骨折后外周血间充质干细胞浓度变化的实验研究

目的]观察骨折后不同时间点外周血间充质干细胞(MSCs)浓度变化,并比较其与骨髓间充质干细胞生物学特性的异同.方法]根据不同处理条件将SD大鼠分为:对照组和骨折组(骨折后1、3、7d共3组),每组20只.分别于骨折后1、3、7d抽取外周血,密度梯度离心法分离培养外周血MSCs,计数成纤维细胞集落形成单位(CFU-Fs)数.流式细胞仪检测细胞表面标记(CD44、CD90、CD34、CD45).成骨、成脂诱导,碱性磷酸酶、茜素红和油红染色检测其分化特性.结果]原代外周血MSCs呈集落生长,骨折组集落数明显多于对照组,其中以骨折后3d组形成的集落数最多,具有显著差异(27.25±11.52 CFU-Fs/cuhure vs 2.80±3.96 CFU-Fs/culture,P＜0.01).外周血MSCs高表达CD44、CD90,低表达CD34、CD45,不同的是CD34小部分呈阳性(＜20％).与对照组骨髓MSCs的诱导结果相同,外周血MSCs成骨诱导后28 d出现钙结节,茜素红染色阳性；成脂诱导后21 d有大量脂滴出现,油红染色阳性.结论]外周血体外密度梯度离心法分离培养的细胞具有多潜能分化的MSCs表面标记且可以向成骨、成脂分化.骨折后外周循环中MSCs数量明显增多,呈一定的时序性变化,可能参与骨折修复.

Change of the number of mesenchymal stem cells in peripheral blood of rats after fracture

Objective]To compare the number of mesenchymal stem cells(MSCs) in the peripheral blood(PB) of rats after fracture at different time points with non-fractured control and evaluate the biological features of MSCs from PB and bone marrow(BM).Method]SD rats were randomly divided into 4 groups:control group,groups of fracture for 1,3,7 d,n=20.At day 1,3,7 after fracture,PB was collected from heart of rats in each groups.MSCs were harvested from PB by density gradient centrifugation.The number of the colony forming unit-fibroblasts(CFU-Fs) was counted when peripheral blood mononuclear cells were cultured at 14d.MSCs were analyzed with a monoclonal antibody panel:CD44,CD90,CD34,CD45 by flow cytometry.Multipotent differentiation was identified through osteogenic and adipogenic induction.Result]The primary MSCs from PB formed colonies.Significant differences in colony numbers of MSCs were found between fracture groups and control group,among which the largest number of colonies were formed by group of fracture at 3 d(P<0.01)(27.25-11.52 CFU-Fs/culture vs 2.80-3.96 CFU-Fs/culture).PB derived MSCs were similar to BMderived MSCs,except for a small population(20%) of CD34 cells among MSCs from PB.Twenty-eight days after osteogenic induction of MSCs,calcium deposition was detected by Alizarin Red,21 days after adipogenic differentiation,lipid droplets were detected by oil red.Conclusion]PB derived MSCs cultured in vitro can differentiate into osteoblasts and adipocytes.Peripheral blood of rats after fracture has more cells with MSC phenotypes than blood from the non-fractured control.MSCs may be involved in fracture repair.