摘要
目的本研究对人骨髓间充质干细胞(hBMMSCs)进行基本生物学特性研究,并探讨以微载体培养技术进行大量增殖的可能性。方法对成人骨髓血用Percoll淋巴细胞分离液进行密度梯度离心,以获取hB-MMSCs。原代和传代培养后描绘细胞增长曲线。以流式细胞术对所获取的hBMMSCs进行细胞表型鉴定。以Cytodex3微载体对hBMMSCs进行动态培养,并与无微载体培养的细胞进行数量对比。结果以Percoll淋巴细胞分离液可成功地对人骨髓血进行间充质干细胞的分离,原代和传代培养后细胞生长良好。以流式细胞术对hBMMSCs进行表型鉴定,其中CD29、CD44、CD73、CD105和CD147呈阳性表达,阳性率高于96%;CD71、CD90的阳性率为86.74%和71.01%;而CD14、CD34、CD38、CD45和HLA-DR呈阴性表达,且大多低于2%。以Cytodex3微载体进行动态培养时,hBMMSCs能够在微载体上迅速贴附、铺展并生长。在相同的时间点,当hBMMSCs在微载体表面长满时,细胞数目均显著多于无微载体的培养方法(P<0.001),其细胞密度和培养速度至少是普通培养法的4.5～9.6倍。Cytodex3上细胞数目的大量增长大约在第8天后进入平台期。结论应用Percoll淋巴细胞分离液能便捷地将hBMMSCs从人骨髓血中分离提取出来,对细胞活性影响小,并可以流式细胞术快速有效地鉴定其表面标记物。已获取的hBMMSCs的纯度较高,而且表型也较均一。与普通培养相比较,Cytodex3微载体培养体系可以在相同时间内获取大量的hBMMSCs,便于进一步开展在心血管外科领域的组织工程学应用。
[Objective] In this trial, human bone marrow mesenchymal stem cells （hBMMSCs） were harvested and cultivated in vitro. Concerning with the basic biological character, we discuss the possibility of massive proliferation of hBMMSCs using the microcarrier cultivating technique, and to facilitate the further tissue engineering experiments. [Method] Pereoll lymphocyte extraction solution was applied with the pretreated human bone marrow blood to harvest the BMMSCs by means of density gradient centrifugation. Cell amount was measured and cell proliferation curves were described after primary and passage culture. Cell surface markers were identified with flow cytometry. Dynamic culture was conducted in part of BMMSCs with Cytodex3 microcarrier, and compared with the non-microcarrier cultivated ceils in quantity. [Result] hBMMSCs could be successfully extracted with the Pereoll solution, and cultivated well in primary and passage culture. The BMMSCs surface markers were identified with flow cytometry which demonstrated that CD29, CD44, CD71, CD73, CD90, CD105, and CD147 expressed positively with a rather higher positive ratio. CD14, CD34, CD38, CD45 and HLA-DR expressed negatively with the ration of lower than 2% of the most. When Cytedex3 microcarrier was utilized in dynamic culture, hBMMSCs could adhere to the microcarrier rapidly and grow. At the same time points, the amounts of hBMMSCs were much greater than that of the culture method （P 〈0.001）. The density and cultivate speed of cells were 4.5-9.6 times of common culture at least. In terms of cell amount curve, a plateau may appear after 8 days of massive proliferation approximately on the Cytodex3 microcarriers. [Conclusion] hBMMSCs Could be conveniently and rapidly extracted using Pereoll solution by means of density gradient centrifugation with less impact on the cell characters. Flow cytometry could afford an effective and fast way to identify the surface markers of BMMSCs. Other than hematopoietie stem cells or fibroblasts, the cells we harvested are definitely hBMMSCs with a rather higher purity and uniformity. Compared with common culture method, massive quantity of hBMMSCs could be obtained with Cytodex3 microcarrier cultivating system in order to develop the further tissue engineering application in the field of cardiovascular surgery.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2009年第12期1761-1766,共6页
China Journal of Modern Medicine
关键词
骨髓间充质干细胞
流式细胞术
微载体
组织工程
bone marrow mesenehymal stem cell
flow cytometry
microcarrier
tissue engineering
