辛伐他汀对体外培养人脐静脉血内皮祖细胞扩增及黏附能力的影响

Effects of simvastatin on the number and adhesiveness of endothelial progenitor cells from umbilical vein blood cultured in vitro

背景：他汀类药物可促进急性缺血后的血管新生、加速球囊损伤血管的再内皮化并减少新内膜下组织的增生，且这两个作用均与血管内皮祖细胞密切相关。目的：通过体外培养人脐静脉血血管内皮祖细胞，观察辛伐他汀对血管内皮祖细胞扩增及黏附能力的影响。设计、时间及地点：以细胞为观察对象，分组对比实验，于2006—02／10在吉林大学基础医学院病理生理教研室完成。材料：人脐静脉血采集来源于吉林大学第二临床医学院妇产科6名正常足月顺产健康产妇。方法：密度梯度离心法分离培养人脐静脉血血管内皮祖细胞。通过免疫荧光法观察内皮细胞吸收乙酰化低密度脂蛋白和荆豆凝血素I情况，流式细胞仪定量榆测血管内皮祖细胞表面CD133、CD34和KDR抗原表达阳性率来鉴定血管内皮祖细胞。倒置显微镜下观察血管内皮祖细胞形态学变化，计数细胞并绘制生长曲线。实验按M199培养基含辛伐他汀纯品浓度不同分5组，分别为0，0．01，01，1，10umol／L辛伐他汀组，其中0umol／L作对照。四甲基偶氮唑盐比色法观察血管内皮祖细胞增殖情况，计数贴壁细胞数法评价血管内皮祖细胞的黏附程度。主要观察指标：原代培养血管内皮祖细胞形态变化；各组细胞增殖及黏附能力。结果：①培养细胞呈长梭形，接种2-4d梭形形态细胞较少，为细胞生长的潜伏期：6～10d贴壁梭形细胞逐渐增多，为对数增殖期：10～14d细胞融合呈集落样生长，梭形细胞明显增多，进入生长平台期。②激光共聚焦显微镜鉴定乙酰化低密度脂蛋白、荆豆凝血素I双染阳性的细胞为正在分化的血管内皮祖细胞。③流式细胞仪检测细胞表达CD133、CD34和KDR抗原的阳性率分别为（730±271）％、（42．00±642）％，（89．30±10．45）％。④四甲基偶氮唑盐比色结果表明各浓度组血管内皮祖细胞增殖活力、贴擘的血管内皮祖细胞数均较对照组增强（P〈0．05）；但以1μmol／L浓度下血管内皮祖细胞增殖活力最强。结论：辛伐他汀能增强体外培养血管内皮祖细胞的扩增及黏附能力。

BACKGROUND： Statins can promote the angiogenesis following acute ischemia, accelerate the re-endothelialization of injured sacculus and reduce the hyperblastosis neointima. These effects are closely associated with vascular endothelial progenitor cells （EPCs）. OBJECTIVE： To isolate, purificate and culture EPCs from human umbilical vein blood, and to observe the effects on expansion and adhesion ability of the EPCs in different concentrations of simvastatin. DESIGN, TIME AND SETTING： Grouping control experiments were carried out in the Department of Pathophysiology, Basic Medical College of Jilin University from February to October in 2006. MATERIALS： EPCs were selected from umbilical vein blood of the parturients underwent full-term normal delivery in the Department of Gynaecology and Obstetrics, the Second Hospital of Jilin University. METHODS： EPCs were isolated by using density gradient centrifugation from human umbilical vein. Immunofluorescence assay was used to detect endothelial cell uptake and absorption acetylated low density lipoprotein and ulex europaeus agglutinin- I. EPCs quantitative detection of surface markers was performed by flow cytometry： CD133, CD34 antigen expression and KDR. Inverted microscope observation was used to assay the morphological changes of EPCs, cell count and the growth curve. Simvastatin concentration was ranked as 0, 0.01, 0.1, 1 and 10 μ mol/L, while 0 μ mol/L served as the controls. EPCs proliferation was observed by MTT method, adhesion of the EPCs was evaluated by counting the number of adherent cell. MAIN OUTCOME MEASURES： The morphological changes of vascular EPCs; the proliferation and adhesion of EPCs in different concentrations of simvastatin. RESULTS： The cultured EPCs were shaped as long fusiform and became smaller at 2-4 days, as latent period; adherent cells increased at 6 10 days, as log proliferation period; ceils showed a colony-like growth into cell fusion and the cells were significantly increased at 10-24 days, as growth platform period. Laser confocal microscopy revealed the differentiating EPCs could uptake acetylated low density lipoprotein and ulex europaeus agglutinin-I, which were stained as red and green fluorescence. Flow cytometry results showed the expression of CD133, CD34 and KDR antigen was （7.30±2.71）%, （42.00±6.42）% and （89.30±10.45）%. MTr colorimetric results showed that the EPCs proliferation activity and number of adherent cells were all enhanced in the simvastatin groups compared with the controls （P 〈 0.05）. However, the EPCs proliferation was the strongest in the concentration of 1 μ moL/L. CONCLUSION： Simvastatin can increase the number and adhesion capacity of vascular EPCs cultured in vitro.