脐带血间充质干细胞移植治疗缺氧缺血性脑损伤新生大鼠的疗效  

Effect of Umbilical Cord Blood Mesenchymal Stem Cell Seeding Treatment for Neonatal Rats with Hypoxic-Ischemic Brain Damage

作　　者：李湘云[1] 胡博[1] 李建斌[2] 单泓[2] 赵林娜[2] 段艳丽[2] 韩小改[2] 杨鹏辉[2] 

机构地区：[1]郑州大学第三附属医院脑瘫康复中心,郑州450052 [2]河南省红十字血液中心,郑州450052

出　　处：《实用儿科临床杂志》2010年第8期590-592,共3页Journal of Applied Clinical Pediatrics

摘　　要：目的探讨混合培养的人脐血间充质干细胞(MSCs)对缺氧缺血性脑损伤(HIBD)新生大鼠的疗效。方法无菌条件下收集20份健康足月新生儿脐血,采用密度梯度离心法分离出脐血单个核细胞,再通过贴壁细胞培养法培养出人脐血MSCs。当细胞传至第3代时,随机取培养的8份脐血MSCs(半量)为A组,8份脐血MSCs(半量)为B组,将A、B组剩余的半量脐血MSCs混合为C组,3组细胞密度均为1×106L-1。混合培养的第3代脐血MSCs用流式细胞仪检测细胞表面抗原CD29、CD34、CD44、CD105的表达。选择P4代人脐血MSCs作为移植细胞,并经5-溴-2-尿嘧啶脱氧核苷(BrdU)体外标记。7日龄SD大鼠45只制备HIBD模型,造模中死亡3只,余42只分为混合移植组(n=16)、单份移植组(n=16)和对照组(n=10)。造模第3天,在立体定位条件下,移植组大鼠经左侧大脑皮质注入人脐血MSCs,对照组在相同部位注入相同体积的PBS。在细胞移植第7天,随机选择6只移植组大鼠处死取脑,采用免疫组织化学方法观察移植细胞在脑内的存活、迁移和分化情况。移植第1、7、14、21、28天采用改良神经功能损害评分(mNSS)对3组大鼠的神经功能进行评价。结果活细胞计数显示,C组细胞增殖较A组、B组快(Pa<0.05)。移植组大鼠脑组织免疫组织化学检测发现移植细胞在脑内能够存活,并以移植点为中心向周围迁移;混合移植组BrdU阳性细胞数多于单份移植组(P<0.05)。移植第1天,移植组大鼠mNSS低于对照组,但差异无统计学意义(P>0.05);第7、14、21、28天,移植组大鼠mNSS低于对照组,差异有统计学意义(P<0.05);第7、14、21、28天,混合移植组大鼠mNSS低于单份移植组,差异有统计学意义(P<0.05)。结论混合培养的人脐血MSCs脑移植对HIBD新生大鼠具有较好的治疗作用。Objective To assess the effect of human umbilical cord blood mesenchymal stem cells （MSCs） by mixed culture of intracerebral transplantation in the neonatal rats with hypoxic-ischemic brain damage（HIBD）.Methods Under sterile conditions,the cord blood of 20 healthy full-term neonates were collected by density gradient centrifugation to isolate mononuclear cells from cord blood and then by adherent cells to develop human umbilical cord blood MSCs.When the cells were in the 3rd generation,8 parts of the cultured umbilical cord blood MSCs （half） were taken out at random for group A;8 parts of umbilical cord blood MSCs （half） for group B;and then mixed the remaining half of the cord blood mesenchymal stem cells from group A and group B as group C.The cell density of the 3 groups was 1×106 L-1.The expressions of the cell surface antigens,CD29,CD34,CD44,CD105 of the 3rd generation of cord blood MSCs cultured in a mixed way were detected by flow cytometry.P4 generation of human umbilical cord blood MSCs was chosen as transplanted cells,which was vitro marked by 5-bromo-2-uracil BrdU.Forty-five 7-day-old SD rats were chosen as HIBD models,3 cases died in the process.The remaining 42 cases were divided into the mixed transplantation group（n=16）,the single transplantation group（n=16） and the control group（n=10）.On the 3rd day after mo-deling,in stereotactic conditions,human umbilical cord blood MSCs were infused into the rats of transplantation group by way of the left cerebral cortex,and the same volume of PBS was infused into the control group by way of the same position.On the 7th day after cell transplatation,the brain of 6 transplanted rats selected randomly were taken out after being killed,and then the brain tissue was used to observe the survival,migration and differentiation of the transplanted cells in the brain by the immunohistochemistry method.On the 1st,7th,14th,21st,and 28th day after transplantation,neurological function of the rats in the 3 groups was evaluated by improved neuro

关 键 词：脐血 间充质干细胞 混合培养 缺氧缺血性脑损伤 移植 分化 

分 类 号：R722.1[医药卫生—儿科]