人间充质干细胞体外扩增及生物学特性的研究

The expansion and biological characteristics of human mesenchymal stem cells

目的 探讨成人骨髓、胎儿骨髓和人脐血 3种不同来源的人类间充质干细胞 (MSC)体外扩增及其生物学特性。方法 观察 3种来源的MSC的生长、增殖和表面标志的表达 ,从而评价MSC的纯化、增殖能力及其免疫学特性。结果 (1) 3种不同来源的人MSC的细胞形态、集落数、集落大小均无差异 ;但成人骨髓MSC在集落形成及集落交错融合时间上均早于胎儿骨髓和脐血MSC ;(2 )3种来源的人MSC传代培养增殖速度无差异 ,但脐血和胎儿MSC融合后无接触抑制 ,而成人骨髓MSC存在接触抑制 ;(3)来源于骨髓单个核细胞 (MNC) 8× 10 6或脐血MSC 2 5× 10 6的MSC ,经体外扩增 3代、5代和 10代后 ,细胞数分别为 10 7、10 8和 10 10 个 ;(4)MSC易纯化 ,P2代细胞均一性为 90 % ,P3代细胞均一性为 95 % ,P5代细胞均一性达到 99% ;(5 )不同来源的MSC表面重要标志 :CD2 9、CD44、CD59、CD90 、CD10 5、CD166表达均为阳性 ,其造血细胞表面标志 :CD11a、CD14 、CD3 3 、CD3 4 、CD2 8、CD45、CD117表达均为阴性 ,与移植免疫排斥发生密切相关的表面标志 :HLA DR、B7 1(CD80 )、B7 2 (CD86)、CD40 和CD40L均为阴性。 3种不同来源的人MSC表面标志 ,差异无显著性。 (6 )两种不同培养体系对人MSC的纯化扩增及生物学特性无影响。结论 (1)

Objective Mesenchymal stem cells (MSCs) were adult stem cells which contribute to the regeneration of mesenchymal tissues such as bone cartilage,muscle, ligament, tendon, adipose and stroma. Due to the multipotential ability and self-renewal capacity, the mesenchymal stem cells can be applied in many fields, such as the seed cells in tissues engineering, cell therapy and gene therapy. To enhance the clinical use of MSCs, the investigators studied the isolation and expansion of MSCs from adult bone marrow, fetal bone marrow and human umbilical cord blood, and investigated their biological identities. Methods Two kinds of incubation systems containing L-DMEM or MSC special culture medium were used to purify and expand MSCs. The growth, purification and proliferative abilities of 3 kinds of MSCs were observed and their immunophenotypes were determined by flow-cytometry. Results (1) The shapes of 3 kinds of cells were same. There was no difference in number and size. The colonies formed early in adult bone marrow MSCs. (2) There was no difference in the expansion speed of the 3 kinds of MSCs, but after the colonies confluenced there had no touching constrain in MSCs from umbilicalcord blood and fetal bone marrow. When the colonies confluenced, the cells also had proliferation ability. But in adult bone marrow, the touching constrain was significant. (3) MSCs had strong self-renewal capacity. After primary culture approximately 5-6×10 5 MSCs were obtained from 8×10 6 MNC of bone marrow and 25×10 6 MNC of umbilical cord blood. After passage 3, passage 5 and passage 10, the investigators could get 10 7,10 8 and 10 10 MSCs, respectively. (4) Along with the increase in the passage and prolonging of culture time, the ability of expansion decreased, but they maintained good puripotantiality. After passage 2, passage 3 and passage 5, the purity of MSCs was 90%, 95% and 99%, respectively. (5)Three kinds of MSCs were all positive for CD_ 29 , CD_ 44 , CD_ 59 , CD_ 90 , CD_ 105 , CD_ 166 and all negative for the markers of hematopoietic cells such as CD_ 11a , CD_ 14 , CD_ 33 , CD_ 34 , CD_ 28 , CD_ 45 . All the important GVHD correlation markers were negative, such as HLA-DR, B7-1(CD_ 80 ), B7-2(CD_ 86 ), CD_ 40 and CD_ 40L .There were no differences in the phenotype among the 3 kinds of MSCs cells. (6) The 2 kinds of culture mediums used did not markedly affect isolation and expansion of MSCs, and the biological properties of MSCs. Conclusions (1) Human MSCs could be isolated from many kinds of human tissues, and they had no difference in their origin; (2) Human MSCs maintained good puripotantiality and self-renewal capacity. Therefore, they could meet with the need of clinical tissue engineering. (3) The negative GVHD correlated markers might result from the fact that MSCs had no HLA barrier but had broad clinical use.