非黏附骨髓间充质干细胞的体外培养与鉴定

Isolation in Vitro and Identification of Non-Adherent Bone Marrow Mesenchymal Stem Cells of Mouse

目的探讨小鼠骨髓中是否存在非黏附间充质干细胞(BM-NA-MSC),体外能否形成成纤维细胞集落形成单位(CFU-F),能否在适当的条件下分化为脂肪细胞、软骨细胞和成骨细胞。方法分离小鼠总骨髓细胞,体外培养,每天转移未贴壁的非黏附骨髓细胞(NA-BMC)到新培养环境中继续培养,每组细胞长满12 d后,亚甲基蓝染色显示形成的克隆,计数克隆个数;在总骨髓细胞培养到第4天时收集未贴壁的NA-BMC到新的培养皿中,待细胞贴壁生长达融合后传代纯化,取第5代细胞接种于向脂肪细胞、软骨细胞和成骨细胞诱导分化的培养液中,2周后分别进行油红染色、抗Ⅱ型胶原的免疫组织化学染色及茜素红染色检测脂肪滴、Ⅱ型胶原和钙化结节的形成和表达。结果小鼠骨髓细胞体外培养条件下不仅总骨髓细胞可以形成CFU-F,而且反复转移的NA-BMC也能不断形成CFU-F;给予适当的诱导条件,非黏附来源的骨髓细胞体外能够分化为产生油滴的脂肪细胞、表达Ⅱ型胶原的软骨和具有钙化能力的成骨细胞。结论小鼠骨髓中存在一类NA-MSC,体外可以形成CFU-F,还可分化为脂肪、软骨和成骨细胞,表现出一定的多分化潜能,为下一步作为种子细胞用于移植实验的研究提供了前提。

Objective To explore whether bone marrow non - adherent mesenchymal stem cells （ BM - NA - MSC ） exist in mouse bone marrow; whether the NA - MSC can form fibroblastic colony units forming（ CFU - F） and whether they have the ability of differentiating into adipocyte, chondrocyte and osteoblast cells,which are typicals of BM - NA - MSC. Methods Bone marrow cells of mouse were isolated and cultured in vitro, non - adherent - bone marrow cells （ NA - BMC ） were repeatedly - transterred into new dishes every day for 5 times, every dish was stained with methylene blue after 12 days cell culture for CFU - F. For the detection of differentiation,NA - BMC were collected at the 4th day of the culture ,3 conditioned medium were used to induce the NA - BMC into adipocyte, chondrocyte and osteoblast cells. Oil red staining,immunohistochemical staining of type Ⅱ collagen and alizarin red staining were developed respectively to detect the differentiation of NA - BMC. Results In mice, not only total bone marrow cell could give rise to CFU - F, but also NA - BMC could give rise to CFU - F continuously. They could differentiate into adipocyte which appeared oil - red positive, chondrocyte expressing type Ⅱ collagen and osteoblast which could calcify after induced with conditioned medium in vitro. Conclusions It indicate that there are NA - MSC in mouse bone marrow. They can give rise to C FU -F continuously and differentiate into adipocyte, chondrocyte osteoblast,which appears some pluripotency and provides a promising for being used into cell therapetics.