Liang Yu-Meng, Wang Xiao-Na, Deng Lei, Wang Lu, Wang Yi, Huang Ya-Jing, Liu Tie-Qiang, Zuo Hong-Li, Sun Qi-Yun, Qiao Jian-Hui, Yu Chang-Lin, Hu Kai-Xun, Ai Hui-Sheng, Guo Mei
Department of Hematology, Affiliated Hospital, Academy of Military Medical Sciences, Beijing 100071,China.
Department of Hematology, Affiliated Hospital, Academy of Military Medical Sciences, Beijing 100071,China. E-mail:
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2017 Aug;25(4):1187-1193. doi: 10.7534/j.issn.1009-2137.2017.04.039.
To explore the biological characteristics of microvesicles(MV) derived from bone marrow mesenchymal stem cells (BM-MSC) and their capability supporting ex vivo expansion of hematopoietic stem cells(HSC).
The MV from cultured BM-MSC supernatant were isolated by multi-step differential velocity contrifugation; the morphological characteristics of MV were observed by electron microscopy with negative staining of samples; the protein level in MV was detected by using Micro-BCA method; the surface markers on MV were analyzed by flow cytometry. The peripheral blood HSC(PB-HSC) were isolated after culture and mobilization; the experiment was diveded into 2 group: in MV group, the 10 mg/L MV was given, while in control group, the same volume of PBS was given; the change of PB-HSC count was observed by cell counting; the change of surface markers on PB-HSC was detected dynamically by flow cytometry; the cell colony culture was used to determin the function change of PB-HSC after co-culture with MV.
MSC-MVs are 20-100 nm circular vesicles under electron microscope. About 10 µg protein could be extracted from every 1×10 MSC. The flow cytometry showed that CD63 and CD44 were positive with a rate of 96.0% and 50.2%, while the HLA-DR, CD34, CD29 and CD73 etc were negative. When being co-cultured with GPBMNC for 2 days, the cell number of MV groups was 1.49±0.15 times of the control group (P>0.05). When being co-cultured for 4 days, the cell number of MV groups was 2.20±0.24 times of the control group(P<0.05). The CD34 cell number of MV groups was 1.76±0.30 times the control group after culture for 2 day and 1.95±0.20 times after culture for 4 day.
The MV has been successfully extracted from MSC culture supernatant by multi-step differential velocity centrifugation. MSC-MV can promote HSC expansion in vitro.
探讨骨髓间充质干细胞（BM-MSC）来源的微泡（MV）的生物学特性及其支持造血干细胞（HSC）体外扩增的能力。
通过多步差速离心法从培养的BM-MSC上清液中分离MV；采用电子显微镜负染观察MV的形态特征；使用微量BCA法检测MV中的蛋白质水平；通过流式细胞术分析MV表面标志物。分离培养并动员后的外周血造血干细胞（PB-HSC）；实验分为2组：MV组给予10 mg/L MV，对照组给予相同体积的PBS；通过细胞计数观察PB-HSC数量的变化；通过流式细胞术动态检测PB-HSC表面标志物的变化；采用细胞集落培养法确定PB-HSC与MV共培养后的功能变化。
电镜下MSC-MVs为20-100 nm的圆形囊泡。每1×10个MSC可提取约10 μg蛋白质。流式细胞术显示CD63和CD44阳性率分别为96.0%和50.2%，而HLA-DR, CD34, CD29和CD73等为阴性。与粒-单核细胞集落形成单位（GPBMNC）共培养2天时，MV组细胞数量为对照组的1.49±0.15倍（P>0.05）。共培养4天时，MV组细胞数量为对照组的2.20±0.24倍（P<0.05）。培养2天后MV组CD34+细胞数量为对照组的1.76±0.30倍，培养4天后为1.95±`0.20倍。
通过多步差速离心成功从MSC培养上清液中提取出MV。MSC-MV可促进体外HSC扩增。
