音猬因子诱导恒河猴间充质干细胞向神经样细胞的分化**☆◆

背景：骨髓间充质干细胞诱导分化为神经细胞，是神经系统疾病细胞治疗的有效手段，但目前的时效尚不完善。 目的：应用神经发育音猬因子诱导恒河猴骨髓间充质干细胞向神经样细胞分化。 方法：应用经典的维甲酸方案与音猬因子方案两种方法诱导恒河猴骨髓间充质干细胞分化为神经样细胞，采用密度梯度离心法分离培养恒河猴骨髓间充质干细胞，倒置相差显微镜观察生长情况，MTT法测定细胞生长曲线，流式细胞仪鉴定细胞表型，免疫组织化学鉴定分化细胞标志，透射电镜和扫描电镜观察分化细胞超微结构。 结果与结论：体外分离培养的恒河猴骨髓间充质干细胞，经流式细胞仪表型鉴定，具有较高均一性。通过音猬因子诱导方案诱导处理7 d后，分化细胞多数表现为NSE、NF-M、Tau和Nestin染色阳性，经图像统计分析发现经音猬因子诱导方案神经干细胞标志物Nestin阳性率显著高于维甲酸诱导方案(P < 0.01)，另一方面经维甲酸诱导方案诱导的细胞表现GFAP阳性率高于音猬因子诱导方案，差异具有显著性意义(P < 0.05)。提示音猬因子诱导方案是一种诱导恒河猴骨髓间充质干细胞向神经样细胞分化的有效途径。

Study on differentiation of rhesus monkeys mesenchymal stem cells into neuron like cells by sonic hedgehog

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) differentiating into neural cells is an effective way of cell therapy of nervous system disease. However, the methods used nowadays still need to be improved. OBJECTIVE: To induce the differentiation of rhesus monkey BMSCs into neuron-like cells by using sonic hedgehog factor. METHODS: Rhesus monkey BMSCs differentiating into neuron-like cells was induced by typical retinoic acid and sonic hedgehog factor. Rhesus monkey BMSCs were isolated and cultured by density gradient centrifugation method. Cell growth was observed under an inverted phase contrast microscope and cell growth curve was determined using MTT assay. Flow cytometry was performed to characterize the phenotype of BMSCs, and immunohistochemistry was utilized to assess differentiated cells. Ultra-structure of the differentiated cells was observed by transmission and scanning electron microscopes. RESULTS AND CONCLUSION: Rhesus monkey BMSCs cultured in vitro were identified by flow cytometry, with high homogenicity. Following sonic hedgehog factor disposal for 7 days, differentiated cells were mainly positive for neurone specific enolase, neurofilament protein, Tau and glial fibrillary acidic protein (GFAP). Image statistical analysis found that in sonic hedgehog factor scheme, neural stem cells marker Nestin positive rate was significantly higher compared with the retinoic acid scheme (P < 0.01). GFAP-positive rate was greater in the retinoic acid scheme than in the sonic hedgehog factor scheme (P < 0.05). Results indicated that sonic hedgehog factor scheme is an effective pathway of rhesus monkey BMSC differentiation into neuron-like cells.