骨髓基质干细胞定向分化为神经细胞及分化后细胞功能特征的研究

Structural and functional characteristics of neural cells orientation of bone marrow stromal cells

目的 探讨骨髓基质干细胞（BMSCs）定向分化为神经细胞的潜能及分化后细胞的功能特征.方法 体外分离、扩增、纯化Sprague-Dawley大鼠BMSCs,取第6代细胞接种并随机分为4组,实验组A、B、C组开始每组加预诱导夜（含预诱导剂碱性成纤维细胞生长因子和表皮生长因子）,6d后A组继续加预诱导夜,其余两组在预诱导后加定向诱导剂（音猬因子和维甲酸）时选择是否保留预诱导液分为B组（保留）和C组（去除） D组不做任何干预,为空白对照组.采用细胞免疫荧光化学法检测分化后神经样细胞的形态和功能标志.结果 接种后的细胞迅速贴壁,加预诱导液6 d细胞增殖迅速,且可以观察到有类似神经干细胞的克隆团形成.在诱导剂的特定组合和时序的诱导下,A、B、C组均出现胞体收缩和突起伸出,呈现神经元细胞样改变.D组细胞形态基本没有变化.6～12 d,B组微管相关蛋白-2（MAP-2）阳性细胞率为59.17%±1.84%,胶质酸性蛋白（GFAP）阳性细胞率为22.10%±1.03% C组MAP-2阳性细胞率为29.35%±4.85%,GFAP为36.35%±4.85%,A组和D组均未检测到上述细胞标志物.12～18 d,A组仅出现少量GFAP阳性细胞 B组MAP-2、GFAP阳性细胞率分别为72.54%±1.02%、21.54%±1.02% C组MAP-2、GFAP阳性细胞率分别为31.02%±2.37%、23.45%±3.74%.神经细胞的功能标志胆碱乙酰转移酶、囊泡乙酰胆碱通道只有B组表达,12～18 d后阳性细胞率分别为45.11%±3.04%、28.22%±1.72%.结论 BMSCs在体外能定向诱导为具有一定功能的神经细胞,为脊髓损伤后细胞移植治疗奠定了基础.

Objective To explore the differentiation of bone marrow stromal cells（BMSCs）into neural cells and their structural and functional characteristics.Methods The BMSCs were isolated from rat posterior femur,amplified and cultivated in a total medium of LG-DMEM containing 15% FBS.The sixth generation of the cells were inoculated in 12 well culture plates and randomly divided into 4 groups：pre-induction medium（EGF＋bFGF）was added in the experimental group A while orientation inducer（SHH＋RA）was added in the remaining groups.The liquid was retained in group B and removed in group C.Group D served as the control group without any intervention.The morphology and function of the nerve-like cells were identified by Chemical cell immunofluorescence.Results After induction by a specific combination of agent and timing,BMSCs showed a neuron-like change in extended cell body contraction and processes in groups A,B and C.The positive cell rates of MAP-2 and GFAP were（72.54 ±1.02）% and（21.54 ±1.02）% respectively in group B,significantly higher than in the other groups.The functional markers ChAT（45.11 ± 3.04）% and VAT（28.22 ± 1.72）% were only expressed in group B.Conclusion As BMSCs are able to differentiate into neuron-like cells that have a certain shape and function in vitro,they may be further explored for the treatment of spinal cord injury.