模拟高住低练对大鼠骨髓源性内皮祖细胞增殖能力的影响

Effects of“Living High-Training Low”on Proliferative Capacity of Endothelial Progenitor Cells from Bone Marrow of Rats

目的:模拟高住低练后,体外原代培养SD大鼠骨髓来源的内皮祖细胞,观察其生长情况,探讨低氧与运动复合刺激对其增殖能力的影响。方法:选用健康雄性SD大鼠24只,随机分为4组:常氧居住组、低氧居住组、常氧训练组和高住低练组。低氧居住组采用递减氧浓度刺激;常氧训练组采用递增负荷跑台训练,每周6天,为期8周。方案结束后12 h,提取大鼠后肢骨髓,运用密度梯度离心法分离得到单个核细胞,以EGM-2培养基进行体外培养诱导分化,获取内皮祖细胞,通过双荧光染色法鉴定内皮祖细胞。倒置显微镜下计数贴壁细胞数量、绘制细胞生长曲线及计算细胞增殖速率。结果:(1)细胞培养48 h后呈圆形贴壁生长,第5 d～7 d呈梭形并出现集落,第10 d集落增多,第20 d细胞分枝状生长、管腔样结构出现。(2)与常氧居住组相比,其余三组贴壁细胞数量显著增加(P<0.05);高住低练组贴壁细胞数量最多(P<0.05);常氧训练组与低氧居住组贴壁细胞数量比较无显著性差异(P>0.05)。各组细胞增殖平均速率无显著差异(P>0.05)。结论:低氧和运动都能促进骨髓源性内皮祖细胞的增殖,低氧和运动复合刺激效果更明显。

Objective To investigate whether or not the proliferative capacity of endothelial progenitor ceils was associated with stimulation of hypoxia and exercise. Methods 24 male Sprague-Dawley rats were randomly and averagely divided into 4 groups ：living at low altitude （LL） ,living and training at low altitude （LLTL）,living at high altitude （LH）,and living at high altitude and training at low altitude （LHTL）. High altitude was simulated through stimulating with decreasing oxygen concentration. Training was performed on a treadmill with incremental load,6 days per week for 8 weeks. 12 hours after the 8- week experiment,the mononuclear cells from bone marrow were obtained by density gradient centrifugation and suspended in EGM-2 for culturing in vitro to induce differentiation,in which endothelial progenitor cells were identified by double fluorescent staining. Inverted microscope was used to assay endothelial progenitor cells count,plot their growth curve, and evaluate the average rate of cellproliferation. Results（ 1 ）Round adherence grow of ceils after 48h culture ,fusiform and colony forming of cells at day 5-7 of culture,and branch and lumen like structure at day 20 of culture were found.（2）The number of adherent cells increased significantly in groups LLTL,LH and LHTL,especially in group LHTL as compared with that in group LL （P 〈 0.05）. There was no significant difference in the number of adherent ceils between groups LH and LHTL （P 〉 0.05）and in the average rate of cell proliferation among all groups （P 〉 0.05）. Conclusion Proliferative capacity of endothelial progenitor cells from bone marrow can be promoted by stimulation of hypoxia or exercise,especially by the combination of the stimulations.