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

Li Zhongxuan; Chen Yundai

doi:10.3969/j.issn.1674-4055.2018.02.23

Abstract：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.