课题背景：新生儿缺氧缺血性脑病是导致新生儿死亡和神经系统后遗症的重要原因，已成为围生期神经病学的一个重要问题。课题拟以人胎脑神经干细胞移植至脑损伤新生鼠，观察其效果。

应用要点：胞浆标记物PKH-26是一种亲脂性染料，实验中用其作为人神经干细胞的示踪剂，可以稳定的整合入细胞膜，不会干扰细胞膜表面标记物的表达，因此能够观察神经干细胞的存活和迁移情况。

偏倚或不足：①因PKH-26属于亲脂性染料，用石蜡切片包埋动物脑组织时，在脱蜡过程中依次使用无水乙醇、体积分数为0.95的乙醇、体积分数为0.80的乙醇会洗掉细胞膜上的PKH，故只有使用冰冻切片才能在荧光显微镜下看到PKH阳性的移植细胞。②移植后2周，移植细胞并不能完全穿越脑室管膜进入脑实质，一部分会在脑室内停留，其是否会拥堵在中脑导水管导致移植失败或继发性脑积水还需在后续实验中深入分析。

摘要
目的：新生儿缺氧缺血性脑病是导致脑性瘫痪的重要原因，至今缺乏有效疗法。将体外培养的人神经干细胞经脑室移植入缺氧缺血性脑损伤新生鼠，观察植入细胞在宿主脑内的存活、迁移及分化。
方法：实验于2005-01/09在解放军海军总医院儿科实验室完成。①对象：神经干细胞来源于孕12周流产的人胎儿脑组织，孕妇签署知情同意书，符合医院伦理委员会规定。清洁级SD新生鼠80只，随机数字表法分为细胞移植组、模型对照组，40只/组，实验过程中对动物的处置符合动物伦理学标准。②实验方法：取人胚胎脑组织，机械分散法分离单个核细胞，接种于添加表皮生长因子、碱性成纤维细胞生长因子、白血病抑制因子的N2培养基中，获取生长旺盛的人神经干细胞球，制成单细胞悬液，浓度约为5.0×1011 L-1，培养6 d后行PKH标记用于植入后示踪。两组新生鼠均建立缺氧缺血性脑损伤模型，造模后3 d，细胞移植组损伤侧脑室缓慢注入5 μL人神经干细胞悬液，模型对照组于相同部位注入等量生理盐水。③实验评估：取未经PKH标记的细胞球， 通过免疫细胞化学染色鉴定巢蛋白的表达及其向神经元、星形胶质细胞的分化情况。分别于细胞移植后1，2，4周及3个月，常规取脑组织，行免疫组织化学和荧光分析，观察植入后细胞存活及分布情况。
结果：在造模及细胞移植过程中，因麻醉、出血细胞移植组新生鼠死亡5只，模型对照组死亡7只，存活率85%~90%。①神经干细胞的鉴定及分化：80％活细胞巢蛋白呈阳性表达，并可分化为神经元、星形胶质细胞。②神经干细胞植入后存活及分布情况：植入后1周，神经丝蛋白阳性细胞多位于损伤侧皮质及海马处，纹状体、脑干、小脑、嗅球也有少量分布。植入后2周，海马和皮质可见神经丝蛋白阳性细胞，胞体伸出的神经微丝更长，细胞数量与1周时基本相似。植入后4周及3个月时PKH阳性细胞数量明显减少。
结论：在含有表皮生长因子、碱性成纤维细胞生长因子、白血病抑制因子的N2培养基中形成的人神经干细胞球，具有良好的增殖能力，可分化为神经元，移植至缺血缺氧新生鼠脑中能够向损伤区迁移，分布范围广。
关键词：神经干细胞；移植；缺氧缺血；新生鼠

王桂芬，栾佐，高宝勤，尹国才，白洁，屈素清.人神经干细胞移植到缺氧缺血性脑损伤新生大鼠脑内的存活及分布[J].中国组织工程研究与临床康复，2008，12(12):2201-2205 [www.zglckf.com/zglckf/ejournal/upfiles/08-12/12k-2201(ps).pdf]

Survival and distribution of human neural stem cells transplanted into cerebral ventricles of neonatal rats with hypoxic-ischemic brain damage

Abstract

AIM： Neonatal hypoxic-ischemic encephalopathy (HIE) is an important cause of cerebral palsy. There is no effective therapy for HIE. This study investigated the survival, migration and differentiation of in vitro cultured human neural stem cells (NSCs) following transplantation into the cerebral ventricles of neonatal rats with hypoxic-ischemic brain damage (HIBD).

METHODS: Experiments were performed at the Laboratory of Department of Paediatrics of Navy General Hospital of Chinese PLA from January to September 2005. ①NSCs were harvested from the brain of a 12-week old abored human fetus. The pregnant woman signed an informed consent. Experiments were accorded with the rules of Hospital’s Ethics Committee. Eighty Sprague-Dawley neonatal rats were equally divided into a NSC transplantation group and a model control group by the random digits table method. Animal interventions in the experiment were accorded with the Animal Ethical Standards. ②Mononuclear cells were collected from human fetal brain tissues by mechanical suspension dispersion and inoculated in the N2 medium containing epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF). Human NSC spheres were prepared into single cell suspension at approximately 5.0×1011 L-1. After 6-days culture, NSCs were labeled with PKH. Neonatal rat models of HIBD were established in both groups. Three days later, 5 μL human NSC suspension was injected into the cerebral ventricle in the NSC transplantation group, and 5 μL saline was injected into the same region in the model control group. ③Nestin expression and differentiation into neurons and astrocytes in human NSC spheres labeled without PKH were identified by immunocytochemistry. At 1, 2, 4 weeks and 3 months after transplantation, the brains of rats were collected and examined by immunohistochemistry and immunofluorescence analysis to determine the survival and distribution of NSCs.

RESULTS: Five neonatal rats in the NSC transplantation group and seven in the model control group died of anesthesia and bleeding during model establishment and cell transplantation, resulting in a survival rate of 85%-90%. ①Identification and differentiation of NSCs: Eighty percent of living cells were positive for Nestin and could differentiate into neurons and astrocytes. ②Survival and distribution of NSCs after transplantation: At 1 week after transplantation, neurofilament positive cells were mainly distributed in the hippocampus and in the cerebral cortex of the injured side, and a few cells were distributed in the striatum, brain stem, cerebellum and olfactory bulb. Compared to that at 1 week, a similar number of neurofilament positive cells and longer neurofilament were observed at 2 weeks.The number of PKH labeled cells was significantly decreased at 4 weeks and 3 months after transplantation.

CONCLUSION: Human NSC spheres cultured in N2 medium with EGF, bFGF and LIF possess a high reproductive activity and can differentiate into neurons. Human NSCs can migrate extensively into injured regions followed transplantation into the brains of neonatal rats with HIBD.

Wang GF, Luan Z, Gao BQ, Yin GC, Bai J, Qu SQ.Survival and distribution of human neural stem cells transplanted into cerebral ventricles of neonatal rats with hypoxic-ischemic brain damage.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(12):2201-2205(China) [www.zglckf.com/zglckf/ejournal/upfiles/08-12/12k-2201(ps).pdf]