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
发布时间：2018-01-06 21:33
本文关键词：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 出处：《中国实验血液学杂志》2017年04期 论文类型：期刊论文
【摘要】：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(P0.05),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(P0.05),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。
[Abstract]:Objective: To investigate the biological characteristics of bone marrow mesenchymal stem cells and microbubbles on hematopoietic stem cell proliferation in vitro. Methods: the separation and purification of bone marrow mesenchymal stem cells by multi-step differential centrifugation (MSC) in the supernatants of microbubbles (MV) method, using negative staining electron microscopy in the sample under the observation of the micro morphological characteristics of bubble; Determination of protein content by Micro-BCA method; using flow cytometric analysis of microbubble surface markers; liquid culture after mobilization of peripheral blood stem cells were divided into two groups, in the same culture system, to join the microbubble group 50 L microbubbles, the control group added volume PBS cells were observed by cell counting; number of changes, changes of stem cell surface markers by flow cytometry and dynamic monitoring of hematopoietic cell colony culture method, observation and microbubble co cultured hematopoietic stem cells change function in vitro. Results: mesenchymal stem cell source 寰场鏄洿寰勫湪20-100 nm涔嬮棿鐨勭被鍦嗗舰鍥婃场,鎻愬彇鍚庣殑寰场娴撳害绾︿负200渭g/ml;鍦ㄩ棿鍏呰川骞茬粏鑳炲井娉′腑CD63琛ㄨ揪鐜囦负96.0%,CD44琛ㄨ揪鐜囦负50.2%,鑰孒LA-DR,CD34,CD29,CD73绛夎〃杈惧潎涓洪槾鎬，
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