【作者】 廖云飞；

【导师】 陈璐璐；

【作者基本信息】 华中科技大学 ， 内科学， 2009， 博士

【摘要】 目的研究不同时期2型糖尿病（T2DM）患者循环内皮祖细胞（EPC）的数目和功能变化以及与血管内皮功能的关系。方法选择498例2007年5月至2008年4月就诊的T2DM患者及我院体检人群。心脑血管并发症诊断根据明确的心脑血管事件和客观的实验室检查资料。所有患者均做血管超声检查外周血管并发症；免散瞳眼底拍照检查糖尿病视网膜病变；6个月内连续两次24h尿白蛋白检查糖尿病肾病；肌电图检查糖尿病周围神经病变。采用流式细胞仪检测循环EPC的数目；体外培养计数集落形成和迁移能力评估EPC的功能。采用血流介导的肱动脉血管舒张功能（FMD）评估血管内皮功能。结果其中正常对照组（NC组）83例，T2DM无血管并发症组（TC组）97例，T2DM合并大血管并发症组（TA组）106例，T2DM合并微血管并发症组（TI组）100例，T2DM合并大血管及微血管并发症组（TAI组）112例。五组循环EPC数目和集落形成能力按顺序排列均为TA＜TAI＜TI＜TC＜NC （532±90、616±93、768±97、1045±106和1724±112个／毫升全血；21±4、28±5、43±7、70±9和85±11个／孔），组间两两比较均有统计学差异（P＜0．05）。TA与TAI组的迁移能力差异无意义（24±6；28±7个／高倍视野），其余组的迁移能力与循环EPC数目变化一致（97±9、115±12和128±14个／高倍视野）。影响大血管并发症患者EPC数目的因素为年龄、糖化血红蛋白（HbA1C）、收缩压（SBP）、BMI和糖尿病患病时间（P＜0．05）；而影响微血管并发症患者EPC数目的因素为年龄、HbA1c和糖尿病患病时间（P＜0．05）。在排除了常见危险因素的影响后，循环EPC数目与FMD成正相关（标准回归系数=0．61，P=0．01）。结论T2DM患者循环EPC数目与功能较正常人群有不同程度的下降，不同血管并发症患者上述改变不尽相同。循环EPC数目与血管内皮功能密切相关，可作为衡量T2DM患者血管内皮功能的标志物之一。目的作为血管内皮细胞的前体细胞，循环内皮祖细胞（EPC）数目和功能的下降是引起2型糖尿病（T2DM）血管病变的重要原因之一。本研究旨在探讨二甲双胍治疗对新诊断T2DM患者循环EPC数目和功能的影响，以期深入了解二甲双胍对糖尿病患者心血管保护作用的具体机制。方法选择2007年9月至2008年5月于我院就诊的T2DM患者及健康体检人群，包括43名新诊断T2DM患者和40名性别、年龄匹配的正常对照者。所有T2DM患者给予口服二甲双胍治疗（1．0-2．0克／天），持续16周。观察治疗前后T2DM患者循环EPC数目和功能的变化，以及与氧化应激的关系。采用流式细胞仪检测循环EPC(CD45^low／CD34⁺／VEGFR2⁺)数目；激光共聚焦显微镜鉴定EPC摄取ac-LDL和结合UEA-1功能；迁移及衰老实验评价EPC功能；硫代巴比妥酸法、黄嘌呤氧化法分别测定血清脂质过氧化产物丙二醛（MDA）水平和总超氧化物歧化酶（SOD）活性。结果二甲双胍治疗能有效降低T2DM患者的血糖水平（P＜0．05），减轻体重（P＜0．05），改善胰岛素抵抗（P＜0．01）。治疗前T2DM患者的循环EPC数目、体外培养ac-LDL⁺／UEA-1⁺细胞数目和迁移能力明显较正常对照者低（P＜0．01），治疗后显著增加（P＜0．05），但仍低于正常水平（P＜0．05）。治疗前T2DM患者EPC的衰老程度较正常人群严重（P＜0．01），治疗后衰老程度较治疗前显著改善（P＜0．01），但仍未达到正常水平（P＜0．05）。血清MDA水平与衰老程度的变化一致（相关系数0．76，P＜0．05），治疗前T2DM患者血清MDA水平较正常人群显著增加（4．82±0．71 vs 2．11±0．35mmol／L，P＜0．01），治疗后血清MDA水平显著下降（2．45±0．56 mmol／L，P＜0．05）。血清总SOD活性与循环EPC数目及迁移能力的变化一致（相关系数分别为0．85、0．73，P＜0．05），治疗前T2DM患者血清总SOD活性较正常人群显著降低（31．49±6．12 vs52．87±5．05U／ml，P＜0．01），治疗后血清总SOD活性较治疗前有所上升（P＜0．05）。结论T2DM患者循环EPC数目明显降低，迁移能力低下，衰老程度严重，与此同时机体氧化应激较重，且氧化应激程度与循环EPC的数目和功能低下具有密切的相关关系。二甲双胍治疗可有效增加新诊断T2DM患者循环EPC数目并改善其功能，同时机体氧化应激状态也得以明显改善，提示二甲双胍可以通过改善氧化应激促进EPC数目和功能。目的前述研究发现二甲双胍治疗能够改善2型糖尿病（T2DM）患者循环内皮祖细胞（EPC）的数目和功能。在此基础上，本研究以高糖培养的健康人外周血EPC为模型，观察二甲双胍能否改善高糖培养导致的EPC功能异常，并初步探讨其机制。方法1．密度梯度离心法获得的正常人外周血单个核细胞培养5天后，采用免疫荧光进行EPC鉴定；加入不同浓度的二甲双胍（0μM、50μM、100μM、200μM）分别干预24、48小时，采用免疫印迹方法检测细胞内腺苷酸活化蛋白激酶（AMPK）及磷酸化水平。2．上述方法培养获得正常人EPC后，随机分为高糖组（加入葡萄糖25mM）、二甲双胍干预组（加入葡萄糖25mM和二甲双胍100mM）和正常对照组培养48小时，观察EPC增殖及迁移能力的变化；同时测定细胞中AMPK的蛋白表达及磷酸化水平。EPC生成一氧化氮（NO）的产量由Griess法测定的NOx （亚硝酸盐和硝酸盐）水平衡量。细胞氧化应激状态用脂质过氧化产物丙二醛（MDA）水平（硫代巴比妥酸法）和超氧化物歧化酶（SOD）活性（黄嘌呤氧化法）评估。结果1．二甲双胍呈时间依赖性促进EPC内AMPKα磷酸化，并在100μM时作用最强。2．较正常对照组，高糖组EPC的增殖和迁移能力显著下降；AMPKα磷酸化水平明显降低，为其30．5±7．4％；MDA水平上升而SOD活性下降；NOx水平仅为其21．8±6．3％（P＜0．05）。在二甲双胍干预组，EPC增殖和迁移能力均较高糖组显著改善；较高糖组，MDA水平下降而SOD活性上升；NOx水平也较高糖组显著增加，为对照组的85．2±8．3％；细胞内AMPK的蛋白表达无明显变化，但其磷酸化水平较高糖组显著增加，恢复到对照组的70．6±9．2％（P＜0．05）。结论二甲双胍可在体外提高EPC的AMPK活性；二甲双胍可直接作用于EPC增加其增殖和迁移能力，其机制与激活AMPK后增加细胞内NO水平以及改善氧化应激有关。更多还原

【Abstract】 Objective To investigate the number and function of circulating endothelial progenitorcell （EPC） in the patients with type 2 diabetes （T2DM） and its association with vascularendothelial function.Methods A total of 498 T2DM patients were recruited from the health examinationpeople and the outpatients and inpatients of the Endocrinology Department in UnionHospital. Assessments of cardiovascular disease and cerebrovascular disease were based oneach patient’s medical records. Peripheral vascular disease was diagnosed byultrasonography bilaterally. Non-mydriatic fundus camera screening was used as a tool toidentify diabetic retinopathy. Urinary albumin exceeding 30mg/24 h which appeared twiceover a period of six months was diagnosed as diabetic nephropathy. An electromyogramwas used to identify diabetic peripheral neuropathy. Circulating EPC was quantified byflow cytometry. Clony forming count （CFU） and migration assay were used for evaluatingthe function of circulating EPC. Vascular endothelial function was assessed byflow-mediated brachial artery dilatation （FMD）.Results There were five groups in the study: Normal control subjects（NC, n=83）, T2DMwithout vascular disease（TC, n=97）, T2DM with macrovascular disease（TA, n=106）,T2DM with microvascular disease（TI, n=100）, T2DM with macro- and micro-vasculardiseases（TAI, n=112）. The sequence of circulating EPC number and CFU in four groupswas TA＜TAI＜TI＜TC （532±90、616±93、768±97、1045±106 and 1724±112 cell/ml;21±4、28±5、43±7、70±9 and 85±11 unit/chamber） and there was a significantdifference between any two groups （P＜0.05）.The results of migration were consistent withcirculating EPC number （97±9、115±12 and 128±14 cell/highpower field） except therewere no significant differences in TA and TAI groups （24±6; 28±7 cell/highpower field）.Age、HbAlc、SBP、BMI and duration of T2DM were the independent risk factors ofcirculating EPC number in T2DM patients with macrovascular disease（P＜0.05）. Age、HbAIC and duration of T2DM were the independent risk factors of circulating EPCnumber in T2DM patients with microvascular disease（P＜0.05）. After the adjustment oftraditional risk factors, the number of circulating EPC had a close correlation with FMD （standardized coefficient=0.61, P=0.01）.Conclusion Compared with healthy subjects, the number and function of circulating EPCin T2DM patients were significant lower and decreased with differenet degrees in T2DMpatients of different vascular diseases. Circulating EPC number was associated withendothelial function and can be considered as a surrogate biological marker of vascularendothelial function for T2DM. Objective As the precursor of vascular endothelial cells, the impaired number andfunction of circulating endothelial progenitor cell （EPC） have been hailed as a novelconcept in the pathogenesis of diabetic vascular complications. In order to understand theunderlying mechanism on vasculoprotective effect of metformin, this study was designed toinvestigate the effect of metformin therapy on the number and function of circulating EPCin patients with newly diagnosed T2DM.Methods 43 newly diagnosed T2DM patients and 40 gender and age matched healthysubjects were recruited from the patients of the Endocrinology Department and the healthexamination people in Union Hospital. All patients were continuously given metformintherapy （1.0-2.0g daily） for 16 weeks. Changes of the number and function of circulatingEPC in T2DM patients before and after treatment and their association with the status ofoxidant stress had been observed. Circulating EPC was defined by the surface expression ofCD45^low/CD34⁺/VEGFR2⁺ and quantified by flow cytometry. DiLDL uptake and UEA-1staining by laser scanning confocal microscope was used as an established second methodto quantify and characterize EPC. Migration and insenecence assays were used forevaluating the functions of circulating EPC. Serum MDA was assayed by thiobarbituricacid adduct with colorimetric method. The activity of total SOD was measured by xanthineoxidase with colorimetric method.Results Metformin treatment can effectively lower the glucose level （P＜0.05）, reducebody weight （P＜0.05） and improve insulin resistance （P＜0.01）. The number of circulatingEPC, ac-LDL⁺/UEA-1⁺ cells and migration capacity in T2DM patients before treatmentwas markedly lower than that in control subjects （P＜0.01） and increased significantly after16 weeks of treatment （P＜0.05）, which was still lower than that in control subjects（P＜0.05）. EPC from T2DM patients before treatment had higher rates of in vitrosenescence than cells from control subjects （P＜0.01） and decreased markedly aftertreatment （P＜0.01）. Consistent with insenecence varieties （correlation coefficient 0.76,P＜0.05）, levels of serum MDA in patients before treatment were significantly higher thanthose in control subjects （P＜0.01） and decreased considerably after treatment （P＜0.05）. Consistent with the varieties of circulating EPC number and migration ability （correlationcoefficient 0.85、0.73, P＜0.05）, activities of total SOD in patients before treatment weresignificantly lower than those in control subjects （P＜0.01） and improved markedly aftertreatment （P＜0.05）.Conclusion Compared with normal subjects, circulating EPC in T2DM patients wascharacteristized as decreased number, lower migration capacity, and more insenecence.Meanwhile, the oxidative stress status in T2DM patients was severe, which had a closerelationship with the decreased number and function of EPC. Metformin treatment couldeffectively improved the number and function of circulating EPC and oxidative stress inpatients with newly diagnosed T2DM, which suggested the role of oxidativestress-mediated pathway in metformin-caused EPC upregulation. Aim It has been shown that metformin could improve the number and function ofcirculating endothelial progenitor cell （EPC） in type 2 diabetes （T2DM） in part 2. Thisstudy was designed to investigate whether metformin had direct effect on EPC and try tofind the underlying mechanism. As the experiment model, circulating EPC from healthysubjects was cultured in high glucose.Methods 1. The mononuclear cells were acquired by density gradient centrifugation fromhealthy subjects. After five days culture, the cells were assessed by immunofluorescence inorder to identify the EPC. Metformin with different concentrations （0μM、50μM、100μM、200μM） and different intervention time （24、48 hours） were added to the cultured EPCfrom healthy subjects. The levels of AMPK protein and its phosphorylation were detectedby WesternBlot analysis. 2. The cultured EPC from new healthy subjects were randomlydivided into three groups: high glucose group （+25mM glucose）, intervention group（+25mM glucose and 100mM metformin）, and control group. After 48 hours culture,proliferation and migration capacities were observed and the levels of AMPK protein andits phosphorylation were assessed as well. NOx （nitrite and nitrate） concentrations wereused as an index of nitric oxide （NO） production and were evaluated by a colorimetricassay based on Griess reaction. The status of oxidative stress was assessed by the level ofmalonaldehyde （MDA） and the activity of superoxide dismutase （SOD） from cellsupernatant. MDA was assayed by thiobarbituric acid adduct with colorimetric method.The activity of SOD was measured by xanthine oxidase with colorimetric method.Results 1. Metformin can promote AMPKαphosphorylation in a time dependentmanner in EPC and has the strongest effect at 100μM. 2. Compared with control group,high glucose group possessed significantly decreased capacities of proliferation andmigration, reduced activity of SOD, increased level of MDA. The phosphorylation level ofAMPKαand the level of NOx in high glucose group were markedly decreased and wasonly 30.5±7.4% and 21.8±6.3%of control group individually （P＜0.05）. Compared withhigh glucose group, proliferation and migration capacities of EPC in intervention group were markedly improved; the level of MDA was decreased and the activity of SOD wasincreased （P＜0.05）. The level of NOx in intervention group was also increased significantlyand was 85.2±8.3% of control group; AMPK protein expression in intervention group hadno obvious changes, however, phosphorylation level of AMPKαwas markedly increasedand was 70.6±9.2% of control group （P＜0.05）.Conclusion In vitro, metformin can markedly stimulate AMPK activity in EPC anddirectly act on EPC to improve proliferation and migration capacities. The possiblemechanism is that metformin can stimulate AMPK activity and subsequently increase NOproduct and improve the imbalance of oxidative stress in EPC.更多还原