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目的:应用体外细胞培养技术,探讨腹侧中脑神经干细胞的生物学特性及分化后TH、DJ-1和Nurr1的表达情况。方法:体外分离培养新生0~1 d C57BL/6仔鼠腹侧中脑神经干细胞,应用倒置相差显微镜观察中脑神经干细胞的生长状况;通过Brd U掺入实验明确神经干细胞的增殖情况;利用免疫荧光细胞染色法观察子代细胞分化趋势和DA分化相关因子TH、DJ-1和Nurr1的阳性表达情况。结果:在条件培养基的作用下,腹侧中脑神经干细胞呈悬浮的神经球生长,增殖速度较慢,干细胞数量较少;低血清自然分化条件下,中脑神经干细胞更易分化为Tuj-1阳性神经元,且分化为TH阳性神经元的数量增加;DJ-1和Nurr1呈上升趋势,且具有时间依赖性,d7与d3组比较,有显著差异(P<0.05)。结论:结果表明腹侧中脑神经干细胞在增殖和分化方面存在区域特异性,提示腹侧中脑神经干细胞特殊的生物学特性。此研究可为干细胞移植治疗帕金森病的优势供体选择提供有利的探索和理论依据。
OBJECTIVE: To investigate the biological characteristics of ventral midbrain neural stem cells and the expression of TH, DJ-1 and Nurr1 after differentiation in vitro using cell culture technique. Methods: The neural stem cells of ventral midbrain of C57BL / 6 offspring were isolated and cultured in vitro. The growth of neural stem cells in midbrain was observed by inverted phase contrast microscope. The proliferation of neural stem cells was detected by Brd U incorporation assay. Fluorescent staining was used to observe the progeny cell differentiation and DA differentiation related factors TH, DJ-1 and Nurr1 positive expression. RESULTS: Under the condition of conditioned medium, the ventral midbrain neural stem cells grew in the suspended neurospheres with a slower proliferation rate and fewer stem cells. Under the conditions of low serum differentiation, the neural stem cells of the mesencephalic brain were more likely to differentiate into Tuj-1 Positive neurons, and the number differentiated into TH-positive neurons increased. DJ-1 and Nurr1 increased in a time-dependent manner, and there was a significant difference between d7 and d3 (P <0.05). CONCLUSIONS: The results show that there is a regional specificity of ventral midbrain neural stem cells in proliferation and differentiation, suggesting specific biological characteristics of ventral midbrain neural stem cells. This study may provide a favorable exploration and theoretical basis for the selection of the dominant donor of stem cell transplantation for the treatment of Parkinson’s disease.