MicroRNA-181b诱导血管内皮细胞衰老和功能异常的机制研究

目的研究micro RNA-181b（mi R-181b）和胰岛素样生长因子1受体（Insulin-like growth factor 1 receptor,IGF1R）参与血管内皮细胞衰老和血管新生的调节机制。方法体外培养人脐静脉内皮细胞（Human umbilical vein endothelial cells,HUVECs）,传代培养计算细胞群体倍增水平（Population doublings,PDL）,将PDL≤8 HUVECs定义为年轻细胞,PDL≥44 HUVECs定义为衰老细胞,并检测mi R-181b和IGF1R在年轻（PDL8）和年老（PDL44）HUVECs中的表达。PDL8 HUVECs过表达或抑制mi R-181b后,分别用MTS比色法、划痕（Wound healing）和成管（Tube formation）实验检测内皮细胞的增殖、迁移、成管等血管新生能力。采用双荧光素酶报告系统法检测mi R-181b对IGF1R转录后水平的调控。此外,给予缺氧刺激,观察缺氧应激条件下mi R-181b对IGF1R表达的调控作用。结果过表达mi R-181b可抑制PDL8内皮细胞的增殖能力22%（P〈0.001）、迁移能力23%（P〈0.001）,对成管能力没有显著影响（P〉0.05）。与PDL8HUVECs比较,mi R-181b在PDL44衰老内皮细胞中上调64%（P=0.046）,IGF1R的m RNA和蛋白水平分别下调39%和45%（P=0.004,P=0.014）。荧光素酶活性实验显示mi R-181b可与IGF1R的3’UTR结合,但过表达mi R-181b对IGF1R的表达水平无显著影响（P〉0.05）。在缺氧应激条件下,mi R-181b可上调IGF1R的表达（P=0.005）。结论 Mi R-181b抑制血管内皮细胞增殖、迁移等血管新生能力,这一作用与mi R-181b在缺氧应激条件下上调血管发育相关基因IGF1R的表达相关,其机制仍需进一步研究。     

阿司匹林抗高糖诱导的内皮细胞衰老的作用

目的 探讨阿司匹林是否具有抗高糖诱导的内皮细胞衰老的作用及其机制。方法 人脐静脉内皮细胞（HUVEC）分别于正常糖浓度培养液 （5.5 mmol/L）、高糖培养液和高糖（33 mmol/L）＋阿司匹林（0.01、0.1、1及3 mmol/L）培养液中培养48 h,观察细胞形态、采用β-半乳糖苷酶染色鉴定衰老细胞,PCR-ELISA检测端粒酶活性,流式细胞仪检测细胞周期和细胞内活性氧水平。结果 高糖培养液作用HUVEC 48 h后,细胞呈现衰老状态,β-半乳糖苷酶染色阳性细胞数明显增加,端粒酶活性明显减低,细胞周期停滞于G0/G1期,S期显著减少,细胞内活性氧水平显著增加。0.01、0.1和1 mmol/L阿司匹林作用后细胞形态改善,β-半乳糖苷酶染色阳性细胞数显著减少,端粒酶活性增强,S期细胞显著增加,细胞内活性氧水平降低（P<0.05）。而3 mmol/L阿司匹林作用后与高糖组相比差异无显著性。结论 高糖环境下阿司匹林（0.01、0.1和1 mmol/L）具有抗内皮细胞衰老的作用,其作用机制可能与氧化应激有关。     

血管内皮细胞衰老与心血管疾病的相关性

血管内皮细胞(VEC)是覆盖于血管内膜表面的单层扁平鳞状上皮细胞,其构成血管壁的生物屏障,不仅属于一种保护性屏障,还能够产生一些自体分泌物用于调节体内平衡和血管紧张度。VEC衰老可导致血管功能受损,是心血管系统(CVS)主要的危险因素,并与心血管疾病(CVD)有着密切的关系。然而,VEC衰老的机制以及VEC衰老对血管功能的影响尚不完全清楚。本综述总结了VEC衰老的特征及其相关分子机制,并对年龄相关CVD进行了阐述。     

氧化型低密度脂蛋白诱导血管内皮细胞基因表达谱的改变

通过对氧化型低密度脂蛋白诱导血管内皮细胞基因表达谱改变的研究，为阐明氧化型低密度脂蛋白致内皮细胞功能障碍及动脉粥样硬化形成的分子机制提供科学依据。采用含有4000条全长已知人类基因cDNA以及96条参照基因cDNA克隆制作的基因表达谱芯片，筛查氧化型低密度脂蛋白（100mg/L)作为24h对人脐静脉血管内皮细胞的基因表达谱改变的影响。结果显示，氧化型低密度脂蛋白可诱导1条基因表达下调（泛肽激活酶），3条基因表达上调（血清和糖皮质激素诱导的蛋白激酶、热休克蛋白70和KDRF）。结果发现，在氧化型低密度脂蛋白作用的早期阶段可诱导内皮细胞相关基因的表达改变；首次发现氧化型低密度脂蛋白可诱导内皮细胞KDRF、泛肽激活酶和血清和糖皮质激素诱导的蛋白激酶基因表达改变，为揭示氧化型低密度脂蛋白引起血管内皮细胞功能障碍的机制提供了研究成果。     

银杏叶提取物对内皮细胞的保护作用

目的 探讨银杏叶提取物(GBE)对血管紧张素Ⅱ(Ang Ⅱ)致体外培养人脐静脉内皮细胞(HUVEC)损伤的保护作用.方法 将处于对数生长期的HUVEC分为正常对照组、AngⅡ组及高、中、低3个GBE处理组,其中正常对照组用含0.5%小牛血清的DMEM培养基培养,Ang Ⅱ组在正常对照组培养基基础上含Ang Ⅱ 10-8mol/L,GBE处理组在AngⅡ组基础上分别加GBE 50、100、200 mg/L做为GBE低、中、高组,采用MTT法测定培养细胞增殖能力.并应用Western印迹法检测各组细胞中磷酸化Akt表达.结果 MTT法检测结果表明,Ang Ⅱ组细胞增殖能力明显高于正常对照组(P＜0.05),除GBE低剂量组外,GBE高、中剂量组A值均明显高于Ang Ⅱ组(P＜0.05);Western 印迹法检测各组培养细胞磷酸化Akt表达结果显示,与正常对照组比较,Ang Ⅱ组磷酸化Akt蛋白表达量明显降低(P＜0.05),GBE高剂量组磷酸化Akt表达明显高于Ang Ⅱ组(P＜0.05),且与正常对照组比较,差异无统计学意义,GBE中、低剂量组磷酸化Akt蛋白表达量明显降低(P＜0.05),但仍显著高于Ang Ⅱ组(P＜0.05).结论 Ang Ⅱ导致血管内皮细胞增殖能力下降可能与其抑制磷酸化Akt表达量有关,GBE可通过上调Akt表达抵抗Ang Ⅱ引起的细胞损伤.     

衰老大鼠急性肺损伤诱导肝功能受损的研究

目的　观察脂多糖 (LPS)致衰老大鼠的急性肺损伤 (ALI)是否可进一步诱发肝功能受损及银杏叶提取物 (GBE)对其是否有保护作用。方法　雄性Wistar大鼠 3 0只复制成衰老模型。再随机分成对照组 (静脉注射生理盐水 ) ;LPS组 (静脉注射LPS)及GBE +LPS组 (注LPS前 7天开始每天GBE灌胃 1次 )。注LPS后 2、6h收集血液并取肺、肝。制备肺、肝组织匀浆待测。结果　衰老大鼠在注射LPS后 2、6h时形成ALI。对照组注射LPS表明 ,2h血中总胆红素含量及谷丙转氨酶 (GPT)活性为(10 9± 0 6)mg/L、(2 6± 3 )U ,LPS 6h组为 (3 0 1± 2 1)mg/L、(88± 12 )U ,两组比较差异有显著性 (P均<0 0 0 1) ;对照组注射LPS表明 ,2h血和肺组织中每毫克蛋白中丙二醛 (MDA)含量分别为 (15 9±1 8) μmol/L、(18 8± 2 1)nmol,LPS 2h组为 (2 2 1± 1 9) μmol/L、(2 8 8± 3 1)nmol,两组比较差异有显著性 (P均 <0 0 0 1) ;而每毫克蛋白血和肺组织中超氧化物歧化酶 (SOD)活性对照组分别为 (2 5 5±2 6)mU/L、(3 6 1± 2 4)U ,LPS 2h组分别为 (2 0 6± 1 9)mU/L、(3 2 0± 2 7)U ,两组比较差异有显著性(P <0 0 1和 0 0 5 )。对照组注射LPS表明 ,2h时肺组织中每毫克蛋白中谷胱甘肽过氧化物酶 (GSH PX)及Na+ K+ ATP酶活性     

山茱萸果核水提取物对D-半乳糖致衰老模型小鼠抗氧化能力的影响

目的探讨山茱萸果核水提取物对D-半乳糖致衰老模型小鼠抗氧化能力的影响。方法选择50只昆明雄性小鼠随机分为正常对照组(10只)、模型组(10只)和提取物组(30只)。其中提取物组依据提取物剂量分为高剂量组(12 g·kg~(-1)·d~(-1))、中剂量组(6 g·kg~(-1)·d~(-1))、低剂量组(3 g·kg~(-1)·d~(-1)),每组10只。正常对照组腹腔注射生理盐水,其余各组通过皮下连续8 w注射500 mg·kg~(-1)·d~(-1)D-半乳糖制备衰老模型。采用Morris水迷宫检测各组小鼠的学习记忆能力,完成测试后采用黄嘌呤氧化酶法测超氧化物歧化酶(SOD)活性,硫代巴比妥酸法测定丙二醛(MDA),比色法测定总抗氧化能力(T-AOC),DTNB法测定谷胱甘肽过氧化物酶(GSH-Px)。统计分析所有小鼠5 d内Morris水迷宫实验逃避潜伏期、穿越平台次数和脑组织MDA、SOD、GSH-Px、T-AOC含量。结果提取物组小鼠平均逃避潜伏期明显低于模型组,前者穿越平台次数高于后者(P0.05);但各剂量组间平均逃避潜伏期,穿越平台次数无显著差异(P0.05)。提取物组小鼠MDA水平明显低于模型组,前者T-AOC、GSH-Px活性高于后两者(P0.05);提取物组小鼠SOD活性高于模型组,但无显著差异性(P0.05)。结论山茱萸果核水提取物可能通过清除D-半乳糖模型小鼠产生过多的自由基,进而提高脑组织的抗氧化能力,从而起到延缓衰老的作用及改善衰老小鼠的学习记忆能力,中等剂量的效果最好。     

银杏叶提取物对脂多糖诱导D-半乳糖致衰老大鼠急性肺损伤的保护作用

目的　观察脂多糖 (LPS)诱导D 半乳糖 (D gal)致衰老大鼠急性肺损伤 (ALI)及银杏叶提取物 (GBE)对其是否有保护作用。方法　大鼠 2 4只随机分成两部分 ,6只为正常对照组 ;18只经腹腔注D gal复制衰老动物模型。后者再随机分成三组 :衰老对照组 (6只 ) ;LPS组 (6只 ,静脉注射LPS诱导形成ALI) ;GBE +LPS组 (6只 ,注LPS前 7天开始每天灌胃给GBE一次 ,按所含黄酮甙计算 ,8mg/kg体重 ,实验当日在给LPS前 2h再给一次GBE)。注LPS后 2h收集标本待测。结果　D gal致衰老大鼠较正常大鼠血中超氧化物歧化酶 (SOD)及肺组织Na+ K+ ATPase活性均显著降低 (P均 <0 0 5 ) ,而血中乳酸脱氢酶 (LDH)活性升高 (P <0 0 5 )。衰老大鼠注LPS后 2h已形成ALI。肺间质及肺泡中有较多炎性细胞 ;肺泡灌洗液中蛋白含量及肺通透指数增加 ;血中乳酸 (LD) ,丙二醛 (MDA) ,一氧化氮 (NO) ,内皮素 1(ET 1) ,肿瘤坏死因子 α(TNF α)含量和LDH活性以及肺组织中髓过氧化物酶 (MPO)活性 ,均显著升高 ;而血中超氧化物歧化酶活性及肺组织Na+ K+ ATPase活性均下降 (P <0 0 5 ,P <0 0 1)。预先给予GBE可显著地缓解除SOD活性外的上述其它指标的变化 (P <0 0 5 )。结论　D gal致衰老大鼠体内抗氧化能力降低。静注LPS可引起衰老大鼠明显的A     

蒲黄提取物对氧化低密度脂蛋白损伤血管内皮细胞的保护作用

目的 研究蒲黄提取物对氧化低密度脂蛋白(ox-LDL)损伤人脐静脉内皮细胞的保护作用.方法　体外培养人脐静脉内皮细胞(HUVEC),采用MTT法测定不同浓度蒲黄提取物对HUVEC的影响.以蒲黄提取物(100,50,10 mg/L)预处理细胞24 h,再加入100 mg/L ox-LDL造成细胞损伤,取培养细胞上清液测定乳酸脱氢酶(LDH)活性,HUVEC用于测定细胞细胞间黏附因子(ICAM-1)、单核细胞趋化蛋白(MCP-1)mRNA的表达.结果 各剂量蒲黄提取物有显著的促进血管内皮细胞增殖的作用.与正常对照组比较,HUVEC经ox-LDL作用后,细胞活性显著下降,LDH的释放量和ICAM-1、MCP-1 mRNA的表达显著升高;100、50、10 mg/L 蒲黄提取物可显著降低LDH活性,下调ICAM-1、MCP-1 mRNA的表达.结论 蒲黄提取物具有促进脐静脉内皮细胞增殖的作用,并可抑制Ox-LDL诱导的HUVECs ICAM-1、MCP-1 mRNA表达的上调和LDH的释放,从而起到保护血管内皮细胞的作用.     

miR-146a调控人脐静脉内皮细胞的衰老及机制

目的探究miR-146a在人脐静脉内皮细胞衰老中的调节作用,并初步探究其机制。方法以人脐静脉内皮ECV-304细胞为研究对象,连续传代建立ECV-304细胞复制性衰老模型,衰老相关β-半乳糖苷酶(SA-β-gal)法检测不同代次(P2、P6、P9、P12、P15代)细胞中衰老细胞比例,实时定量PCR法检测不同代次(P2、P6、P9、P12、P15代)细胞中miR-146a表达。靶基因预测软件预测miR-146a靶基因。细胞转染建立miR-146a表达上调的P12代细胞(Pre-miR146a组)和miR-146a表达下调的P12代细胞(Anti-miR146a组),并建立相应对照组,SA-β-gal法检测衰老细胞比例,Western blot检测NADPH氧化酶4(NOX4)蛋白表达。结果 P6、P9、P12和P15代细胞的衰老细胞比例明显高于P2代细胞(P0.05),miR-146a表达则明显低于P2代细胞(P0.05)。靶基因预测miR-146a的靶基因为NOX4;转染后,Pre-miR146a组衰老细胞比例明显低于对照组(P0.05),Anti-miR146a组衰老细胞比例明显高于对照组(P0.05); Pre-miR146a组NOX4蛋白表达明显低于对照组(P0.05),Anti-miR146a组NOX4蛋白表达明显高于对照组(P0.05)。结论 miR-146a在衰老的人脐静脉内皮细胞ECV-304中表达下降,上调miR-146a表达能够抑制ECV-304衰老,其机制与调节NOX表达有关。     

Growth inhibition of hepatocellular carcinoma tumor endothelial cells by miR-204-3p and underlying mechanism

AIM:To investigate the mechanism by which miR-204-3p inhibits the growth of hepatocellular carcinoma(hCC)tumor endothelial cells(TECs).METHODS:Flow cytometry was used to identify hCCTECs and analyze their purity.Differentially expressed miRNAs in hCC TECs as compared to normal hepatic sinusoidal endothelial cells(hSECs)were examined using the hmiOA v4 human miRNA OneArray?microarray.miR-204-3p showed the most significant decrease in expression and was further studied.Over-expression of miR-204-3p was achieved using lentiviral transduction into TECs of hCC.The biological changes in hCC TECs before and after transduction were detected using MTT and apoptosis assays.The association between miR-204-3p and fibronectin 1(FN1)was determined using the dual luciferase activity assay.Changes in FN1protein expression before and after transduction were detected using Western blot analysis.RESULTS:Microarray results showed that compared to normal hSECs,15 miRNAs were differentially expressed in hCC TECs,including 6 miRNAs with increased expression and 9 miRNAs with decreased expression.Among them,miR-204-3p showed the most significant decrease in expression(log2=-1.233477,P=0.000307).Over-expression of miR-204-3p in hCC TECs via lentiviral transduction significantly inhibited the proliferation of hCC TECs and promoted apoptosis.Results from the dual luciferase activity experiment showed that the luciferase intensity in the wild type FN1 group was significantly inhibited(P<0.05),while that in the mutant FN1 group was not obviously affected.This observation indicated that FN1 was one of the potential targets of miR-204-3p.After over-expression of miR-204-3p in hCC TECs,Western blot analysis showed that the expression of FN1 protein was significantly inhibited.CONCLUSION:MiR-204-3p acts on its potential target gene,FN1,and inhibits its expression,thus blocking the adhesion function of FN1 in promoting the growth of TECs.     

巴曲酶对内皮祖细胞功能的影响及其机制的探讨

目的研究巴曲酶(DF-521)对体外培养下内皮祖细胞(EPCs)的增殖、分化、成血管能力以及NO分泌能力的影响,并探讨其可能的机制。方法从人外周血中提取单个核细胞,用流式细胞术检测细胞表型CD34、CD31、血管内皮生长因子2和血管性假血友病因子(vWF),激光共聚焦显微镜观察细胞经过FITC标记的荆豆凝集素I和DiI标记的乙酰化低密度脂蛋白双染色后的情况,由此鉴定EPCs。实验分为:DF-521的低、中、高剂量干预组和对照组。检测各组EPCs的CD31和vWF表达,观察EPCs在基质胶上的成管情况以及测定培养液中NO的浓度。透射电子显微镜鉴定分化后的细胞。结果低、中、高剂量干预组EPCs的数量、CD31、vWF表达及NO浓度均明显高于对照组(P<0.05),而且这些效应随DF-521浓度升高而增加(P<0.05)。高剂量干预组EPCs形成条梭状和管腔样结构。电子显微镜观察到怀布尔-帕拉德小体。结论体外培养务件下.DF-521促进EPCs的增殖和成血管功能,并诱导其向内皮细胞分化,改善分泌NO的能力。DF-521的这些作用可能与内皮型一氧化氮合酶有关。     

Effect of propionyl-L-carnitine on human endothelial cells

A possible protective effect of propionyl-L-carnitine on human endothelial cells was studied both under basal culture conditions and in the presence of agents capable of influencing oxidative damage, such as glucose/glucose oxidase and oxidized low-density lipoproteins. Propionyl-L-carnitine had no significant effect on the observed decline in various parameters of cell viability, e.g., cell detachment, release of lactate dehydrogenase, and the rate of protein synthesis. Propionyl-L-carnitine progressively decreased the fluorescence intensity in fura-2-loaded endothelial cells obtained during excitation at 340 nm. A similar effect was observed with propionyl-L-carnitine and acetyl-L-carnitine, but not with L-carnitine and D-carnitine. These results suggest that propionyl-L-carnitine and acetyl-L-carnitine decrease the cytoplasmic calcium level in endothelial cells.     

Possibility of the regression of atherosclerosis through the prevention of endothelial senescence by the regulation of nitric oxide and free radical scavengers

In the elderly, atherosclerotic diseases such as stroke and myocardial infarction occupy a major part of their causes of death and care. The elderly always have atherosclerosis in their aorta and other arteries and are exposed to risk of attacks. It is the elderly who should receive its safe, harmless and advanced treatment. Advanced stage of atherosclerosis in the elderly is progressed by complicated risk factors such as dyslipidemia and diabetes mellitus and specific risk factors for the elderly, aging (and menopause). Treatment of atherosclerotic disease may need special ones targeted for the elderly. Recent studies reported that frequencies of dyslipidemia were not decreased in the older oldest. In the elderly, impaired glucose tolerance occurrs and it progresses atherosclerosis. Endothelial dysfunction like impairment of nitric oxide (NO) bioavailability also progresses atherosclerosis. Although we tried to regress the high cholesterol diet‐induced atherosclerosis in rabbit aorta with a normal diet with or without statin, regression could not be achieved. NO targeting gene therapy (adenovirus endothelial nitric oxide synthase [eNOS] gene vector) regressed 20% of atherosclerotic lesions through reduction of lipid contents, however, a more integrated strategy is important for complete regression. We paid attention to NO bioavailability and developed two ways of increasing it in atherosclerosis: citrulline therapy and arginase II inhibition by estrogen. Further, we found a close relation between atherosclerosis and endothelial senescence and that NO can prevent it, especially in a diabetic model. Taken together, regression of atherosclerosis can be achieved by not only regulation of various risk factors but regulation of the cross‐talk of NO and free radicals.     

富硒麦芽粉水提取物通过细胞衰老和凋亡抑制人白血病K562细胞的增殖

目的 研究富硒麦芽粉水提取物能否抑制K562细胞增殖的作用.方法 Coulter细胞计数法检测细胞增殖的抑制作用,流式细胞仪检测细胞周期分布以及细胞内活性氧自由基的水平,DNA特异染料Hoechst 33342染色检测染色质凝集,免疫印迹法检测凋亡相关蛋白的表达,衰老相关的β-半乳糖苷酶染色检测细胞衰老.结果 富硒麦芽粉水提取物能抑制K562细胞的增殖,IC50值为0.5 mg/mL.进一步研究发现：富硒麦芽粉水提取物使细胞阻滞在G2/M期、增加细胞内活性氧自由基水平,染色质发生凝集,引起细胞凋亡标志蛋白的切割.此外,4 mg/mL的水提取物能引起60％的细胞发生衰老.结论 富硒麦芽粉水提取物具有明显的抑制K562细胞增殖的作用,其机制与诱导细胞衰老和凋亡有关.     

In vitro senescence of immune cells

Immune cells are eminently suitable model systems in which to address the possible role of replicative senescence during in vivo aging. Since there are more than 10⁸ unique antigen specificities present within the total T lymphocyte population of each individual, the immune response to any single antigen requires massive clonal expansion of the small proportion of T cells whose receptors recognize that antigen. The Hayflick Limit may, therefore, constitute a barrier to effective immune function, at least for those T cells that encounter their specific antigen more than once over the life course. Application of the fibroblast replicative senescence model to the so-called cytotoxic or CD8 T cell, the class of T cells that controls viral infection and cancer, has revealed certain features in common with other cell types as well as several characteristics that are unique to T cells. One senescence-associated change that is T cell-specific is the complete loss of expression of the activation signaling surface molecule, CD28, an alteration that enabled the documentation of high proportions of senescent T cells in vivo. The T cell model has also provided the unique opportunity to analyze telomere dynamics in a cell type that has the ability to upregulate telomerase yet nevertheless undergoes senescence. The intimate involvement of the immune system in the control of pathogens and cancer as well as in modulation of bone homeostasis suggests that more extensive analysis of the full range of characteristics of senescent T cells may help elucidate a broad spectrum of age-associated physiological changes.     

右心导管术对静脉血中循环内皮细胞的影响及临床意义

目的 :观察右心导管术对静脉内皮的影响 ,明确其机制及临床意义。方法 :1 2例行右心导管术的患者 ,手术前后分别采静脉血 4.5 ml,用五步法分离脱落的静脉内皮细胞 ,免疫荧光法鉴别 ,光镜计数。结果 :右心导管术后循环内皮细胞计数明显高于术前 (P <0 .0 0 1 ) ,但其数量变化与手术时间无线性关系。结论 :右心导管术对静脉内皮有机械损伤。     

抵抗素对内皮细胞NO生成的影响及Akt信号机制

目的探讨抵抗素对内皮细胞NO生成的影响及其可能的信号机制。方法分离、培养人脐静脉内皮细胞（HUVECs）,以不同浓度抵抗素（15、50、100ng／ml）干预。荧光显微镜检测各组细胞中N0的生成,RT-PCR检测eNOS mRNA表达水平,Western blot检测Akt和eNOS磷酸化水平。结果15、50、100ng／ml抵抗素干预HUVECs 24h后,胰岛素刺激的内皮NO生成显著降低（三组分别为4．01±0．69、3．764±0．71、3．73±0．45,vs对照组P均〈0．05）,同时伴有内皮Akt和eNOS磷酸化水平的降低（us对照组P均〈0．05）,而eNOS mRNA表达无显著改变。结论抵抗素可通过P13K／Akt途径影响HuVECs eNOS磷酸化水平,进而调节内皮细胞NO生成,但该影响并非Akt依赖性。