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  

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

作　　者：白洁[1] 栾佐[2] 汪兆艳[2] 

机构地区：[1]北京大学第一医院儿科,北京市100034 [2]解放军海军总医院儿科,北京市100037

出　　处：《中国组织工程研究与临床康复》2008年第8期1401-1405,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

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7d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3d,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=-1mm,ML=-1.5mm,DV=-4.0mm,缓慢注入2×106L-1细胞悬液5μL。移植后1h,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15min,最终达压力为1.8个绝对大气压,稳压60min,1次/d,连续10d。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12d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�AIM： Research has proved that hyperbaric oxygenation （I-IBO） 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 carded 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＋I-IBO 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 μ L hNSCs suspension （2× 10^6 L^-1 concentration） was implanted into the left cerebral ventricle gradually. The rats in transplantation＋HBO group were administered I-IBO 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 ne

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 

分 类 号：R394.2[医药卫生—医学遗传学]