单核细胞趋化蛋白-1在系膜增生性肾小球肾炎中的表达

目的用免疫组化的方法来分析单核细胞趋化蛋白-1在系膜增生性肾小球肾炎中表达的变化规律,揭示单核细胞趋化蛋白-1是否可做为病理条件下系膜细胞增生程度评判的一项指标。方法应用链霉素抗卵白素一过氧化酶连接法（S—P法）检测正常肾组织、42例轻至重度系膜增生性肾小球肾炎肾组织标本肾小球区单核细胞趋化蛋白-1表达的相对面积的变化。利用HPIAS—1000采集的图像对肾小球区的阳性表达区域所占相对面积进行测算。结果在正常肾组织切片中,仅部分肾小球内系膜细胞、肾小管上皮细胞及灶件炎细胞浸润区域单核细胞趋化蛋白-1阳性表达,绝大多数没有表达,染色阴性。不同程度增生肾小球区单核细胞趋化蛋白-1表达的相对面积不同,单核细胞趋化蛋白-1阳性表达的相对面积随系膜增生病变程度加重而增加,存在统计学差异。结论单核细胞趋化蛋白-1在肾小球区的表达随系膜增生病变程度加重而增加。在不同病变程度系膜增生性肾小球肾炎的肾小管、间质存在强、弱不等的表达。正常肾组织中,仅有少量单核细胞趋化蛋白-1表达。     

亚砷酸钠对HaCaT细胞中DNMT1、HDAC1基因mRNA转录及蛋白表达的影响

目的 了解不同剂量的亚砷酸钠(NaAsO2)对人皮肤角质形成细胞(HaCaT细胞)中DNA甲基转移酶1(DNMT1)、组蛋白去乙酰化酶1(HDAC1)基因mRNA转录和蛋白表达的影响.方法 分别以0、3.13、6.25、12.5和25μmol/L NaAsO2溶液重复间隔处理HaCaT细胞72 h(NaAsO2处理24...     

染料木黄酮对ox-LDL诱导人静脉平滑肌细胞MCP-1表达的影响

目的: 研究染料木黄酮(Gen)对氧化型低密度脂蛋白(ox-LDL)诱导的人脐静脉血管平滑肌细胞(hUVSMC)单核细胞趋化蛋白-1(MCP-1)mRNA和蛋白表达的影响。方法: 以hUVSMC为对象,采用逆转录聚合酶链反应和酶联免疫吸附试验观察不同浓度Gen(1.0×10-5、3.0×10-5、9.0×10-5 mol/L)对ox-LDL诱导的MCP-1 mRNA和蛋白表达的影响。结果:三种浓度的Gen均能明显降低ox-LDL诱导的hUVSMC MCP-1mRNA 和蛋白的表达,且高剂量与生理浓度的17b-雌二醇有相近的作用。结论:染料木黄酮能下调ox-LDL诱导的hUVSMC MCP-1mRNA和蛋白的表达,这可能是染料木黄酮抗动脉粥样硬化的重要机制之一。     

人细胞间黏附分子1cDNA的克隆及表达载体的构建

目的 构建人细胞间黏附分子-1真核表达重组体pcDNA3．1hisB-ICAM-1。方法 设计、合成ICAM-1基因序列特异性引物，从肝癌组织中提取总RNA，以此为模板经RT-CR扩增人ICAM-1的cDNA片段；然后将其克隆到pGEM-T Easy Vector，构建中介重组体(pGEM-ICAM-1)，再克隆，构建真核表达重组体pcDNA3．1HisB-ICAM-1，经菌落PCR和限制性酶切有目的片段出现，进行DNA序列分析。结果 RT-PER扩增得到人成熟ICAM-1的cDNA片段大小为1622bp，中介重组体(pGEM-ICAM-1)，酶切后与真核表达载体连接，根据酶切鉴定和DNA序列分析得到真核表达重组体pcDNA3.1HisB-ICAM-1；结论：成功构建了真核表达重组体pcDNA3．1HisB-ICAM-1。     

来氟米特对转化生长因子β_1诱导的体培养的大鼠肾小球系膜细胞中Smad2表达的影响

目的观察来氟米特(leflunomide,LEF)在转化生长因子(transforming growth factor,TGF)-β_1刺激大鼠肾小球系膜细胞(glomerular mesangial cell,GMC)表达Smad2过程中发挥的作用。方法建立体外培养大鼠肾小球系膜细胞模型,经鉴定造模成功后第7代用于实验。按照对模型大鼠体外培养的肾小球系膜细胞的处理方式不同,将其分为TGF-β_1组(TGF-β_1 5 ng/mL),LEF-1组(LEF 5μg/mL+TGF-β_1 5 ng/mL),LEF-2组(LEF 50μg/mL+TGF-β_1 5 ng/mL)和对照组(无血清RPMI 1640培养液+20%胎牛血清)。分别于15 min,30 min,1 h,2 h和6 h收集标本,用间接免疫荧光法检测各组磷酸化Smad2(phosphorylation-Smad2,p-Smad2)蛋白表达变化;采用荧光半定量RT-PCR法检测各组Smad2mRNA表达变化。结果对照组磷酸化Smad2蛋白少量表达,阳性细胞率为(21.40±1.51)%。TGF-β_1组阳性细胞率为(70.00±3.23)%,30 min时表达开始升高,1 h及2 h时表达明显增高,6 h表达开始下降。各时间点LEF-1组和LEF-2组磷酸化Smad2蛋白表达下降,阳性细胞率分别为(23.60±2.80)%和(25.81±1.29)%,细胞荧光强度呈减弱趋势。与TGF-β_1组相比,LEF-1组和LEF-2组磷酸化Smad2蛋白表达下降,差异有显著意义(P0.05)。但LEF-1组和LEF-2组与对照组比较,差异无显著意义(P0.05)。肾小球系膜细胞中Smad2mRNA相对表达量,TGF-β_1组显著高于对照组,2 h时达高峰。LEF-1组和LEF-2组Smad2mRNA显著低于TGF-β_1组,差异有显著意义(P0.01);但LEF-1组和LEF-2组间比较,差异无显著意义(P0.05)。LEF-1组和LEF-2组Smad2mRNA相对表达量1h时较低,之后逐渐升高,说明随时间延长,来氟米特对体外培养的肾小球系膜细胞干预作用逐渐减弱。结论经转化生长因子-β_1刺激后,体外培养的大鼠肾小球系膜细胞磷酸化Smad2蛋白和Smad2mRNA相对表达量分别增加,来氟米特可降低Smad2蛋白和Smad2mRNA表达水平,为来氟米特的肾脏保护作用提供理论依据。     

转化生长因子-β_1/Smad信号通路在肾小球系膜细胞的表达及血管紧张素转化酶抑制剂对其影响

目的探讨转化生长因子(transforming growth factor,TGF)-β1/Smad信号传导通路在肾小球系膜细胞(glomerular measanial cell,GMC)中的作用,观察血管紧张素转化酶抑制剂(angiotensin-converting enzyme inhibitor,ACEI)福辛普利(fosinopril,Fos)在该通路中对磷酸化Smad2/3(phosphorylationSmad2/3,P-Smad2/3)和Ⅰ型胶原蛋白(collagenⅠ,ColⅠ)表达的影响,阐明福辛普利的肾保护作用机制。方法采用经典方法进行大鼠肾小球系膜细胞复苏、培养和传代后,将其按处理方式分为3组:TGF-β1组(TGF-β15ng/mL处理);②Fos组(TGF-β15ng/mL+Fos1×10-4mol/L处理);③对照组(空白)。甲基噻唑基四唑(methyl thiazolyl tetrazolium,MTT)比色法分别检测3组肾小球系膜细胞增殖光密度(optical density,OD)值,计算细胞抑制率;间接免疫荧光法检测磷酸化Smad2/3表达情况;酶联免疫吸附测定(enzyme linkedi mmunosorbent assay,ELISA)法测定Ⅰ型胶原蛋白的变化,计算抑制率。结果各时间点肾小球系膜细胞增殖荧光强度比较,TGF-β1组明显高于对照组,Fos组低于TGF-β1组,差异有显著意义(P<0.05)。磷酸化Smad2/3在对照组仅极微量表达,TGF-β1组在刺激30min后,荧光强度显著增强,且集中于细胞核。与TGF-β1组相比,Fos组磷酸化Smad2/3表达下降,细胞荧光强度较弱,差异有显著意义(P<0.05)。对照组Ⅰ型胶原蛋白仅少量基础性分泌。与对照组相比,TGF-β1组在各时间点Ⅰ型胶原蛋白分泌量显著上升,差异有显著意义(P<0.05);与TGF-β1组比较,Fos组Ⅰ型胶原蛋白在15min和6h的表达量比较,差异无显著意义(P>0.05);在12h和24h比较,差异有显著意义(P<0.05)。结论人重组转化生长因子-β1能激活体外培养的大鼠肾小球系膜细胞磷酸化-Smads表达,并激活转化生长因子-β/Smads信号途径,该途径激活能致Ⅰ型胶原分泌增多,并能诱导肾小球系膜细胞增殖,其作用呈一定的时间依赖性关系。福辛普利能部分阻断转化生长因子-β/Smads信号传导通路在大鼠肾小球系膜细胞的激活,抑制Ⅰ型胶原分泌及肾小球系膜细胞增殖,其抑制作用呈一定的时间依赖性关系。     

姜黄素抑制人肝癌细胞BEL-7402中VEGF和HIF-1α表达通过PI3K／AKT／FRAP信号传导通路

目的探讨MAPK和PI3K信号传导通路在姜黄素调节VEGF和HIF-1α表达中的作用。方法分别加入LY29400225μmol／L、50μmol／L，U012610μmol／L、20μmol／L，rapamycin 5ng／ml、10ng／ml处理人肝癌细胞BEL-7402，30min后加入姜黄素10μmol／L，对照组单独加入0、10μmol／L姜黄素，缺氧环境中培养6h后，行RT—PCR和Western Blot检测VEGF蛋白、mRNA和HIF-1α蛋白表达；分别加入不同浓度的的姜黄素以及LY294002 25 μmol／L处理人肝癌细胞BEL-7402，缺氧环境中培养6h后，行Western Blot检测磷酸化AKT和总AKT蛋白表达。结果姜黄素10μmol／L＋LY29400225μmol／L组、姜黄素10μmol／L＋LY294002 50 μmol／L组、姜黄素10μmol／L＋rapamycin 5 ng／ml组、姜黄素10μmol／L＋rapamycin 10 ng／na组HIF-1α蛋白、VEGF蛋白、mRNA表达分别与姜黄素10μmol／L组相比降低，差异有统计学意义（P〈0．01）；而姜黄素10μmol／L＋U012610μmol／L组、姜黄素10μmol／L＋U0126 20 μmol／L组HIF—1α蛋白、VEGF蛋白、mRNA表达分别与姜黄素10μmol／L组相比差异无统计学意义（P〉0.05）；不同浓度的姜黄素、LY294002 25 μmol／L处理的人肝癌细胞BEL-7402缺氧6h后磷酸化AKT蛋白表达逐渐降低，LY294002 25 μmol／L可以基本阻断磷酸化AKT蛋白的表达，而对总AKT蛋白表达无明显变化。结论姜黄素对人肝癌细胞BEL-7402中HIF-1α蛋白和VEGF的表达通过P13K／AKT／FRAP信号传导通路。     

MiRNA-194 Regulates Palmitic Acid-Induced Toll-Like Receptor 4 Inflammatory Responses in THP-1 Cells

There is strong evidence to suggest that inflammatory responses link obesity and diseases, and the understanding of obesity-induced inflammatory mechanisms is central to the pathogenesis of diseases such asnonalcoholic fatty liver disease(NAFLD) and atherosclerosis that are modified by obesity. Based on this, anti-inflammatory treatments become a potential therapies for obesity-related diseases like NAFLD.A critical role of toll-like receptor (TLR) and its downstream molecules such as tumor necrosis factor receptor-associated factor 6(TRAF6) has been documented in inflammatory response induced by fatty acid. TLR pathway regulation provides a new insight to controlling the inflammatory response induced by fatty acid. Taken together, our study was aimed to understand the mechanism of fatty acid-mediated inflammation and look for an effective target which can prevent the inflammatory response induced by obesity. In this study, we used the saturated fatty acid palmitic acid (PA) to activate TLR4 signal pathway in human monocyte cells THP-1 that established an intracellular inflammatory model. Followed with activated TLR4, downstream molecular TRAF6 was upregulated and ultimately induced proinflammatory cytokine production. Based on this model, we also found that PA downregulated miR-194 expression with TLR4 activation. Moreover, our results showed that key signal molecular TRAF6 is a target of miR-194, overexpression of miR-194 directly decreased TRAF6 expression and attenuated the release of proinflammatory cytokine TNF-α in PA-activated monocyte THP-1. We conclude that miR-194 negatively regulates the TLR4 signal pathway which is activated by PA through directly negative TRAF6 expression.     

Vitamin C Prevents Hydrocortisone-Induced Injury in HMEC-1 through Promoting Bestrophin-3 Expression

Objective: To investigate the protective effects and underlying mechanisms of Vitamin C (VC) on hydrocortisone (HC)-induced cell injury in human microvascular endothelial cells (HMEC).

Methods: Cell viability was measured by CCK-8 assay and the expression of Best-3 was detected by Western blotting assay. The experiment was divided into normal control, HC injury group, VC treatment groups, HC?+?Best-3 siRNA group, HC?+?VC?+?Best-3 siRNA group, HC?+?pcDNA3.1 Best-3 group, and HC?+?VC?+?pcDNA3.1 Best-3 group.

Results: HC inhibited HMEC-1 cell viability was balanced with lower expression of Best-3 in a dose-dependent manner. Conversely, VC promoted HMEC-1 cell viability was paralleled to higher expression of Best-3 in a dose-dependent manner. Silencing Best-3 with Best-3 siRNA inhibited HMEC-1 cell viability, however, over-expression of Best-3 with pcDNA3.1 Best-3 promoted HMEC-1 cell viability. Moreover, VC and over-expression of Best-3 prevented HC-induced HMEC-1 cell apoptosis; however, silencing Best-3 further enhanced HC-induced HMEC-1 cell apoptosis. HC reduced Best-3 expression, which was alleviated by VC treatment. HC treatment decreased Bcl-2 expression, facilitated Bax expression. Both of VC and over-expression of Best-3 promoted Bcl-2 expression and decreased Bax expression. Additionally, VC and Best-3 expression have a synergistic effect.

Conclusions: VC can efficiently attenuate HC-induced HMEC-1 cell injury, which may be related to promote Best-3 expression.     

Leptin对THP-1细胞增殖和吞噬功能的影响及机制的初步研究

目的探讨瘦素对人单核细胞系THP-1增殖和吞噬活性的影响,为阐明瘦素在抗感染免疫中的作用提供依据。方法采用CCK-8法检测瘦素处理前后THP-1细胞的增殖活性,采用流式细胞术测定瘦素处理前后THP-1细胞对白色念珠菌的吞噬情况,RT-PCR检测甘露糖受体(MR)的表达情况。结果经瘦素处理后,THP-1细胞出现明显增殖(P0.01),且呈现明显的剂量-效应关系。瘦素处理后的THP-1细胞对白色念珠菌的吞噬也明显增强,但RT-PCR结果显示这种增强与MR表达之间无必然联系。结论瘦素可以明显刺激THP-1细胞增殖和吞噬活性的增强。     

雄激素对THP-1巨噬细胞源性泡沫细胞胆固醇含量的影响

目的探讨雄激素对THP-1巨噬细胞源性泡沫细胞胆固醇含量的影响。方法 0.4μmol/L睾酮处理THP-1巨噬细胞源性泡沫3、6、12和24h以及不同浓度的睾酮处理THP-1巨噬细胞源性泡沫24h,采用高效液相色谱检测细胞内总胆固醇和游离胆固醇的含量。结果 0.4μmol/L睾酮作用12和24h其细胞内总胆固醇、游离胆固醇和胆固醇酯明显增加,与各自对照组比较差异有统计学意义(P0.05);0.4μmol/L和4μmol/L睾酮处理24h组其细胞内总胆固醇、游离胆固醇和胆固醇酯明显增加,与对照组比较差异有统计学意义(P0.05)。结论超生理浓度的雄激素可能通过影响细胞胆固醇含量而参与和/或促进AS的发生发展。     

Piceatannol and Its Metabolite,Isorhapontigenin, Induce SIRT1 Expression in THP-1 Human Monocytic Cell Line

Piceatannol is a phytochemical that is present in large amounts in passion fruit (Passiflora edulis) seeds, and is an analog of resveratrol. Recently, the absorption and metabolism of piceatannol were investigated in rats, and isorhapontigenin, O-methyl piceatannol, was detected as a piceatannol metabolite in rat plasma. To elucidate the function of piceatannol and its metabolites, we investigated the expression of sirtuin 1 (SIRT1) in THP-1 monocytic cells after treatment with piceatannol and its metabolites, and compared their effects with those of resveratrol and its metabolites. Piceatannol and resveratrol upregulated the expression levels of SIRT1 mRNA and SIRT1 protein. An extract of passion fruit seeds, which contained high levels of piceatannol, also upregulated SIRT1 mRNA expression. As for the metabolites, isorhapontigenin upregulated SIRT1 mRNA expression, whereas resveratrol glucuronides and sulfate did not affect SIRT1 expression. These findings indicate that after intake of piceatannol, not only piceatannol itself, but also its metabolite, isorhapontigenin, contributed to the upregulation of SIRT1 expression.     

Ethanol Prevents Oxidant-Induced Mitochondrial Permeability Transition Pore Opening in Cardiac Cells

Aims: The purpose of this study was to determine if ethanolprevents the mitochondrial permeability transition pore (mPTP)opening via glycogen synthase kinase 3β (GSK-3β).Methods: Cardiac H9c2 cells were exposed to ethanol (10–1000µM) for 20 min. GSK-3β activity was determined bymeasuring its phosphorylation at Ser⁹. Mitochondrial membranepotential (_m) was assessed by imaging (confocal microscopy)H9c2 cells loaded with tetramethylrhodamine ethyl ester (TMRE).To activate GSK-3β, cells were transfected with constitutivelyactive GSK-3β (GSK-3β-S9A-HA) mutant plasmid. Results:Treatment of cardiac cells with low doses of ethanol (10–500µM) significantly enhanced GSK-3β phosphorylation,indicating that ethanol can inactivate GSK-3β in H9c2 cells.The effect of ethanol on GSK-3β activity was reversed bythe phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002and ethanol could enhance Akt phosphorylation, implying thatthe PI3K/Akt pathway accounts for the action of ethanol. Ethanolprevented oxidant (H₂O₂)-induced loss _m, an effect that wasreversed by LY294002, indicating that ethanol can modulate themPTP opening caused by oxidant stress through the PI3K/Akt pathway.Ethanol failed to preserve _m in cells transfected with the constitutivelyactive GSK-3β (GSK-3β-S9A-HA) mutant, suggesting thatethanol prevents the mPTP opening by inactivating GSK-3β.Conclusions: These data suggest that ethanol prevents the mPTPopening through inactivation of GSK-3β. The PI3K/Akt signalingpathway is responsible for inactivation of GSK-3β by ethanol.     

Sex-specific association of ACAT-1 rs1044925 SNP and serum lipid levels in the hypercholesterolemic subjects

Background  

Acyl-CoA:cholesterol acyltransferase (ACAT) is a key enzyme in cellular cholesterol homeostasis and in atherosclerosis. The cellular cholesterol efflux correlated with serum high-density lipoprotein cholesterol (HDL-C) concentrations has shown to be impaired in hyperlipidemic mice. The present study was carried out to clarify the association of ACAT-1 rs1044925 single nucleotide polymorphism (SNP) and serum lipid levels in the hyperlipidemic subjects.     

牛磺酸拮抗氯化镉致肝细胞氧化损伤的保护作用

目的观察牛磺酸拮抗氯化镉致大鼠肝细胞氧化损伤的保护作用。方法实验分5组:①阴性对照组;②氯化镉组:分别加入终浓度为10、20、40、100μmol/L的氯化镉;③同时处理组:终浓度为100 mmol/L的牛磺酸与氯化镉10、20、40、100μmol/L同时加入;④后处理组:先加入终浓度为10、20、40、100μmol/L的氯化镉,45min后加入100 mmol/L的牛磺酸;⑤预处理组:先加入终浓度为100 mmol/L的牛磺酸,45 min后加入10、20、40、100μmol/L的氯化镉。所有的处理组均培养2 h。结果①肝细胞中10、20、40、100μmol/L的氯化镉组GSH-Px酶活性明显低于阴性对照组;随着镉浓度增加,GSH-Px酶活性下降呈一定的剂量-效应关系。②预先处理组(65.26±14μmol/L)及同时处理组(49.65±1.54μmol/L)细胞GSH-Px酶活性明显升高,与相应的氯化镉组(27.11±1.09μmol/L)比较有显著性差异(P<0.05)。③当后处理组镉浓度达到40～100μmol/L时,细胞内SOD活性显著降低(23.08±1.49 U/ml),与相应的氯化镉组...     

Inositol Prevents Expression of a Genetic Model of Neural Tube Defects in Mice

Although up to 70% of neural tube defects (NTDs) can be prevented by adequate folate consumption, some NTDs are unrelated to folate, myo-Inositol was found to reduce the incidence of spinal NTDs that are unrelated to folate in curly tail mice.