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目的优化培养条件,获得体外克隆化培养的成年大鼠骨髓多能成体祖细胞(rMAPC),并对其进行表型分析及分化潜能鉴定。方法选择低血清条件培养基全骨髓直接贴壁法初步分离大鼠骨髓干细胞,用极限稀释法进行克隆化培养;用流式细胞术及免疫细胞化学法对克隆化培养的 rMAPC 进行表型分析,并进行体外成脂肪、成骨及成神经细胞诱导,RT-PCR 检测 Oct 4及三个胚层早期标志物,确定其表型及分化潜能。结果单个细胞来源 rMAPC 在体外扩增至20代仍保持良好的生长状态;流式细胞术及细胞免疫组化检测结果显示 CD71、α-SMA 及 Vimentin 表达阳性;CD34、CD44、CD45表达阴性;细胞周期分析显示 S+G_2+M 期细胞约占17%,而处于 G_0/G_1期的细胞占83%:rMAPC 体外可被诱导向脂肪细胞、骨细胞及神经细胞分化;RT-PCR 检测到 Oct 4及三个胚层早期标志物的表达。结论体外克隆化培养的 rMAPC 能维持良好的干细胞生物学特性。
Objective To optimize the culture conditions and obtain the adult rat bone marrow multipotent adult progenitor cells (rMAPC) cloned and cultured in vitro. The phenotypic characteristics and differentiation potential of adult rat bone marrow progenitor cells (rMAPCs) were determined. METHODS: Bone marrow stem cells were isolated from rat bone marrow by direct adherent method in the condition of low serum culture medium and cloned by the limit dilution method. The phenotypes of cloned rMAPC were analyzed by flow cytometry and immunocytochemistry, The adipogenic, osteogenic and neuronal cells were induced in vitro. The early markers of Oct 4 and the three germ layers were detected by RT-PCR and their phenotype and differentiation potential were determined. Results The single cell-derived rMAPC still maintained a good growth state after 20-d expansion in vitro. The results of flow cytometry and immunohistochemistry showed that the expression of CD71, α-SMA and Vimentin were positive, while the expressions of CD34, CD44 and CD45 were negative. Periodic analysis showed that about 17% of cells in S + G_2 + M phase and 83% in G_0 / G_1 phase: rMAPC could be induced to differentiate into adipocytes, osteocytes and neurons in vitro. Oct 4 And the expression of three early germ layer markers. Conclusion The rMAPC cultured in vitro can maintain good stem cell biology.