不同浓度葡萄糖对大鼠骨髓来源内皮祖细胞增殖、迁移及血管形成的影响

Influence of glucose in different concentrations on proliferation, migration and angiogenesis of rat bone marrow-derived endothelial progenitor cells

目的探究大鼠内皮祖细胞(endothelial progenitor cells,EPCs)在不同浓度葡萄糖条件下的增殖、迁移及血管形成情况,为后续高糖环境EPCs病理生理机制探究提供参考。方法通过密度梯度离心法分离大鼠骨髓来源单个核细胞,经过培养纯化后得到原代EPCs。将EPCs分为5组,EGM-2培养基含有5mM葡萄糖,将5mM组设为对照组,添加葡萄糖使10mM组、15mM组、20mM组、25mM组达到相应的浓度。通过吞噬结合实验鉴定EPCs,CCK-8试剂盒检测不同时间点EPCs的增殖能力以及绘制生长曲线。Transwell实验检测EPCs迁移能力。观察EPCs体外血管形成情况。结果首次更换培养基后,贴壁细胞为圆形,7 d呈纺锤形,14 d呈铺路石样。所培养细胞具有吞噬Dil-ac LDL与FITC-UEA-I结合的特性,为EPCs,细胞阳性率为(93.10±0.85)%。同一时间点随着葡萄糖浓度升高,吸光度值降低,增殖下降,培养72 h后,在25mM达到最大抑制程度,差异有统计学意义(P<0.01)。同一浓度,各组培养12 h开始进入对数生长期,48 h进入平台期。随着葡萄糖浓度升高,迁移至下室EPCs比例逐渐减少(5mM:1.00±0.05;10mM:0.68±0.06;15mM:0.56±0.03;20mM:0.40±0.04;25mM:0.23±0.03),与5mM组对比,25mM组迁移比例最低[(0.23±0.03)vs.(1.00±0.05)],差异有统计学意义(P<0.01)。葡萄糖浓度为25mM时抑制EPCs迁移最明显。随着葡萄糖浓度升高,体外血管形成能力逐渐降低,5mM时所形成管腔完整,至25mM时无完整管腔(5mM:1.00±0.04;10mM:0.81±0.04;15m M:0.70±0.04;20mM:0.63±0.03;25mM:0.56±0.03)。与5mM组相比,25mM组被明显抑制,[(0.56±0.03)vs.(1.00±0.04)],差异有统计学意义(P<0.01)。高糖抑制EPCs体外血管形成,在25mM时最明显。结论随着葡萄糖浓度升高,大鼠内皮祖细胞增殖、迁移以及血管形成被抑制,在25m M时最明显,可作为后续探究高糖环境下内皮祖细胞病理生理机制的参考浓度。

Objective To investigate the proliferation, migration and angiogenesis of endothelial progenitor cells （EPCs） under condition of higher glucose in different concentrations in rats, and provide reference for study on pathophysiological mechanism of EPCs in high glucose environment. Methods Mononuclear cells were separated from bone marrow of rats by using density gradient centrifugation, and primary EPCs were obtained after culture and purification. EPCs were divided into 5 groups. EGM-2 medium contained 5 mM glucose, so 5 mM group was set as the control group, and glucose was added to reach corresponding concentration in 10 mM group, 15 mM group, 20 mM group and 25 mM group. EPCs were identified by using phagocytosis assay, EPCs proliferation was detected by using CCK-8 detection kit at different time points and growth curve was drawn. EPCs migration was detected by using transwell assay, and EPCs angiogenesis in vitro was observed. Results After the first change of medium, the parietal cells showed round, spindle-shaped appearance on the 7th d and cobblestone appearance on the 14th d. The cultured cells, which were EPCs, had the characteristics of phagocytosing Dil-acLDL and combining with FITC-UEA-I, and the positive cell rate was （93.10 ＋ 0.85）%. At the same time point, both OD value and EPCs proliferation decreased along with the increase of glucose concentration. After 72-h cultivation, the maximum inhibition of EPCs proliferation was reached in 25 mM group （P〈0.01）. EPCs began to enter logarithmic growth period after 12-h cultivation and platform stage after 48-h cultivation in all groups in the same glucose concentration. Along with the increase of glucose concentration, the proportion of EPCs migrated to lower chamber decreased gradually （1.00±0.05 in 5 mM group, 0.68±0.06 in 10 mM group, 0.56±0.03 in 15 mM group, 0.40±0.04 in 20 mM group and 0.23±0.03 in 25 mM group）. The proportion of EPCs migration was the lowest in 25 mM group compared with 5 mM group （0.23±0.03） vs. （1.00±0.05）, P〈0.01. When glucose concentration was 25 mM, EPCs migration was most significantly inhibited. EPCs angiogenesis in vitro decreased gradually along with the increase of glucose concentration, and lumen was complete when glucose concentration was 5 mM and there was almost no complete lumen when glucose concentration was 25 mM （1.00±0.04 in 5 mM group, 0.81±0.04 in 10 mM group, 0.70±0.04 in 15 mM group, 0.63±0.03 in 20 mM group and 0.56±0.03 in 25 mM group）. Compared with 5 mM group, EPCs angiogenesis in vitro was inhibited significantly in 25 mM group （0.56±0.03） vs. （1.00±0.04）, P〈0.01. The inhibition of EPCs angiogenesis was the most significant in 25 mM group. Conclusion EPCs proliferation, migration and angiogenesis are inhibited along with the increase of glucose concentration, which is most significant when glucose concentration is 25 mM. The glucose concentration of 25 mM can be taken as a reference for further study on pathophysiological mechanism of EPCs in high glucose environment.