骨髓间充质干细胞微泡生物学特性及其促进造血干细胞体外扩增作用的研究

Biological characteristics of Microvesicles Derived from Bone Marrow Mesenchymal Stem Cells and Their Capacities Supporting ex vivo Expansion of Hematopoietic Stem Cells

目的:探讨骨髓间充质干细胞微泡的生物学特性及其对造血干细胞体外扩增的作用。方法:用多步差速离心法分离提纯骨髓间充质干细胞(MSC)培养上清中的微泡(MV),采用样本负染的方法在电子显微镜下观察微泡的形态特征;用Micro-BCA法测定其蛋白含量;用流式细胞术分析微泡表面标志;液体培养动员后外周血造血干细胞实验分为两组,在相同培养体系下,给微泡组加入50μl微泡,对照组加入等体积PBS;采用细胞计数观察细胞数目的变化,应用流式细胞术动态监测造血干细胞表面标志的变化,细胞集落培养法观测与微泡共培养后造血干细胞在体外的功能变化。结果:间充质干细胞来源的微泡是直径在20-100 nm之间的类圆形囊泡,提取后的微泡浓度约为200μg/ml;在间充质干细胞微泡中CD63表达率为96.0%,CD44表达率为50.2%,而HLA-DR,CD34,CD29,CD73等表达均为阴性;微泡与GPBM NC共培养2 d后,微泡组细胞数是对照组的1.49±0.15倍(P>0.05),CD34^+细胞数(3.93±0.60)×10~4是对照组(2.30±0.64)×10~4的1.76±0.30倍;4 d时微泡组细胞数(10.19±0.65)×10~6是实验组细胞数(4.67±0.70)×10~6的2.20±0.24倍(P<0.05),微泡组CD34^+细胞数(7.82±0.41)×10~4是对照组(4.03±0.35)×10~4的1.95±0.20倍。结论:通过多步差速离心法能够成功从MSC上清中提取微泡,微泡在体外对造血干细胞具有促进增殖的作用。

Objective： 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）. Methods： 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. Results： MSC-MVs are 20-100 nm circular vesicles under electron microscope. About 10 μg protein could be extracted from every 1 × 10^6 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. Conclusion： The MV has been successfully extracted from MSC culture supernatant by multi-step differential velocity centrifugation. MSC-MV can promote HSC expansion in vitro.