人脐血间充质干细胞移植与神经节苷脂注射治疗脑瘫大鼠的对比研究

目的：建立稳定的人脐血间充质干细胞（mesenchymal stem cells，MSCs）分离培养体系，观察其移植对脑瘫(CP)大鼠功能恢复的影响及细胞的存活、迁移、向神经细胞分化的情况，并与神经节苷脂（GM1）注射对CP鼠神经功能恢复进行对比。方法：采集足月新生儿脐带血100ml，分离出单个核细胞，体外培养并予5 溴脱氧嘧啶尿苷（Brdu）标记72h；受孕17d孕鼠腹腔注射脂多糖（LPS）0.4mg/(kg·d)，12h后置于缺氧环境2～2.5h，4h后再次腹腔注射同剂量LPS，孕鼠自体分娩，生后4周，通过行为学评分选择CP模型动物80只，随机分为4组：CP组（CP模型）、假移植组（CP模型+PBS）、MSCs移植组（CP模型+MSCs）、GM1注射组（CP模型+GM1），每组20只。移植后第1、2、3周应用免疫荧光法观察Brdu标记的MSCs的存活、迁移，移植后第4周，通过行为学评价观察大鼠神经功能恢复情况，应用免疫荧光法检测其胶质纤维酸性蛋白(GFAP)和神经元特异性烯醇化酶（NSE）的表达情况。结果：MSCs移植比GM1注射使大鼠握持时间更长，足错误次数更少(P<0.05)；移植的MSCs可在大鼠脑组织中存活, 并向四周脑组织迁移, (0.45±0.68)个MSCs表达GFAP,(0.15±0.36)个MSCs表达NSE。结论：人脐血MSCs移植比神经节苷脂注射较好的提高CP大鼠神经功能的恢复, 移植细胞可在大鼠脑缺血区域中存活、迁移，并部分向星形胶质细胞或神经元分化。

Comparative study on transplantation of human umbilical cord-derived mesenchymal stem cells vs. injection of gangliosides for cerebral palsy in rats

Objective: To establish the isolation and steady cultivation system of mesenchymal stem cells (MSCs) derived from human umbilical cord blood and observe the neurological functional recovery after transplanting MSCs, and detect the survival time, migration and neural differentiation of MSCs; to compare the neurological functional recovery between MSCs transplantation vs. injection of gangliosides (GM1). Methods: The mononuclear cells isolated from 100 mL of cord blood of full-term babies were cultured in vitro and labeled with 5-bromodeoxyuridine (Brdu) for 72 h; pregnant rats of 17 days were injected with LPS 0.4 mg/(kg.day) intraperitoneally. After 12 h, the rats were placed in the oxygen-free environment for 2-2.5 h. Four h later, the rats were given LPS once again. Eighty 4-week-old new born cerebral palsy (CP) rats were chosen by behavioral evaluation. The rats were randomly divided into 4 groups: CP group (model of CP), false transplantation group (transplantation of PBS after CP), MSCs transplantation group (transplantation of MSCs after CP), GM1 group (injection of GM1 after CP). At first, 2nd, and 4th week after transplantation, immumofluorescence method was used to check the survival, migration and differentiation of MSCs labeled with Brdu after transplantation of MSCs. Neurological functional recovery was observed according to the evaluation of behaviors at the forth week, and the expression of glial fibriliary acidic protein (GFAP) and neuron-specific enolase (NSE) in BrdU-labeled MSCs was detected by immumofluorescence method. Results: In MSCs transplantation group, the length of holding time was longer, while the errors of feet were less than in GW1 group (P<0.05). The transplanted MSCs could survive in the brain and migrate to the surrounding brain tissue. About (0.45±0.68) MSCs expressed GFAP and (0.15±0.68) MSCs expressed NSE. Conclusion: For the transplantation of MSCs in rats, the neurological functional recovery was better than the injection of GM1 (P<0.05). The transplanted MSCs could survive in the brain, migrate to the surrounding brain tissue and differentiate into astrocytes or neurons.