应用要点：①实验通过与胚胎中脑多巴胺能神经元组织体内移植疗效的比较，探讨神经干细胞诱导分化的多巴胺能神经元移植治疗帕金森病大鼠的作用，目前国内外相关的研究还很少。②实验结果表明，神经干细胞诱导分化的多巴胺能神经元与胚胎中脑多巴胺能神经元移植治疗帕金森病大鼠具有相同的疗效。③实验为进一步开展神经干细胞诱导分化的多巴胺能神经元移植治疗帕金森病提供了实验基础及理论依据。

同行评价：神经干细胞具有分化为神经元和胶质细胞的潜能，相关的基础研究为临床治疗神经系统疾病带来新希望。文章应用神经干细胞体外诱导分化的多巴胺能神经元移植治疗帕金森病大鼠，立项依据充分，内容新颖，有学术创新和临床应用前景。如能明确移植后胚胎中脑细胞存活率及诱导分化的多巴胺能神经细胞移植存活率则更有说服力。

相关链接：随着神经干细胞技术的飞速发展，来源于胚胎和成体神经干细胞的成功分离、纯化、培养、传代、建立细胞系以及体外定向诱导分化等技术的成熟，中脑神经干细胞在体外特定细胞因子的作用下可定向诱导分化成多巴胺能神经元。应用体外诱导分化的多巴胺能神经元替代胚胎中脑多巴胺能神经元组织移植治疗帕金森病，既解决了胎脑移植数量不足的问题，又避免了伦理学方面的争论，目前已成为帕金森病治疗研究的热点。

摘要
目的：目前对于体外诱导分化的多巴胺能神经元的生物学功能、体内存活状况，以及与胚胎中脑多巴胺能神经元移植治疗帕金森病大鼠的疗效比较方面尚缺乏深入的研究。观察神经干细胞诱导分化的多巴胺能神经元移植治疗帕金森病大鼠的作用，并与胚胎中脑多巴胺能神经元组织移植治疗帕金森病大鼠的疗效进行比较。
方法：实验于2006-06/2007-09在中山大学附属第一医院完成。①实验材料：健康成年雄性SD大鼠及孕14~15 d的SD大鼠由中山大学动物实验中心提供，实验过程中对动物处置符合动物伦理学标准。②实验方法：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。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，并进行免疫细胞化学鉴定和流式细胞仪检测。参考Sauer和Lee等方法制备帕金森病大鼠模型，并将造模成功大鼠随机分组，每组9只：分化细胞组向每一坐标点注入1×1011 L-1神经干细胞诱导分化的多巴胺能神经元；胚胎中脑组注入 1×1011 L -1胚胎中脑多巴胺能神经元；手术对照组注入DMEM/F12细胞培养液。③实验评估：移植术后10、20、40、60 d诱发大鼠不对称旋转行为以观察治疗效果，并对移植区进行酪氨酸羟化酶免疫组织化学检测以了解移植细胞体内存活的情况。
结果：①大鼠神经干细胞球在诱导分化液中呈贴壁生长，球内细胞从球体中央逐渐向四周分化扩展出形态各异的细胞。免疫细胞化学染色显示，分化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6 d的细胞中酪氨酸羟化酶染色阳性细胞的比率为(16.7±2.8)%。②移植后20 d分化细胞组大鼠的不对称旋转行为开始明显下降（P < 0.05）。移植后40 d和60 d，分化细胞组大鼠的旋转圈数与胚胎中脑组比较差异不显著（P > 0.05）。③分化细胞组和胚胎中脑组大鼠纹状体移植区有酪氨酸羟化酶染色阳性细胞，多数细胞位于移植针道边缘。
结论：神经干细胞诱导分化的多巴胺能神经元与胚胎中脑多巴胺能神经元移植治疗帕金森病大鼠具有相同的疗效。
关键词：帕金森病；神经干细胞；多巴胺能神经元；神经移植；细胞移植

柯春龙，陈白莉，金华伟，郭少雷.神经干细胞诱导分化为多巴胺能神经元与胚胎中脑多巴胺能神经元移植治疗帕金森病效果比较[J].中国组织工程研究与临床康复，2008，12(16):3019-3023
[www.zglckf.com/zglckf/ejournal/upfiles/08-16/16k-3019(ps).pdf]

Transplantation of dopaminergic neurons differentiated from neural stem cells versus embryonic mesencephalic dopaminergic neurons for treating Parkinson’s disease

Abstract

AIM：Biological function and in vivo survival of differentiated dopaminergic neurons (DNs), and comparison to the transplantation of embryonic mesencephalic DNs for treating Parkinson’s disease rats are seldom studied. This study investigated the effect of transplantation of dopaminergic neurons differentiated from neural stem cells (NSCs) into Parkinson’s disease rats, and compared to the transplantation of embryonic mesencephalic DNs.
METHODS: Experiments were performed at the First Affiliated Hospital of Sun Yat-sen University between June 2006 and September 2007. ①Healthy adult male SD rats and pregnant 14-15 day SD rats were provided by Animal Experiment Center of Sun Yat-sen University. Animal intervention met the animal ethical standards.②NSCs derived from embryonic rats were cultivated in serum-free culture solution containing epidermal growth factor and basic fibroblast growth factor. The cells were passaged and differentiated to DNs in differentiate solution containing interleukin (IL) 1a, IL11, leukemia inhibitory factor (LIF) and glial cell line-derived neurotrophic factor (GDNF). The differentiated cells were detected by immunocytochemistry and flow cytometry. Rat models of Parkinson’s disease were established according to the methods of Sauer and Lee et al. Successful models were randomly divided into 3 groups, 9 in each group. Differentiated DNs (1×1011 L-1) were injected into rats of differentiated DNs group. Embryonic mesencephalic DNs (1×1011 L-1) were injected into rats of embryonic mesencephalic DNs group. DMEM/F12 medium was injected into rats of control group. ③The rotational asymmetry of Parkinson’s disease rats was observed 10, 20, 40 and 60 days after the surgery to assess the effect. In transplanted areas, Tyrosine Hydroxylase (TH) was detected with immunocytochemistry to assess the survival of transplanted cells in vivo.
RESULTS: ①In differentiate solution, the rat neural stem cell globes became attaching the dish, cells inside the globes grew out gradually and became irregular in shape. There were some TH positive cells in differentiated cells when detected with immunocytochemistry. The ratio of TH positive cells in cells differentiated for 6 days was (16.7±2.8)% when detected by flow cytometry. ②The rotational asymmetry was decreased obviously 20 days after transplantation of differentiated DNs into Parkinson’s disease rats (P < 0.05). 40 and 60 days after the surgery, the rotational frequencies had no significant difference between differentiated DNs group and embryonic mesencephalic DNs group（P > 0.05）. ③There were TH positive cells in the transplanted areas of Parkinson’s disease rats of differentiated DNs group and embryonic mesencephalic DNs group, most of them located in the margin of transplanted pin hole.
CONCLUSION: When grafted into the striatum of Parkinson’s disease rats, DNs differentiated from NSCs had the same effect as embryonic mesencephalic DNs.

Ke CL, Chen BL, Jin HW, Guo SL.Transplantation of dopaminergic neurons differentiated from neural stem cells versus embryonic mesencephalic dopaminergic neurons for treating Parkinson’s disease.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(16):3019-3023(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-16/16k-3019(ps).pdf]