脐血间充质干细胞的分离扩增及向成骨及脂肪细胞的分化

目的探讨新生儿脐血间充质干细胞（mesenchymal stem cells，MSCs）体外分离、纯化、扩增，以及向成骨及脂肪细胞定向诱导分化的方法与条件。方法无菌条件下收集新生儿脐血60～120ml，枸橼酸钠抗凝，以Ficoll—Hypaque淋巴细胞分离液密度梯度法、沉降红细胞后密度梯度法及CD34^＋免疫磁珠负选法分离单个核细胞（mononuclear cells，MNCs）。分离获得的MNCs采用L—DMEM培养基或Mesencult^TM培养基／10％胎牛血清进行MSCs培养传代，获得第3代集落生长细胞作流式细胞仪表面抗原测定，并向成骨及脂肪细胞定向诱导分化，成骨细胞钙沉积经茜素红染色鉴定，脂肪细胞胞浆油滴经油红染色鉴定。结果经沉降红细胞后分离的MNCs，使用Mesencult^TM培养基／10％胎牛血清培养成功率高，第3代可出现明显的集落生长，而另两种方法分离培养的细胞则难以形成集落；集落细胞表面抗原测定表达CD29、CD59、CD71而不表达CD34、CD45及HLA—DR等分子。成骨定向诱导分化的集落细胞经茜素红染色胞浆中出现有大量的钙沉积；成脂肪定向诱导分化的集落细胞油红染色示胞浆充满油滴空泡。结论新生儿脐血中可分离出MSCs，并可在体外进行培养扩增。以甲基纤维素沉降红细胞后密度梯度离心分离的MNCs培养较为有效，集落细胞表达基质细胞表面抗原，能够向成骨细胞及成脂肪细胞定向诱导分化。

METHOD AND CONDITIONS OF ISOLATION AND PROLIFERATION OF MULTIPOTENT MESENCHYMAL STEM CELLS

Objective To investigate the method and conditions of isolation,proliferation of multipotent mesenchymal stem cells(MSCs)from human umbilical cord blood in vitro, and to induce osteogenic and adipogenic differentiation directly for identification. Methods Human umbilical cord blood was collected in asepsis condition, isolated by density gradient centrifugation, or sedimented red cell with methylcellulose, and then the same centrifugation was done, or obtained by negative immunodepletion of CD34+. These isolated mononuclear cells were used to carry on plastic adherent culture. To obtain single cell-derived colonies, these cells were proliferated clonally in medium which consists of L-DMEM or Mesencult TM medium and 10% fetal calf serum(FCS) respectively, then their differentiation potentiality to osteoblasts and lipoblasts was tested. Results The mononuclear cells isolated by sedimented and centrifugated way cultured in Mesencult TM medium and 10%FCS were most available. These adhesive cells could become obviously short rod-shape or shuttle-shape cells after 5-7 days.The colonies form well in 3rd-passage cells. The mononuclear cells obtained by only centrifugalized in density gradient were hard to form colony, isolated by immunomagnetic beads were hard to culture. The surface antigens of these colonies cells presented CD29, CD59, CD71 but not CD34,CD45 and HLA-DR etc. The colony cells differentiating into osteoblasts that produce mineralized matrices, stained by alizarin red, and differentiating into adipocytes that accumulate lipid vacuoles, stained by oil red. Conclusion MSCs can be isolated from human umbilical cord blood and proliferate it in vitro. The way that mononuclear cells are sedimented red cell by methylcellulose and cultured by Mesencult TM medium and 10% FCS is the valid method of isolation. Proliferation colonies cells present matrix cell immunophenotypes, and can differentiate into osteoblasts and adipocytes.