人骨髓和脐带来源间充质干细胞体外支持造血能力的比较研究

本研究分离鉴定人骨髓和脐带来源的间充质干细胞（mesenchymal stem cells，MSC），比较两种间充质干细胞体外支持长期造血的能力。用密度梯度离心或酶消化方法分离骨髓和脐带间充质干细胞，通过贴壁培养的方法进一步纯化Msc。检测骨髓和脐带MSC的表型以及成脂、成骨分化潜能。通过LTC—IC（long—term culture—initiating cells）实验，检测骨髓和脐带间充质千细胞体外支持长期造血的能力，并在培养的第3、5、7周分析两种MSC组悬浮细胞的表型。结果表明，骨髓和脐带间充质干细胞体外培养时均呈纺锤形、贴壁生长，2种MSC均表达CD90、CD105、CD73、CD29、CD54、CD166和HLA—ABC，不表达HLA—DR、CD34和CD45。化学染色证实，2种MsC体外能分化为脂肪细胞和成骨细胞。LTC—IC实验第5周脐带间充质干细胞组CFC（colony forming cell）的产率与骨髓间充质干细胞组比较无统计学差异（P〉0．05）；第6、7、9周，脐带MSC组CFC的产率均低于骨髓MSC组（P〈0．05）。第3、5、7周悬浮细胞表型检测结果显示，随着培养时间的延长，2种MSC组CD34和CD117的阳性率均明显下降，而CD33、CD13、CD11b的阳性率逐渐上升。结论：从人骨髓和脐带组织中成功分离并鉴定出间充质干细胞，脐带间充质干细胞能够在体外支持长期造血，但其造血支持能力弱于骨髓间充质干细胞。

Comparative Study of In Vitro Hematopoietic Supportive Capability of Human Mesenchymal Stem Cells Derived from Bone Marrow and Umbilical Cord

The present study was aimed to isolate and identify human mesenchymal stem cells from adult bone marrow （BM-MSC） and umbilical cord （UC-MSC）, and to compare their ability to support in vitro long-term hematopoiesis. MSC from bone marrow and umbilical cord were isolated by using density gradient centrifugation or enzyme digestion. MSC were further purified by adherent culture. Immunophenotype, adipogenic and osteogenic differentiation potential of BM-MSC and UC-MSC were detected. The hematopoietic supporting capacity of BM-MSC and UC-MSC was assessed by LTC-IC assay. Nonadherent cells in each group were collected for phenotypic analysis at 3, 5 and 7th week of culture. The results showed that BM-MSC and UC-MSC in culture shared a similar spindle-shaped morphology and adhered to the tissue culture substrate. They were both positive for CD90, CD105, CD73, CD29, CD54, CD166, HLA-ABC, and negative for HLA-DR, CD34 and CD45. BM-MSC and UC-MSC could differentiate into adipocytes or osteoblasts confirmed by oil red O staining and von Kossa staining, separately. LTC-IC assay showed that at 5th week of culture, the difference of the CFC yields between UC-MSC group and BM-MSC group was not statistically significant （p 〉0.05）. At 6, 7, 9th week of culture, the CFC yields in the UC-MSC group were lower than those of BM-MSC （p 〈 0.05 ）. The phenotypic analysis of nonadherent cells at 3, 5, 7th week of culture indicated that along with prolongation of time, the percentages of CD34 ^＋ cells and CD117 ^＋ cells in each group decreased markedly, and the percentages of CD33 ^＋ cells, CD13 ^＋ cells and CD11b ^＋ cells increased gradually. It is concluded that MSC from human adult bone marrow and umbilical cord can be successfully isolated and identified. UC-MSC are able to support long-term hematopoiesis in vitro, but its hematopoietic supportive capacity is weaker than those of BM-MSC.