体外培养中脑神经干细胞的生物学特性鉴定及分化后TH、DJ-1和Nurr1的表达

The neurotrophic property of midbrain neural stem cells of postnatal mouse in vitro and the expression of TH,DJ-1 and Nurr1 after conditioned differentiation

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0～1dC57BL／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BrdU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 neurotrophic property of midbrain neural stem cells （Mid-NSCs） and the posi- tive expression of TH, DJ-1 and Nurrl of Mid-NSCs after conditioned differentiation in vitro. Methods： Mid-NSCs isola- ted from newborn C57BL/6 mice were cultured in vitro and cells growth observed by phase contrast microscope. Mixed with BrdU in culture medium, the proliferation of Mid-NSCs was identified by immunocytochemistry. The differentiation tendency and expression levels of TH, DJ-1 and Nurrl with related DA differentiation were examined after conditioned in- duction by immunofluorescent staining. Results： Under the function of conditioned cultural medium, ventral Mid-NSCs grow in suspension of neurospheres, with lower speed and fewer stem cells. In the natural differentiation of low serum, Mid-NSCs might be more likely to differentiate into Tuj-1-positive neurons, with increased number of TH-positive neu-rons. Meanwhile, there appeared the uptrend of DJ-1 and Nurrl, expression, and Nurrl with the time dependence, and the comparison between d7 group and d3 group showed the remarkable difference （P 〈 0.05）. Conclusion： The result showed Mid-NSCs in vitro had the region specificity in aspects of proliferation and differentiation, which indicated the spe- cial biological characteristics of Mid-NSCs. The study provides the advantageous theoretical basis in choosing superiority donors for stem cell transplantation in the treatment of Parkinson＇s diseases.