诱导时间对体外培养大鼠神经干细胞向多巴胺能神经元分化的影响(英文)

Effects of induction time on dopaminergic neuronal differentiation from rat neural stem cells cultured in vitro

背景：近年来通过筛选不同细胞因子的诱导分化作用发现，某些特定的细胞因子配伍可明显诱导中脑神经干细胞体外定向分化成多巴胺能神经元，研究还发现神经干细胞体外诱导分化的多巴胺能神经元可以有效应用于移植治疗帕金森病，为提高其体内移植的疗效，目前迫切需要深入研究神经干细胞诱导分化的生物学特性。目的：探讨大鼠神经干细胞在分化液中诱导不同时间后，其体外分化成多巴胺能神经元的效应。设计：单一样本观察。单位：中山大学附属第一医院神经外科。材料：实验于2007-05／10在中山大学附属第一医院完成。选择清洁级孕14d的健康SD大鼠6只，体质量350～400g，由中山大学动物实验中心提供（许可证号码SCXK（粤）2007．0034）。实验过程中对动物处置符合动物伦理学标准。方法：①在含表皮生长因子及碱性成纤维细胞生长因子的无血清培养液中培养胚胎大鼠中脑神经干细胞。②经传代扩增后，在含白细胞介素1a、白细胞介素11、白血病抑制因子、胶质细胞源性神经营养因子的诱导分化液中向多巴胺能神经元分化。③诱导分化2，4，6，8，10d后，流式细胞仪检测分化细胞中酪氨酸羟化酶阳性细胞的比值。主要观察指标：①大鼠神经干细胞诱导分化后细胞形态的变化。②诱导不同时间的大鼠神经干细胞分化成酪氨酸羟化酶阳性细胞的比值。结果：在诱导分化液中大鼠中脑神经干细胞球呈贴壁生长，球形结构开始塌陷，球内细胞从球体中央逐渐向四周分化扩展出较多形态各异的细胞，分化6d后，多数细胞已生长出一两个长突起及多个短突起。免疫细胞化学显示分化的细胞中含有酪氨酸羟化酶染色阳性细胞，流式细胞仪检测诱导分化2，4，6，8，10d的分化细胞中酪氨酸羟化酶染色阳性细胞的比例分别为（3．2±0．9）％，（6．8±1．6）％，（16．7±2．6）％，（14．8±1．8）％，（12．2±2．5）％，各组间差异有显著性意义（F=26．449，P〈0．05）。结论：诱导时间对大鼠中脑神经干细胞体外分化成多巴胺能神经元的能力存在影响，诱导6d的神经干细胞分化成多巴胺能神经元的比例最高。

BACKGROUND： Recently, several scientists have found that differentiation of neural stem cells （NSCs） towards dopaminergic neurons may be increased in vitro by combination of some special cytokines. They have also found that dopaminergic neurons differentiated from NSCs can be used for the treatment of Parkinson＇s disease. To improve the therapeutic effects of in vitro transplantation, we should further study the biologic characteristics of NSCs at the induction and differentiation. OBJECTIVE： To explore the differentiation of NSCs which were incubated in differentiation solution for different time towards dopaminergic neurons in vitro. DESIGN： Single sample observation. SETTING： Department of Neurosurgery, First Affiliated Hospital of Sun Yat-sen University. MATERIALS： This study was performed at the Department of Neurosurgery, First Affiliated Hospital of Sun Yat-sen University from May to October 2007. Six healthy Sprague Dawley （SD） rats, gestational age 14 days, of clean grade, weighing 350-400 g, were provided by the Laboratory Animal Center, Sun Yat-sen University[permission No. SCXK （yue）2007-0034]. The protocol was performed in accordance with ethical guidelines for the use and care of animals. METHODS： NSCs derived from rat embryonic mesencephalon were cultured in serum-free culture medium containing epidermal growth factors and basic fibroblast growth factors. After passage, the NSCs were induced to differentiate towards dopaminergic neurons in the differentiation medium supplemented with interleukin lu, interleukinl 1, human leukaemia inhibitory factors, and glial cell line-derived neurotrophic factors. The percentage of tyrosine hydroxylase positive neurons in differentiated cells was detected with flow cytometer when NSCs were cultured in differentiation solution for 2, 4, 6, 8 and 10 days, respectively. MAIN OUTCOME MEASURES： Cellular morphological alteration of rat NSCs after differentiation. The percentage of tyrosine hydroxylase positive neurons in differentiated cells derived from NSCs. RESULTS： In differentiation medium, NSC spheres attached the bottom of plates and began to collapse. Cells inside the spheres grew out gradually and became irregular in shape. Six days later, most of the cells had 1 or 2 long processes and a few short processes. The percentage of tyrosine hydroxylase positive neurons in differentiated cells was respectively （3.2±0.9）%, （6.8±1.6）%, （16.7±2.6）%, （14.8±1.8）% and （12.2±2.5）% after culture for 2, 4, 6, 8 and 10 days, with significant differences （F = 26.449, P 〈 0.05）. CONCLUSION： Induction time influences the differentiation of NSCs towards dopaminergic neurons in vitro. The percentage of dopaminergic neurons is the highest in differentiated cells derived from NSCs which are cultured in differentiation solution for 6 days.