共培养条件下嗅鞘细胞对骨髓间充质干细胞向神经样细胞分化的影响

The effect of olfactory ensheathing cells on neural cell-like differentiation of bone mesenchymal stem cells in vitro co-culture system

中文关键词:  骨髓间充质干细胞  嗅鞘细胞  共培养  分化

英文关键词:Bone mesenchymal stem cell  Olfactory ensheathing cell  Co-culture  Differentiation

中文摘要:

  【摘要】 目的：观察体外骨髓间充质干细胞（BMSC）和嗅鞘细胞（OEC）非接触共培养时，OEC对BMSC向神经样细胞诱导分化的影响。方法：采用5~6周龄、体重100g左右的SPF级雄性Sprague Dawley（SD）品系大鼠20只，分离培养大鼠BMSC和OEC，BMSC取自双侧股骨，OEC取自嗅上皮。使用6孔0.4?滋m的Transwell共培养系统进行培养。将2×104/cm2的第3代OEC置于共培养系统上层，3×104/cm2的第3代BMSC置于下层。对共培养7d和14d后的BMSC行免疫组化染色，检测神经细胞特异性蛋白巢蛋白（Nestin）、神经元类型Ⅲβ-微管蛋白（Tuj-1）和神经胶质纤维酸性蛋白（GFAP）的表达情况。流式细胞仪检测Nestin、Tuj-1和GFAP阳性细胞的分布和所占的比例。结果：免疫组化检测显示，共培养7d和14d后BMSC均表达Nestin、Tuj-1和GFAP。流式细胞仪分析，共培养7d后Nestin、Tuj-1和GFAP阳性细胞比例分别为46.3%、76.5%和23.2%，共培养14d后分别为27.7%、89.1%和50%。结论：非接触共培养条件下，OEC能诱导BMSC向神经样细胞分化。

英文摘要:

  【Abstract】 Objectives: To investigate the differentiation of bone mesenchymal stem cell(BMSC) induced by co-culturing with olfactory ensheathing cell(OEC). Methods: BMSC and OEC were isolated and cultured from 20 5-6-week-old 100g male rats. BMSC was isolated from rat femur, and OEC was isolated from rat olfactory epithelium. The co-culture system used was 6-well 0.4m Transwell co-culture system. The third generation cells were co-cultured within this system at a density of 2×104/cm2 and 3×104/cm2 for OEC and BMSC respectively. BMSC was processed for immunofluorescence and flow cytometry at 7d and 14d for the specific marker Nestin, neuronal class Ⅲ β-tubulin(Tuj-1) and glial fibrillary acidic protein(GFAP). Results: Immunocytochemistry showed BMSC could express Nestin, Tuj-1 and GFAP after 7d and 14d co-culture. Flow cytometry revealed that the positive rate of Nestin, Tuj-1 and GFAP was 46.3%, 76.5% and 23.2% after 7d co-culture; and the positive rate of Nestin, Tuj-1 and GFAP was 27.7%, 89.1% and 50% after 14d co-culture. Conclusions: BMSC can efficiently differentiate into neural-like cells when coculturing with OEC.