恒河猴骨髓基质细胞向成熟神经细胞分化的实验研究

The differentiation of bone marrow stroma cells of rhesus monkey into maturation neural cells

目的研究恒河猴骨髓基质细胞(BMSCs)在体外向神经细胞诱导分化过程中神经递质分泌和神经细胞抗原表型表达情况。方法无菌条件下密度梯度法分离猴BMSCs,在体外应用神经干细胞培养液进行体外培养和诱导分化,分阶段用高效液相色谱法检测培养基中单胺类生物活性物质的含量,免疫细胞化学方法检测神经细胞抗原表型。结果高效液相检测神经干细胞培养基未测到单胺类生物活性物质,而经体外培养增殖10d、14d、30d的细胞培养基可检测到去甲肾上腺素 (NE)和多巴胺(DA)。Asp方差分析显示:随着BMSCs培养天数的增加,培养基中所含的NE和DA尤显著性差异(P>0．05)。同期细胞免疫组化检测出酪氨酸羟化酶(TH)、巢蛋白抗体(nestin)、β-微管蛋白 (β-tublin)、胶质原性纤维酸性蛋白抗体(GFALP)和神经元特异烯醇化酶(NSE)抗原表达。结论恒河猴BMSCs能在体外增殖,在适宜条件下能分化成神经元样细胞,并合成、分泌NE和DA;部分细胞表达神经干细胞、成熟神经元、胶质细胞和DA能神经元的抗原表型,提示在一定条件下,诱导分化的 BMSCs经过神经干细胞阶段可向成熟神经组织细胞分化。

Objective To study the monoamine biological activity materials and the antigen phenotype of neural stem cells derived from bone marrow of Rhesus monkey in the course of their induced differentiation into nerve cells in vitro. Mehtods Under the sterile condition, bone marrow stem cells, derived from bone marrow of Rhesus monkey, were isolated by density gradient method and differentiated into neural stem cells in vitro, followed by evaluating the antigen phenotype of differentiation neuron by immunocytochemical method and detecting the content of monomine materials in culture medium by high performance liquid chromatograpy （HPLC） in different phases. Results No monoamine biological activity materials in nerve stem cells culture medium were detected. However, the contents of Noradrenaline （NE） and Dopamine （DA） were detected in the culture mediums which were cultured for 10, 14 and 30 days by HPLC. Asp analysis of variance showed that the contents of NE and DA in the culture mediums were not significantly different following the elongation of culture time （P〉0.05）. Meanwhile, part of cells presented Nestin, β-tublin NSE, GFAP and TH-positive antigen by immunocytochemical method. Conclusion BMSCs of Rhesus may proliferate and differentiate into the neuron-like cells and even synthesize and excrete neurotransmitters like NE and DA on desirable conditions. Some of them may express nestin, tublin, NSE, GFAP and TH-positive antigen in vitro, indicating that the neural stem cells induced from BMSCs of Rhesus may differentiate to mature nerve cells.