人羊膜间充质干细胞联合神经生长因子及纤维蛋白胶移植治疗大鼠脑损伤  

Human amniotic membrane-derived mesenchymal stem cells combined with nerve growth factor and biologic fibrin glue transplantation in the treatment of bran injury in rats

作　　者：王辉[1] 关方霞[2] 杨波[1] 齐义军[2] 宋来君[3] 杜英[4] 胡祥 胡炜[1] 焦红亮[1] 李远[1] 

机构地区：[1]郑州大学第一附属医院神经外科,河南省郑州市450052 [2]郑州大学生物工程系,河南省郑州市450001 [3]河南大学医学院生物化学教研室,河南省开封市475100 [4]郑州大学基础医学院微生物与免疫学教研室,河南省郑州市450052 [5]深圳市北科细胞工程研究所,广东省深圳市518000

出　　处：《中国组织工程研究与临床康复》2008年第51期10005-10009,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：河南省医学科技创新人才工程项目(2005018)~~

摘　　要：背景:以往对间充质干细胞分化的报道多为体外培养,甚少涉及宿主体内间充质干细胞的分化及辅助因子对其分化的影响。目的:探讨人羊膜间充质干细胞移植对脑损伤大鼠行为学和空间学习记忆能力的影响,以及神经生长因子、纤维蛋白胶在人羊膜间充质干细胞移植中的作用。设计、时间及地点:细胞学体内对照观察,于2006-07/2007-01在郑州大学医学院完成。材料:正常足月剖腹产胎儿的胎盘由郑州大学第一附属医院妇产科提供。Wistar大鼠90只,随机分为5组:假手术组、模型对照组、细胞移植组、细胞+神经生长因子组、细胞+纤维蛋白胶组,18只/组。方法:无菌条件下钝性分离胎盘脐带面的羊膜,经胰酶消化制成单细胞悬液,贴壁法纯化扩增,传至第3代的人羊膜间充质干细胞备用。假手术组只开颅钻孔不进行硬膜外撞击,其余4组大鼠均采用自由落体硬膜外撞击法建立脑损伤模型。造模1d后,模型对照组在损伤区分点注射生理盐水40μL;细胞移植组注射含1×107个人羊膜间充质干细胞的等量生理盐水;细胞+神经生长因子组在细胞移植组基础上于造模后连续2周每日腹腔注射0.5μg神经生长因子;细胞+纤维蛋白胶组注射人羊膜间充质干细胞与纤维蛋白胶混合液40μL;假手术组不行细胞移植。主要观察指标:行为学评分,Morris水迷宫检测潜伏期,脑组织切片免疫组化染色检测神经元特异性烯醇化酶和胶质纤维酸性蛋白的表达。结果:移植后7d,与模型对照组比较,细胞移植组、细胞+神经生长因子组、细胞+纤维蛋白胶组行为学评分均明显升高,但细胞移植组升高幅度明显低于后2组(F=155.322,P<0.05)。移植后3周,与模型对照组比较,细胞移植组、细胞+神经生长因子组、细胞+纤维蛋白胶组潜伏期均明显缩短,但细胞移植组缩短幅度明显小于后2组(F=22.678,P<0.05)。移植后4周,人羊膜间充质干细BACKGROUND： Previous studies are on in vitro culture of mesenchymal stem cell differentiation. Few studies on mesenchymal stem cell differentiation in vivo or effects of cofactor on their differentiation. OBJECTIVE： To study the amniotic membrane-derived mesenchymal stem cell （AD-MSC） transplantation on behavior, spatial learning and memory in traumatic brain injury rats, and the abilities to nerve growth factor （NGF）, biologic fibrin glue （BFG） in AD-MSC transplantation. DESIGN, TIME AND SETTING： The cytology in vivo controlled study was performed at the Medical College of Zhengzhou University from July 2006 to January 2007. MATERIALS： The placenta from healthy normal full-term fetus was obtained from Department of Gynaecology and Obstetrics, First Affiliated Hospital, Zhengzhou University. A total of 90 Wistar rats were equally and randomly assigned into a sham operation group, a model control group, a cell transplantation group, a cell ＋ NGF group, a cell ＋ BFG group. METHODS： Fetal amniotic membrane was harvested from the placenta by blunt dissection under a sterile condition, made into monoplast suspension by trypsinization, and purified by adherece. At the third passage, AD-MSCs were used. Rats in the sham operation group underwent perforation. Rats in other groups were established into models of traumatic brain injury using free-falling epidural impact method. At 1 day following model induction, 40 u L saline was infused into injury sites in the model control group. An equal volume of saline containing 1×10^7 AD-MSCs were injected into rats in the cell transplantation group. Rats in the cell ＋ NGF group were injected with 1×10^7 AD-MSCs and 0.5 u g NGF for successively for 2 weeks. Rats in the cell ＋ BFG group were infused with AD-MSCs and 40 u L BFG. Rats in the sham operation group did not receive cell transplantation. MAIN OUTCOME MEASURES： Ethology score; Latency was measured using Morris water maze test. Neuron specific enolasc and glial fibrillary acidic protein expr

关 键 词：羊膜间充质干细胞 移植 神经生长因子 纤维蛋白胶 

分 类 号：R394.2[医药卫生—医学遗传学]