大鼠静脉多次注射人脐带间充质干细胞安全性的研究  

Safety study of repeatedly intravenous infusion with human umbilical cord mesenchymal stem cells in rats

作　　者：张颖[1] 李娜[1] 林筝[1] 辛毅[1] 崔巍[1] 刘飒[1] 王忠[1] 陈雄[1] 赵颂军[1] 袁惠 黄益民[1] 周玉杰 罗毅[4] 张兆光[1] 

机构地区：[1]首都医科大学附属北京安贞医院-北京市心肺血管疾病研究所,100029 [2]检验科 [3]心内科 [4]首都儿科研究所

出　　处：《心肺血管病杂志》2015年第3期221-227,共7页Journal of Cardiovascular and Pulmonary Diseases

基　　金：国家自然科学基金支持项目(81370228);北京市自然科学基金支持项目(5042006;7072012);北京市优秀人才培养资助项目(2012D003034000009)

摘　　要：目的：观察多次静脉输注人脐带间充质干细胞（hu MSCs）对大鼠的毒性反应。方法：1贴块法培养hu MSCs;流式细胞仪鉴定第4代hu MSCs特异性表面抗原;标准试剂盒鉴定第4代hu MSCs向脂肪细胞和成骨细胞分化的潜能;2182~203g的SD大鼠分为对照组、0.9%氯化钠组和干细胞组,各8只,雌雄各50%。3干细胞组分别于第1、4和15天时经尾静脉注射（2.5×106）个/kg体质量的第4代hu MSCs,0.9%氯化钠组于同等时间注射与细胞悬液等量0.9%氯化钠液。4第3次注射转染RFP基因（中文）的hu MSCs后11天,腹主动脉取血,检测和比较血常规和生化指标;取脑、心、肺、肝及肾进行病理学检查;荧光显微镜观察器官组织中表达RFP的hu MSCs细胞分布情况,流式细胞仪检测骨髓中表达RFP的hu MSCs;ELISA检测血清γ-IFN、IL-2、IL-4、IL-6、IL-8、IL-10及IL-12含量。结果：1第4代hu MSCs特异性表面抗原CD73、CD90和CD105表达强阳性,纯度分别为98.7%、99.6%和98.6%,而CD34、CD45、CD11b、CD19、CD79a和HLA-DR的表达水平为阴性,CD14表达水平为23.1%;hu MSCs分化成脂细胞和成骨细胞的比例均为99%;2注射3次干细胞后实验动物全部存活,干细胞组部分血常规和生化指标与0.9%氯化钠组相比有差异,但与对照组相比,只有PLT明显降低、CHOL明显升高;3干细胞组脑、心、肺、肝、肾的形态和组织病理学无明显改变;（4）干细胞组的器官组织和骨髓中未检测到表达RFP的hu MSCs;（5）干细胞组血清IL-8水平明显升高。结论：多次尾静脉注射hu MSCs对大鼠没有明显的毒性反应。Objective：To investigate the influence and toxicity of repeatedly intravenous infusion with the human umbilical cord mesenchymal stem cell （huMSCs） in SD rats. Methods：（1） The umbilical cord was collected from a mature male infant, and huMSCs were aseptically isolated from the umbilical Wharton＇ s jelly tissue and expanded in vitro. The specific surface markers of 4th huMSCs passage cells were analyzed by flow cytometer. The osteogenic and adipogenic differentiation potential of the cells were evaluated by using osteogenesis and adiposis kit. （2）24 SD rats weighting 182 -203g were divided into three groups： control, saline and experimental groups. Eaeh group includes 4 male and 4 female rats. （3）At the day of 1,4 and 15, animals in experimental group were repeatedly tail intravenous infused with （2. 5 ~ 106） mL huMSCs in 0. 5ml saline solution. huMSCs infused at the day 15 were transfected RFP gene and expressed RFP protein. （4）At the 11 day after infused RFP transgenic huMSCs, the blood samples were drew through the abdominal aorta. The blood routine examination and the biochemical indexes were measured in the clinical lab. The brain, heart, lung, liver and kidney were collected and HE dye for pathological observation. Fluorescence microscopy was used to observe distribution of huMSCs expressing RFP in organ tissue slides, and flow cytometry was used to survey huM- SCs expressing RFP in femur bone marrow. Serum γ-IFN, IL-2, IL4, IL-6, IL-8, IL-10, IL-12 were detected by ELISA methods. All parameters were compared between three groups. Results：（1） The 4th huMSCs passage cells positively expressed the specific surface markers CD73 ,CD90 and CD105 （ positivity were 98.7% ,99. 6% and 98.6% ） ; and CDSg,CIM5,CD11 b, CD19, CD79a and HLA-DR were negatively expressed. Respectively ; CD14 expression is 23.1% postive. The differentiation rate of the huMSCs towards the osteoblasts and adipo- cytes were over 99%. （2）All experimental animals survived after repeated huMSCs treatm

关 键 词：人脐带间充质干细胞 大鼠 毒性反应 

分 类 号：R54[医药卫生—心血管疾病]