人神经干细胞移植联合环孢素A对小鼠局灶性脑缺血模型的作用  

Protective effect of human neural stem cell transplantation combined with cyclosporine A on focal cerebral ischemia in mice

作　　者：马文超 王超慧 曹凡 刘建国[2] 孙辰婧[2] 王志伟 戚晓昆[2] Ma Wenchao;Wang Chaohui;Cao Fan;Liu Jianguo;Sun Chenjing;Wang Zhiwei;Qi Xiaokun(Naval Clinical College of Anhui Medical University,Hefei 230032,Anhui Province,China)

机构地区：[1]安徽医科大学海军临床学院,合肥230032 [2]解放军总医院第六医学中心神经内科

出　　处：《中国脑血管病杂志》2021年第6期395-404,共10页Chinese Journal of Cerebrovascular Diseases

基　　金：国家重点研发计划(2018YFA0108601)。

摘　　要：目的评估人胚神经干细胞(NSCs)移植在小鼠脑缺血模型中的作用,并探讨其联合使用免疫抑制剂对脑缺血模型疗效的影响。方法选用雄性清洁级C57B6/L小鼠50只,对动物进行标准饲养。造模前,对实验小鼠进行行为学评价,包括转棒实验和角落实验,小鼠在转棒仪上的运动时间≥300 s为完成了转棒实验测试,可纳入进行后续的实验;角落实验共进行10次测试,10次中连续3次未连续向左或右转向的小鼠为完成了角落实验测试,可被纳入进行后续的实验。用线栓法对完成行为学训练的C57B6/L小鼠制作脑缺血模型。造模术后72 h对实验小鼠进行头部MR T2加权成像检查,将梗死体积≥同侧半球20%的小鼠纳入实验。50只C57B6/L小鼠,排除未完成行为学训练2只、造模术后死亡6只、梗死体积<同侧半球20%17只,最终纳入25只小鼠,根据实验需要,将其完全随机分为人胚NSCs移植组(A组,8只)、环孢素A+人胚NSCs移植组(B组,9只)和脑缺血模型对照组(C组,8只)。实验所用的人胚NSCs来自解放军总医院第六医学中心妇产科孕12周自然流产的胎儿脑组织。造模成功后4 d,即人胚NSCs移植前3 d,B组小鼠经腹腔注射环孢素A,剂量为10 mg/(kg·d),连续注射至处死小鼠。造模成功后7 d,经A、B组小鼠脑实质内行立体定位注射移植人胚NSCs,移植位点分别选择缺血侧纹状体及缺血侧大脑皮质。移植后第1、4周,通过转棒实验、角落实验评价不同组小鼠的运动平衡功能。在小鼠造模后3 d及移植后第4周分别对其进行MR T2加权成像检查,并计算小鼠脑梗死体积。行为学评价及影像学评价完成后,灌注断头取脑行免疫荧光染色,以观察植入人胚NSCs的存活数量、分布以及分化情况。stem121抗体与4′,6-二脒基-2-苯基吲哚(DAPI)双阳性的细胞数量代表移植核心区域植入人胚NSCs的存活数量;stem121抗体与微管蛋白Ⅲ抗体作为混合一抗双染,其双阳Objective To evaluate the role of human neural stem cells(NSCs)transplantation in the model of ischemic stroke in mice,and to explore the effect of combined immunosuppressant therapy on ischemic stroke.Methods Fifty clean male C57B6/L mice were used with standard feeding.Before modeling,behavioral evaluation was conducted on the experimental mice,including the rotarod test and corner test.If the movement time of mice on the rotarod test instrument was≥300 s,the rotarod test was completed and the mice could be included in subsequent experiments.A total of 10 tests were conducted in the corner test.Mice that did not turn to the left or right continuously for 3 consecutive times in 10 tests was considered completion of the test and could be included in the subsequent experiments.The ischemic model of C57B6/L mice was made by suture method.Cranial MRI T2-weighted imaging was performed 72 h after modeling,and mice with infarct volume≥20%of ipsilateral hemisphere were included in the experiment.Of the 50 mice,2 mice did not complete the behavioral training,6 mice died after the modeling,and 17 mice had infarction volume<20%of the ipsilateral hemisphere.Finally,25 mice were included.The mice were randomly divided into NSCs transplantation group(group A;n=8),NSCs+cyclosporin A transplantation group(group B;n=9),and cerebral ischemic model control group(group C;n=8).The human embryo NSCs used in the experiment were obtained from fetal brain tissues spontaneously aborted at 12 weeks of gestation in the Department of Obstetrics and Gynecology of the Sixth Medical Center of the People′s Liberation Army(PLA)General Hospital.Mice in group B were intraperitoneally injected with cyclosporin A at a dosage of 10 mg/(kg·d)at 4 days after successful modeling,also 3 days before NSCs transplantation,until the mice were killed.One week after modeling,NSCs of human embryos were injected with stereotactic guidance into the striatum and cerebral cortex of mice brain parenchyma in group A and B.At 1 week and 4 weeks after transplant

关 键 词：神经干细胞 环孢素A 缺血性卒中 立体定位技术 行为学评价 

分 类 号：R743.3[医药卫生—神经病学与精神病学]