【作者】 周学凯；

【导师】 郭文怡； 李飞；

【作者基本信息】 第四军医大学 ， 内科学， 2011， 硕士

【摘要】 背景与目的随着近年来我国经济的发展,人民生活水平提高,2型糖尿病的发病率也迅速升高。作为一种慢性代谢性疾病,患者常表现为糖、脂等物质的代谢紊乱。但其最主要的致残和致死原因是并发的大血管疾病。多个研究也显示,糖尿病患者的冠心病发病率明显高于非糖尿病患者。近年来研究发现,在冠心病等大血管疾病的发生和发展中,血管平滑肌祖细胞（smooth muscle progenitor cells, SPCs）发挥了十分重要的作用。SPCs是一类能增殖并分化为平滑肌细胞的前体细胞,存在于骨髓、外周血及血管外膜等多个组织器官中。其中分布于外周循环血的SPCs主要表达CD14⁺/CD105⁺。有研究表明,在多种心血管危险因素的影响下,血管内膜受到损伤,局部组织细胞通过释放趋化因子和细胞因子能动员骨髓中的SPCs进入外周循环血,并归巢于受损血管段,参与新生内膜的形成,最终导致动脉粥样硬化的发生和发展。然而,对于SPCs与2型糖尿病患者心血管并发症间的关联性却知之甚少。本研究旨在通过观察2型糖尿病患者外周血平滑肌祖细胞数量的变化特点,揭示其在体内的影响因素。此外,通过体外实验观察高糖对SPCs的功能影响,为进一步探索糖尿病患者发生心血管并发症的机制奠定基础。实验方法第一部分:成人外周血SPCs的体外分离及诱导分化。无菌条件下,采集成人外周血,应用密度梯度离心法分离获得单个核细胞,接种到含有血小板源性生长因子（platelet-derived growth factor-BB,PDGF-BB）和碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF）的培养基中进行诱导扩增,12d后利用流式细胞仪分析贴壁细胞中SPCs的含量并分选纯化,继续在转移生长因子-β1（transforming growth factor-β₁,TGF-β₁）的作用下诱导分化。倒置显微镜观察SPCs在增殖、分化过程中的形态变化,免疫荧光染色法观察其平滑肌肌动蛋白-α（α-smooth muscle actin,α-SMA）的表达,同时应用Western blot技术检测调宁蛋白和平滑肌肌球蛋白重链（smooth muscle myosin heavy chain,SM-MHC）的表达情况。第二部分:统计分析外周血中SPCs数量的变化及相关因素。选择2010年1月至2011年1月在西京医院心血管内科诊治的2型糖尿病患者35例及20名健康志愿者。分为:单纯性2型糖尿病组（DM组,n=20）、2型糖尿病伴冠心病组（DM+CAD组,n=15）和健康对照组（Con组,n=20）。应用流式细胞仪对外周血SPCs行计数分析,同时检测空腹血糖（FPG）、糖化血红蛋白（HbA1c）、收缩压（SBP）、舒张压（DBP）、总胆固醇（TC）、甘油三酯（TG）、低密度脂蛋白胆固醇（LDL-C）及高密度脂蛋白胆固醇（HDL-C）等临床指标。通过多元线性逐步回归法分析外周血SPCs数量与各临床指标的相关性。第三部分:不同浓度葡萄糖对SPCs功能的影响。分离外周血单个核细胞,进行诱导扩增后应用流式细胞分选仪获得SPCs。随机分为5组给予不同浓度葡萄糖干预:对照组（5.5mmol/L）,11mmol/L组,22mmol/L组,44mmol/L组和渗透压对照组（5.5mmol/L葡萄糖+38mmol/L甘露醇）。分别用MTT比色法,Transwell小室迁移实验以及粘附能力测定实验检测各组干预6天后,SPCs增殖、迁移和粘附能力的变化。此外,培养SPCs8天,期间用22mmol/L葡萄糖分别干预0、2、4、8天,观察各组细胞增殖、迁移和粘附能力的变化。实验结果1.成人外周血单个核细胞诱导培养4d时开始出现细胞集落,12d时细胞呈明显梭形。流式细胞仪分析显示,CD14⁺/CD105⁺的SPCs占贴壁细胞的（71.8±7.2）%。经分选纯化后的SPCs培养到28d呈旋涡状生长。间接免疫荧光染色显示,α-SMA表达呈阳性。Western blot检测显示,调宁蛋白和SM-MHC分别于14,21d开始表达,并逐渐增加。2.单纯性2型糖尿病患者外周血中SPCs数量明显高于健康对照组（26.20±11.36 vs. 14.30±7.61,P<0.05）,而2型糖尿病伴冠心病组又明显高于单纯2型糖尿病组（42.00±8.93 vs. 26.20±11.36,P<0.05）。多元线性逐步回归分析显示:HbA1c、SBP、HDL-C是影响成人外周血SPCs数量的独立相关因素。其回归方程为y=13.433+2.435 x₂-21.625 x₈+0.188 x3。Spearman相关分析显示:外周血SPCs数量与HbA1c、SBP呈明显正相关（r=0.684, r=0.379,P<0.01）,而与HDL-C呈明显负相关（r=-0.654,P<0.01）。3.在高糖干预实验中,与对照组相比,高糖各组均能明显促进外周血SPCs的增殖、迁移和粘附能力,其中22mmol/L葡萄糖组的影响最为显著,44mmol/L葡萄糖组的促进作用有所下降（P<0.05）。用22mmol/L葡萄糖分别干预SPCs 0、2、4、8天,其增殖、迁移和粘附能力随着作用时间延长而增强,以干预8天组最显著（P<0.05）。结论:1.通过对成人外周血单个核细胞的诱导扩增,可以获得表达CD14+/CD105+的SPCs,并证实了SPCs具有向平滑肌细胞进一步分化的能力;2. 2型糖尿病患者体内糖、脂代谢紊乱可能导致外周血SPCs数量升高,从而促进冠心病的发生和发展。HbA1c、SBP、HDL-C作为独立相关因素,可能影响了外周血中SPCs的数量,并能通过其预测SPCs的数量变化趋势;3.高糖能在一定范围内增强外周血中SPCs的增殖、迁移、粘附能力,并呈现浓度和时间依赖性。可以推测长期高血糖通过促进SPCs的功能参与受损血管过度修复,引起部分心血管疾病的发生发展。更多还原

【Abstract】 Background and ObjectiveAs the economy of China is developing and the quality of life has improved, the incidence of type 2 diabetes is rapidly increasing. Diabetic patients often have the metabolic disorders of glucose and lipid. However, the most important cause of disability and mortality in diabetic patients is complicated by large vessel diseases. It has been shown that the incidence of coronary heart disease in diabetes is significantly higher than non-diabetic patients. Recent studies have found that SPCs plays an important role in the development of atherosclerosis.Circulating-derived SPCs express CD14/CD105 double positive, which can proliferate and differentiate into smooth muscle cells. In addition, SPCs can also be found in the bone marrow and adventitia. Studies have shown that a variety of cardiovascular risk factors cause the vascular endothelium damage. The chemokines and cytokines released from local vascular cells mobilize the bone marrow derived-SPCs into the peripheral circulation, and SPCs home in the damaged vessels involving in the formation of neointima, leading to the development of atherosclerosis.The aim of the current study is to observe the changes of circulating derived-SPCs in type 2 diabetes, and find the factors to these changes in the body. It will help explore the mechanism of the cardiovascular complications in diabetic patients.MethodsPart one: Isolation ,culture and differentiation of adult human circulating- derived SPCs in vitro. Mononuclear cells were isolated from adult human peripheral blood by density gradient centrifugation. The isolated cells subsequently were plated onto dishes in medium supplemented with PDGF-BB and bFGF. After 12 days of culture in vitro, SPCs were （identified）characterized as adherent cells and sorted by flow cytometry. SPCs were then induced to differentiate in response to TGF-β1. Inverted microscope was used to observe morphological changes of SPCs. Smooth muscle cells specific markers （α-SMA, Calponin, SM-MHC） were then checked with immunofluorescent staining and Western blotting.Part two: To investigate the correlation within the number of circulating- derived SPCs and factors in the patients with type 2 diabetes mellitus. we selected 55 type 2 diabetic patients enrolled in Xijing Hospital from January 2010 to January 2011 and 20 healthy volunteers. They were divided into 3 groups: 20 type 2 diabetic patients without coronary artery disease （DM group）, 15 type 2 diabetes patients with coronary artery disease （DM+CAD group） and 20 healthy volunteers （Con group）. We detected peripheral blood CD14⁺/CD105⁺ cells by flow cytometry. In addition, we detected other parameters （FPG, HbA1c, SBP, DBP, TC, TG, LDL-C, HDL-C）.The relationship between the number of SPCs and the parameters were analyzed by multivariate regression analysis. Part three: Effects of high glucose on biological characteristics of smooth muscle progenitor cells. Mononuclear cells were isolated from adult human peripheral blood by density gradient centrifugation and cultured. After 12 days of culture in vitro, SPCs were sorted by flow cytometry. They were randomly divided into 5 groups （control group, 11 mmol/L group , 22 mmol/L group , 44 mmol/L group and osmotic pressure control group）. The SPCs were incubated with glucose in a series of concentrations for 6 days. Proliferation and migration of SPCs were assayed by MTT assay and Transwell chamber assay respectively. SPCs adhesion assay was performed by replating the cells on fibronectin-coated dishes and the adherent cells were then counted. In addition, the SPCs were incubated with glucose （22mmol/L） for different durations （0 , 2 , 4 , 8 days）. The SPCs proliferation and migration ability were abserved and adhesion assay was performed.Results1. Four days later, cell colonies were presented. Twelve days later, most adherent cells were showing spindle-shape. CD14/CD105 double positive cells were SPCs, which accounted for （71.8±7.2）% of adherent cells. Twenty-eight days later, cells arranged in order, showing whirlpool-shape. On day 28, immunostaining of adherent cells was positive forα-SMA. Expression of calponin and SM-MHC was found at 14 d and 21 d with Western blotting respectively, and they gradually enhanced.2. The number of circulating-derived SPCs in patients of DM group was significantly increased compared with controls （26.20±11.36 vs. 14.30±7.61,P<0.05）, and it was higher in patients of DM+CAD group than in the DM group （42.00±8.93 vs. 26.20±11.36,P<0.05）.Multiple regression analysis showed that HbA1c, HDL-C and SBP were independent related factors influencing circulating-derived SPCs levels. The equation was y=13.433+2.435 x₂-21.625 x₈+0.188 x₃ . Linear correlation analysis show that the number of SPCs in peripheral blood was correlated positively with HbA1c and SBP （r=0.684, r=0.379, P<0.01）, and negatively with HDL-C （r=-0.654,P<0.01）.3. Compared with the control group, high glucose accelerated proliferation , migration and adhesion ability. Especially when the SPCs were incubated with glucose in the final concentration of 22mmol/L for 8 days, the effects were the most prominent.ConclusionsOur results demonstrated that:1. SPCs could be harvested through culturing mononuclear cells from adult human peripheral blood. They could differentiate into smooth muscle cells in vitro.2. The number of circulating-derived SPCs was associated with metabolism disorder. And the increasing SPCs may contribute, in part, to the pathogenesis of atherosclerosis in type 2 diabetic patients. As the independent related factors, the level of HbA1c、SBP and HDL-C can predict the number of circulating SPCs.3. Within a certain range, high glucose could significantly improve the function of SPCs in a concentration and time dependent manner. It can be speculated that long-term hyperglycemia in patients could stimulate the function of SPCs, which plays an important role in the the development of cardiovascular disease.更多还原