抗坏血酸诱导新生鼠神经干细胞向多巴胺能神经元的分化

Differentiation into dopaminergic neurons from neonatal rat neural stem cells induced by ascorbic acid

背景：针对帕金森病进行细胞移植治疗的研究进程中，除寻求合适的细胞系外，如何将各种有分化潜能的细胞诱导为含量丰富、有功能的多巴胺能神经元尤为关键。目的：观察不同浓度抗坏血酸对神经干细胞向多巴胺能神经元分化的影响。设计、时间及地点：细胞学体外观察，于2007-01／12在广西医科大学科学实验中心完成。材料：清洁级新生24h内SD大鼠30只，由广西医科大学实验动物中心提供。抗坏血酸为Sigma产品。方法：取新生鼠脑组织，组织块胰蛋白酶消化法体外分离培养神经干细胞，传至第2代以5×10^8L^-1密度接种。设立4组：空白对照组不给予抗坏血酸，仅加入含体积分数为10％的胎牛血清、2％B27的DMEM／F12培养基：剩余3组在此基础上分别加入50，100，200μmol／L抗坏血酸进行诱导，10d后终止诱导。主要观察指标：RT-PCR检测诱导后细胞酪氨酸羟化酶基因mRNA的表达，免疫细胞化学染色鉴定神经干细胞，检测神经干细胞向多巴胺能神经元分化率。结果：各浓度抗坏血酸诱导组均得到795bp的扩增片断，即均表达酪氨酸羟化酶mRNA。神经球具有自我更新和表达巢蛋白的能力，诱导分化后的细胞能表达神经元、星形胶质细胞及少突胶质细胞特异性抗原。与空白对照组比较，50，100，200μmol／L抗坏血酸诱导组酪氨酸羟化酶阳性细胞率均明显升高（P〈0．05）；且100，200μmol／L抗坏血酸诱导组升高幅度明显高于50μmol／L（P〈0．05），100，200μmol／L抗坏血酸诱导组间比较无明显差异（P〉0．05）。结论：从新生鼠脑组织分离出的神经干细胞在体外具有自我更新、多向分化潜能及表达巢蛋白的能力。50～200μmol／L抗坏血酸均能促进神经干细胞向多巴胺能神经元分化，抗坏血酸浓度从100μmol／L增加至200μmol／L时酪氨酸羟化酶阳性细胞率无明显变化。

BACKGROUND： Studies regarding cell transplantation for Parkinson＇s disease focuses on searching appropriate cell lines and induction of abundant functional dopaminergic （DA） neuron from cells with differentiation potential. OBJECTIVE： To investigate the effects of ascorbic acid （AA） on the differentiation of neural stem cells （NSCs） into DA neurons in vitro. DESIGN, TIME AND SETTING： In vitro observation of cytology. The experiment was performed at the Laboratory Center of Guangxi Medical University from January to December 2007. MATERIALS： A total of 30 Sprague Dawley rats （within 24 hours after birth） were provided by the Experimental Animal Center of Guangxi Medical University. AA was purchased from Sigma, USA. METHODS： The primary NSCs were isolated from whole brain of rats, suspended and cultured in serum-free medium in vitro at a density of 5×10^8/L （the second passage）. Control cells were not given AA but only cultured in DMEM/F12 containing 10% fetal bovine serum and 2% B27; other cells were additionally induced with 50, 100, 200μmol/L for 10 days. MAIN OUTCOME MEASURES： Tyrosine hydroxylase （TH） mRNA was detected by RT-PCR; NSCs and differentiation from NSCs to DA neurons were detected by immunocytochemistry. RESULTS： Three AA groups had 795 bp amplified fragment, i.e. expressing TH mRNA. The neurospheres could proliferate and passage in vitro. Immunocytochemistry study indicated that the neurospheres were nestin-positive, had the capacity of self-renewing and proliferation, and could differentiate into multi-directions, specific antigens of neurons, astrocytes, and oligodendrocytes were expressed also. Compared with blank control group, AA increased the proportion of TH-positive cells （P 〈 0.05）, in particular 100 and 200μmol/L was significantly higher than 50 μmol/L （P 〈 0.05）, while no significant difference was found between 100 and 200 μmol/L AA （P 〉 0.05）. CONCLUSION： The NSCs isolated from neonatal rats show the capacity of self-renewing and proliferation, multi-differentiation, and expressing nestin antigen in vitro. AA （50-200 μmol/L） can promote NSCs differentiate into DA neurons, and TH positive rate minimally changes when AA increases from 100 to 200μmol/L.