课题背景：在解放军海军总医院栾佐主任带领下，课题组在前期基础研究成果上结合神经干细胞移植治疗新生儿缺氧缺血性脑损伤临床工作的实际需要而开展的，主要研究高压氧对缺氧缺血性新生大鼠外源性神经干细胞移植的影响，其内容包括高压氧对植入细胞存活、分化的影响，并从不同方面探讨其作用机制。从目前的研究成果看，高压氧可能有助于植入细胞的存活和分化。

创新要点：研究表明高压氧具有保护和促进内源性神经干细胞增殖的作用，还未见高压氧对移植后外源性神经干细胞作用的报道，本课题具有实用性和创新性。

偏倚或不足：①实验仅从移植后宿主脑组织神经元病理改变的角度来阐释高压氧对神经干细胞移植的影响，需进一步论证说明相关数据的统计学分析结果。②如能在此实验基础上增加模型组作对比，可更加体现单纯细胞移植及其联合高压氧治疗的效果差异。③实验只观察了短期疗效，如能设立多个不同观察时间点则更加完善。

摘要
目的：研究证明高压氧治疗能明显减轻缺氧后宿主脑区深部水肿，减少内源性神经元变性与坏死，并增加围产期鼠新生神经元数量与碱性成纤维生长因子活性。实验拟进一步观察缺氧缺血性脑损伤大鼠移植人神经干细胞后予高压氧治疗对脑组织神经元病理改变的影响。
方法：实验于2007-04在解放军海军总医院完成。①细胞来源及动物：经医院伦理委员会授权，孕妇知情同意下留取孕12周流产的人胎儿脑组织。新生7 d龄SD大鼠20只，购自北京维通利华实验动物技术有限公司，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：取胎儿脑组织，在无菌条件下培养扩增并制备成人神经干细胞单细胞悬液。20只大鼠均建立缺氧缺血性脑损伤模型，3只死亡，剩余鼠随机数字表法分为2组，细胞移植+高压氧组9只，单纯细胞移植组8只。造模后3 d，两组大鼠均于左侧侧脑室进行细胞移植，注射位点以前囟为参照点，AP= -1 mm，ML= -1.5 mm，DV= -4.0 mm，缓慢注入2×106 L-1细胞悬液5 μL。移植后1 h，将细胞移植+高压氧组大鼠放入高压氧舱内，给予高压氧通气，升压及降压过程各15 min，最终达压力为1.8个绝对大气压，稳压60 min，1次/d，连续10 d。两组大鼠麻醉后断头取脑，制备组织切片。③实验评估：免疫荧光法检测神经干细胞巢蛋白的表达、人神经干细胞植入后神经丝蛋白表达及其向神经元分化情况。尼氏染色检测移植后脑组织皮质、海马神经元形态、结构的变化。
结果：移植过程中细胞移植+高压氧组有1只鼠因麻醉死亡，高压氧通气过程中没有动物死亡。①人神经干细胞的鉴定：人胎儿脑组织体外培养12 d获取生长旺盛的人神经干细胞小集落，即神经球。倒置显微镜下观察可见每个小集落为数十个细胞构成，细胞折光性强，周边有清晰的光晕。免疫荧光标记大部分活细胞表达巢蛋白。②人神经干细胞植入后神经丝蛋白表达及其向神经元分化：植入后10 d，两组在皮质及海马区均可见神经丝蛋白阳性细胞，即植入细胞分化形成的神经元，其细胞形态与宿主内源性细胞相似。③神经元尼氏染色：两组大鼠均没有发现肿瘤形成。移植后10 d与单纯细胞移植组相比，细胞移植+高压氧组海马及皮质区组织细胞水肿程度明显减轻，且海马CA1区、CA3区、齿状回神经元排列更整齐，组织结构更完整。
结论：缺氧缺血性脑损伤新生大鼠移植人神经干细胞后，高压氧治疗可减轻损伤易感区组织细胞水肿程度，并使海马区神经元结构更加完整，推测有可能促进细胞的成活、迁移及分化。
关键词：神经干细胞移植；高压氧；缺氧缺血性脑损伤；尼氏染色

白洁，栾佐，汪兆艳.高压氧对外源性人神经干细胞移植治疗损伤脑组织神经元病理状态的改善效应[J].中国组织工程研究与临床康复，2008，12(8):1401-1405 [www.zglckf.com/zglckf/ejournal/upfiles/08-8/8k-1401(ps).pdf]

Effect of hyperbaric oxygenation on neuronal pathological improvement after exogenous human neural stem cell transplantation for treating brain damage

Abstract

AIM：Research has proved that hyperbaric oxygenation (HBO) therapy is good for relieving brain edema after hypoxia attack, and helpful of reducing endogenetic neurons degeneration and death, increasing the amount of regenerated neurons and basic fibroblast growth factor activity of perinatal rats. Therefore, this study investigated the effect of HBO on neuronal pathological changes after human neural stem cells (hNSCs) transplantation for neonatal rats with hypoxic-ischemic brain damage (HIBD).
METHODS: This experiment was carried out in April 2007 at Naval General Hospital of Chinese PLA.①Cell resource and animals: Authorized by the Ethics Committee of the hospital, and with the informed consent of gravida, we obtained the fetal brain tissue from 12-week abortus. Twenty 7-day-old SD rats were bought from Beijing Weitong Lihua Experimental Animal Technological Company. Any manipulation of the animal was consistent with the standard of Ethics.②Methods: Human fetal brain tissues were obtained to make NSCs single cell suspension under sterile condition. Twenty neonatal rats were used for HIBD model, 3 died during procedure. Rest of them divided randomly into two groups: transplantation+HBO group (n=9) and transplantation group (n=8). The rats received hNSCs implantation on 3 days after modeling. Coordinates as follows: AP= -1 mm, ML=-1.5 mm, DV=-4.0 mm, 5 μL hNSCs suspension (2×106 L-1 concentration) was implanted into the left cerebral ventricle gradually. The rats in transplantation+HBO group were administered HBO treatment (1.8 atmosphaera absolutus, 15 minutes procedure for boosting pressure and underpressure, 1 hour daily for 10 days) 1 hour after hNSCs transplantation. Brains were removed following decapitation to prepare sections for further use.③Evaluation: Immunofluorescence analysis for detecting the survival and neurons differentiation of the transplanted hNSCs by Nestin and neurofilament (NF) expression as well as Nissl staining for neuronal pathological changes in cortex and hippocampus.
RESULTS: One rat of implantation+HBO group died during the implantation procedure, and no death was found during HBO therapy.①Identification of hNSCs: After cell culture in vitro for 12 days, the eugonic neurospheres with high refraction and clear halation peripherally were gained under inverted microscopy. Most of them expressed Nestin by immunofluorescence.②NF expression and neurons differentiation after implantation: Ten days after implantation, NF-positive cells, that was the differentiated neurons could be detected in cortex and hippocampus of both groups, with similar form of endogenetic cells.③Nissl staining of neurons: Tumor was observed in neither group. Ten days after implantation, cell and tissue edema was much relieved in hippocampus and cortex area, and neurons were well-arranged and intact in CA1, CA3 and gyrus dentatus in hippocampus of transplantation+HBO group compared with those in transplantation group.
CONCLUSION: HBO therapy can relieve edema in hippocampus and cortex, and improve neurons arrangement in hippocampus after hNSCs transplantation for neonatal rats with HIBD. Therefore, HBO therapy is probably helpful for survival, migration and neurons differentiation after hNSCs implantation.

Bai J, Luan Z, Wang ZY.Effect of hyperbaric oxygenation on neuronal pathological improvement after exogenous human neural stem cell transplantation for treating brain damage.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(8):1401-1405(China) [www.zglckf.com/zglckf/ejournal/upfiles/08-8/8k-1401(ps).pdf]