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

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.

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

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.