧条件下,体外培养的人视网膜Müller细胞上水通道蛋白4(AQP4)和血管内皮生长因子(VEGF)的表达情况。方法:酶消化法培养人视网膜Müller细胞,免疫荧光和透射电镜进行细胞鉴定,CoCl2诱导细胞缺氧,RT-PCR测定视网膜Müller细胞AQP4和VEGF基因的表达。结果:免疫荧光显示95%以上细胞谷氨酰胺合成酶、神经胶质纤维酸性蛋白、α-平滑肌肌动蛋白、中间丝波形蛋白呈阳性。Müller细胞AQP4 mRNA和VEGFmRNA的表达在缺氧条件下均升高,VEGF更明显,且呈部分浓度和时间相关性。缺氧12 h时VEGF mRNA表达达高峰,24 h时AQP4 mRNA达高峰。结论:AQP4和VEGF均与缺氧时的细胞水肿有关,二者关系密切,VEGF的作用较早且强。     

高糖对体外培养兔视网膜Müller细胞AQP4表达的影响

目的：观察体外培养兔视网膜Müller细胞在高糖条件下水通道蛋白-4（AQP4）表达的变化。方法：采用体外培养的兔视网膜Müller 细胞，分为正常对照组及高糖组（葡萄糖浓度：30、40和50 mmol•L^-1），分别培养1、3和5 d；采用免疫细胞化学、流式细胞术及RT-PCR方法对Müller 细胞AQP4的表达情况进行检测。结果：高糖组Müller 细胞AQP4表达的绿色荧光明显弱于正常对照组（P<0.01）；流式细胞仪检测中波峰位置明显提前，AQP4表达强度较对照组明显减弱（P<0.01），且随着葡萄糖浓度的增高和作用时间的延长，AQP4表达强度逐渐减弱；血糖浓度50 mmol•L^-1培养3 d时，RT-PCR半定量分析显示，AQP4 mRNA的表达比对照组明显降低（P<0.01）。结论：高糖能降低视网膜Müller细胞AQP4的表达，且随着葡萄糖浓度的增高和作用时间的延长，AQP4的表达强度也逐渐减弱；高糖时AQP4的表达下降可能是机体对糖尿病视网膜病变的一种保护性反应。     

高糖和缺氧对体外培养视网膜M üller细胞VEGF表达的影响

目的 :观察体外培养兔视网膜 Müller细胞在高糖和缺氧条件下血管内皮生长因子( vascular endothelial growth factor,VEGF)表达的变化。方法 :采用免疫细胞化学、原位杂交和ELISA方法对高糖和缺氧条件下体外培养兔视网膜 Müller细胞 VEGF的表达进行定性定量测定。结果 :高糖能刺激 VEGF表达 ,且通过转录水平调节 ,这种调节呈时间和剂量依赖性。 Co Cl2 能造成 Müller细胞不完全缺氧 ,刺激 VEGF的分泌。结论 :Müller细胞在高糖、缺氧条件下 VEGF表达增强 ,VEGF表达水平的升高可能是高糖及缺氧条件下促进糖尿病视网膜病变的因素之一。     

高糖对体外培养兔视网膜Müller细胞AQP4表达的影响

目的:观察体外培养兔视网膜M櫣ller细胞在高糖条件下水通道蛋白-4(AQP4)表达的变化。方法:采用体外培养的兔视网膜Müller细胞,分为正常对照组及高糖组(葡萄糖浓度:30、40和50mmol.L-1),分别培养1、3和5d;采用免疫细胞化学、流式细胞术及RT-PCR方法对Müller细胞AQP4的表达情况进行检测。结果:高糖组M櫣ller细胞AQP4表达的绿色荧光明显弱于正常对照组(P<0.01);流式细胞仪检测中波峰位置明显提前,AQP4表达强度较对照组明显减弱(P<0.01),且随着葡萄糖浓度的增高和作用时间的延长,AQP4表达强度逐渐减弱;血糖浓度50mmol.L-1培养3d时,RT-PCR半定量分析显示,AQP4 mRNA的表达比对照组明显降低(P<0.01)。结论:高糖能降低视网膜M櫣ller细胞AQP4的表达,且随着葡萄糖浓度的增高和作用时间的延长,AQP4的表达强度也逐渐减弱;高糖时AQP4的表达下降可能是机体对糖尿病视网膜病变的一种保护性反应。     

化学乏氧人肝癌细胞SMMC-7721中HIF-1α和miR-210的表达

目的探讨不同浓度CoCl2对人肝癌细胞SMMC-7721乏氧诱导因子-1α（HIF-1α）和miR-210表达的影响以及RNA干扰HIF-1α基因对miR-210表达的影响。方法采用Western blot法检测不同浓度CoCl2培养24 h后SMMC-7721细胞HIF-1α蛋白表达变化,实时定量PCR检测miR-210相对表达量。脂质体介导靶向HIF-1α基因的RNA干扰质粒pGenesil-HIF转染SMMC-7721细胞后,乏氧培养24、48和72 h,分别采用Western blot法和实时定量PCR检测细胞HIF-1α蛋白表达变化和miR-210相对表达量。结果 50和100μmol/L CoCl2培养24 h后细胞HIF-1α蛋白表达和miR-210相对表达量与0μmol/L组相比,差异均无统计学意义（均P〉0.05）;150和200μmol/L CoCl2培养24 h后细胞HIF-1α蛋白表达和miR-210相对表达量均显著高于0μmol/L组（P〈0.05或P〈0.01）。转染质粒pGenesil-HIF后乏氧培养24、48和72 h,细胞HIF-1α蛋白表达和miR-210相对表达量显著低于阴性干扰组（均P〈0.01）。结论化学乏氧人肝癌细胞SMMC-7721中HIF-1α和miR-210表达明显上调,RNA干扰HIF-1α基因可明显下调乏氧细胞miR-210表达。     

培哚普利对U937泡沫细胞血管内皮生长因子表达的影响

目的：研究培哚普利对氧化修饰的低密度脂蛋白（ox-LDL）诱导的U937泡沫细胞血管内皮生长因子（VEGF）mRNA、蛋白表达的影响。方法：U937细胞与80μg/mLox-LDL孵育48h,建立U937泡沫细胞模型,以不同浓度的培哚普利（0.01、0.10、1.00μmol/L）预处理U937细胞24h再加入80μg/mL的ox-LDL孵育48h,通过逆转录-聚合酶链式扩增反应（RT-PCR）和酶联免疫吸附试验（ELISA）分别检测U937细胞VEGF mRNA和蛋白的表达情况。结果：U937泡沫细胞组VEGF mRNA的表达较U937细胞对照组明显增加[（2.371±0.253）vs（0.954±0.245）（P〈0.01）],培哚普利干预后,随着浓度（0.01、0.10、1.00μmol/L）的增加,U937泡沫细胞的VEGF mRNA表达水平呈浓度依赖性下降[（2.168±0.270）vs（1.533±0.248）vs（1.022±0.189）（P〈0.01）];U937泡沫细胞组VEGF蛋白表达较U937细胞对照组明显增加[（1804.18±177.59）pg/mL vs（716.19±60.82）pg/mL（P〈0.01）],培哚普利干预后,随着浓度（0.01、0.10、1.00μmol/L）的增加,U937泡沫细胞VEGF蛋白表达呈浓度依赖性降低（1601.46±154.68）pg/mL vs（1377.09±110.36）pg/mL vs（1017.89±147.18）pg/mL（P〈0.01）。结论：培哚普利能够浓度依赖性地下调ox-LDL诱导的U937泡沫细胞VEGF mRNA、蛋白的表达。     

六味地黄汤含药血清对HK-2细胞PHD2/HIF-1α信号途径的影响

目的以缺氧诱导的HK-2细胞为研究对象,探讨脯氨酸羟化酶2/缺氧诱导因子1α(PHD2/HIF-1α)信号途径对肾间质纤维化的影响,以及六味地黄汤含药血清干预作用。方法 40只SPF级雄性SD大鼠分为空白组和给药组,每组20只,空白组以10 m L/(kg·d)生理盐水,给药组以67.5 g/(kg·d)六味地黄汤灌胃连续1周,腹主动脉取血,离心后分离血清。体外培养HK-2细胞,将实验分为空白对照组(10%胎牛血清)、正常血清组(10%正常大鼠血清)、氯化钴(Co Cl2)处理组(150μmol/LCo Cl2+10%正常大鼠血清)和含药血清组(150μmol/LCo Cl2+10%六味地黄汤含药血清)。体外培养HK-2,分组处理24 h后,用Western Blot法和RT-PCR法分别检测六味地黄汤含药血清对缺氧诱导的HK-2细胞中PHD2、HIF-1α、结缔组织生长因子(CTGF)的蛋白水平和mRNA表达的影响。结果与正常血清组比较,Co Cl2处理组HK-2细胞中PHD2的蛋白水平和mRNA表达均降低(P0.05),而HIF-1α、CTGF的蛋白水平和mRNA表达均升高(P0.05);与Co Cl2处理组比较,含药血清组HK-2细胞中PHD2的蛋白水平和mRNA表达均升高(P0.05),而HIF-1α、CTGF的蛋白水平和mRNA表达均降低(P0.05)。结论 PHD2/HIF-1α信号途径参与肾间质纤维化,六味地黄汤可能通过上调PHD2的表达,促进HIF-1α的降解,后者下调CTGF的表达,从而抑制肾间质纤维化。     

尼氟灭酸对氯化钴诱导血管内皮细胞凋亡的保护作用

目的：探讨尼氟灭酸（NFA）对氯化钴（CoCl2）诱导血管内皮细胞凋亡的保护作用,阐明其可能的机制。方法将血管内皮细胞随机分为对照组、100μmol／L CoCl2损伤组和20、40μmol／L NFA保护组,四甲基偶氮唑蓝比色（ MTT）法检测各组细胞增殖活性,酶联免疫吸附试验（ ELISA ）检测细胞内相关凋亡蛋白Bcl-2、Bax及Caspse-3的表达情况。结果 MTT检测,与对照组比较,100μmol／L CoCl2组在24 h细胞增殖活性明显降低（P＜0．01）;与CoCl2损伤组相比,20、40μmol／L NFA保护组在24 h细胞增殖活性升高（P＜0．05,P＜0．01）,40μmol／L NFA保护组更加明显。 ELISA法检测显示,与对照组比较,100μmol／L CoCl2组在24 h细胞Caspse-3、Bax的表达增多,Bcl-2表达减少（ P＜0．05,P＜0．01）。与损伤组相比,NFA处理缺氧后血管内皮细胞Caspse-3、Bax的表达减少,Bcl-2表达增多（P＜0．05,P＜0．01）。结论 CoCl2能诱导血管内皮细胞缺氧损伤,引起细胞的凋亡;NFA可以保护CoCl 2诱导的血管内皮细胞的缺氧损伤,这可能与上调Bcl-2、下调Bax和Caspse-3抑制其凋亡有关。     

rhEPO对高糖大鼠视网膜 Müller细胞凋亡及 NF-κB表达影响

目的 探讨重组人促红细胞生成素（rhEPO）对高浓度葡萄糖（高糖）作用下大鼠视网膜Müller细胞凋亡的保护作用及对核因子-κB （NF-κB）表达的影响。方法 将Müller细胞随机分为空白对照组、高糖培养组和不同浓度rhEPO组（分别在高糖培养液中加5、10、20、40 kU/L的rhEPO）。培养48 h后MTT法检测各组Müller细胞活力,并通过SP法检测rhEPO干预下NF-κB蛋白表达的变化。结果 高糖培养组Müller细胞活力较空白对照组显著下降（F=70.39,q=9.13,P〈0.01）;rhEPO干预后与高糖培养组相比细胞活力明显提高（q=4.30～7.08,P〈0.05）。高糖培养组NF-κB表达量较空白对照组明显增高（F=49.76,q=4.37,P〈0.05）,而各rhEPO组的NF-κB表达量与高糖培养组相比明显增高（q=5.74～27.08,P〈0.01）。结论 rhEPO对高糖作用下Müller细胞的凋亡具有保护作用,并能增强NF-κB的表达,其机制可能是通过上调NF-κB的表达来发挥作用。     

新蛋白质合成介导知母活性成分对M2受体稳定性的调节作用

目的 探讨知母活性成分ZMS提高CHOm2细胞毒蕈碱样乙酰胆碱2型受体 (M2受体) mRNA表达的机制.方法 体外培养至80% ～90%融合的CHOm2细胞分为ZMS 1组(单纯添加1×10-5 mol/L ZMS作用24 h)、ZMS 2组(添加1×10-5 mol/L ZMS作用24 h后加入1 μg/mL环己酰亚胺作用12 h)、ZMS 3组(加入1 μg/mL 环己酰亚胺预处理4 h后加入1×10-5 mol/L ZMS作用24 h),以上各组分别设立对照组(以等体积DMSO替代ZMS,其他处理与相应ZMS组相同).各组细胞培养基中均加入放线菌素D抑制mRNA合成,于不同时间点收集CHOm2细胞,Real-time PCR检测M2受体mRNA相对表达量并计算半衰期.结果 与相应对照组比较,ZMS 1组和ZMS 2组CHOm2细胞M2受体mRNA的半衰期明显延长,分别为(4.75±0.54)h vs(2.13±0.23)h和(5.43±1.13)h vs (2.46±0.09) h(均P<0.05);添加1 μg/mL 环己酰亚胺预处理的ZMS 3组CHOm2细胞M2受体mRNA半衰期的(3.06±0.23)h与其相应对照组的(3.00±0.20)h比较,差异无统计学意义(P>0.05).结论 ZMS提高M2受体mRNA的稳定性需要有新蛋白质合成的参与.