摘要
骨髓是间充质干细胞(MSC)的主要来源。在大多数研究中,先用密度梯度离心法分离出单个核细胞(MNC),再将MNC培养得到MSC。然而,近年来的研究显示,这种标准方法分离MSC的效率并不很高。本研究探讨骨髓抽提物小粒是否是造成该法低效的原因及如何更好地从骨髓抽提物小粒中分离出MSC。选取了20例患者,用标准贴壁法培养MNC,用组织块培养法培养骨髓小粒。对于1-10号患者,先从骨髓抽提物中分离得到MNC,再从剩余的骨髓抽提物中过滤出骨髓小粒,比较从MNC和骨髓小粒培养得到的成纤维样细胞的克隆数量和形态;对于11-20号患者,分离顺序正好相反,比较从MNC和骨髓小粒培养得到的贴壁细胞的免疫表型和功能,对剩余的骨髓抽提物也进行了培养。结果表明:骨髓小粒中的贴壁细胞为MSC。如果先过滤出骨髓小粒,再用密度梯度离心法分离出MNC,然后将二者分别进行培养,则可将骨髓抽提物中的MSC完全分离出来。在密度梯度离心法中大多丢掉的骨髓小粒,是骨髓中MSC的又一重要来源。骨髓小粒可用组织块培养法进行培养。结论:将骨髓抽提物小粒和MNC分别培养可以从单次骨髓抽提物中富集更多的MSC。
Bone marrow （BM） is the major source of mesenchymal stem cells （MSC）. In most experiments, MSC were classically cultured from mononuclear cells （MNC） isolated by density gradient centrifugation method. However, several studies have demonstrated that this method was less efficient for MSC recovery. This study was aimed to investigate whether BM particles were the cause resulting in less efficiency of this method and how to isolate them. A total of 20 patients were enrolled in this study. MNC were cultured by standard adherence and BM particles were cultivated by primary explant culture. For BM from patients 1-10, MNC were first isolated and BM particles were then filtered out. The morphology and the fibroblastic colony number were compared between cultures of MNC and BM particles. For BM from patients 11-20, MNC isolation and BM particle filteration were processed in opposite order, then the immunophenotype and function between adherent cells expanded from MNC and BM particles were compared. In addition, for patients 11-20, the left BM aspirates were cultured too after BM particles and MNC were isolated separately. The results showed that adherent cells from BM particles were MSC. After BM particles were filtered out and cultured separately, MSC could be recovered completely from MNC isolated by density gradient centrifugation and no MSC were left in the residual BM aspirates. BM particles, which have been mostly discarded by the method of density gradient centrifugation, are another important source of MSC and they can be cultivated reliably by primary explant culture. It is concluded that more MSC are recovered from a single BM sample by culturing BM particles and MNC separately.
出处
《中国实验血液学杂志》
CAS
CSCD
2010年第6期1552-1559,共8页
Journal of Experimental Hematology
基金
supported by the fund from Ministry of Education of China (20070023087)
National Natural Science Foundation of China (30570357,30600238 and 30700285)
863 project of the Ministry of Science and Technology of China (2006AA02A110)
关键词
间充质干细胞
骨髓小粒
单个核细胞
密度梯度离心
组织块培养
mesenchymal stem cell
bone marrow particle
mononuclear cell
density gradient centrifugation
primary explant culture
