中国农业科学 ›› 2008, Vol. 41 ›› Issue (1): 229-236 .doi: 10.3864/j.issn.0578-1752.2008.01.031

• 畜牧·资源昆虫 • 上一篇    下一篇

小鼠MSCs体外定向诱导分化神经及脂肪细胞的研究

赵 兴,柏学进,封纪武,董雅娟   

  1. 青岛农业大学胚胎工程中心
  • 收稿日期:2006-09-06 修回日期:2007-10-12 出版日期:2008-01-10 发布日期:2008-01-10
  • 通讯作者: 董雅娟

inducing differentiation of mouse bone marrow mesenchymal stem cells into neurocytes and adipocytes in vitro

  

  1. 青岛农业大学胚胎工程中心
  • Received:2006-09-06 Revised:2007-10-12 Online:2008-01-10 Published:2008-01-10

摘要: 【目的】建立昆明小鼠骨髓间充质干细胞的分离、培养体系,并鉴定其定向诱导分化为神经及脂肪细胞的能力。【方法】采用贴壁培养法分离培养昆明小鼠骨髓间充质干细胞(MSCs),通过相差倒置显微镜对其进行形态学特征观察;通过碱性磷酸酶(AKP)染色、Hoechst 33258染色、瑞士-姬姆萨复合染色检测MSCs的表型特征;通过β-ME诱导MSCs定向分化为神经样细胞,诱导后的细胞进行H.E染色,观察其形态学特性;通过NF-200、NSE免疫荧光细胞化学方法对已分化的神经样细胞进行鉴定和分化率分析;通过马血清诱导MSCs定向分化为脂肪细胞,观察其体外生长过程,并对诱导后的脂肪细胞进行油红O染色鉴定和分化率分析。【结果】分离得到的MSCs为成纤维样细胞,可见多个核仁,AKP染色阳性率为93.2%±1.5%;β-ME诱导后,MSCs分化为多种表型的神经细胞与原代培养的小鼠全脑细胞的外形相似,诱导后的神经细胞NF-200、NSE免疫荧光细胞化学染色均呈阳性,NF-200阳性率为85.4%±1.8%,NSE阳性率为82.7%±2.1 %;马血清诱导后,MSCs可100%分化为脂肪细胞,油红O染色为阳性。【结论】 采用贴壁培养法能够成功分离和培养昆明小鼠的MSCs,分离得到的MSCs具有定向分化成多种神经细胞和脂肪细胞的能力。

关键词: 小鼠, 骨髓间充质干细胞, 分离培养, 诱导分化, 神经细胞, 脂肪细胞

Abstract: Objective To establish a system for isolation and culture of bone marrow mesenchymal stem cells (MSCs) from Kun Ming mouse in intro and to identify characteristic of the cells, directional neuronal and adipocytic induction after culture expansion. Methods MSC were isolated and cultivated from the bone marrow of Kun Ming mice by investigated their adherence-dependent growth characters; The morphology characters of MSCs were observed under inverted phase contrast microscope; Shape characteristics of MSCs were studied by AKP staining, Hoechst33258 staining, White Giemsa staining; Inducing MSCs into neurone-like cells by β-ME, and staining with HE after inducing, then observe their characteristics under inverted phase contrast microscope; differentiation ratio analysis of differentiated neurocytes were detected by immunocyte-fluorescence of NF-200 and NSE; inducing MSCs into adipocytes by horse serum, observing they process of growth in vitro, differentiation ratio analysis of differentiated adipocytes were detected by Oil Red O stain. Result Separated MSCs were fibroblast-like cells, their have several nucleoluses, positive rates of AKP staining were 93.2%±1.5%. MSCs could differentiate into several phenotype characteristic neurocytes after inducing by β-ME, their shape characteristics were similar with primer passage of mouse full-brain cells. Both NF-200 and NSE immunocyte-fluorescence of Neurones after inducing were positive, positive rates of NF-200 and NSE were 85.4%±1.8% and 82.7%±2.1% respectively; MSCs could differentiate into adipocytes in 100 percent after inducing by horse serum, Oil Red O stain was positive. Conclusion Mesenchymal stem cells of Kun Ming mouse can be successfully isolate and cultivate from mouse bone marrow by keeps its adherence-dependent growth characters. They have directional trans-differentiation potentiality to various neurocytes and adipocytes.

Key words: mice, MSCs, separation and culture, induction and differentiation, neurocytes, adipocytes