TIMP2基因修饰间充质干细胞治疗阿尔兹海默症的实验小鼠模型研究  

TIMP2 modified human umbilical cord mesenchymal stem cells improve the behavior of Alzheimer′s disease mice

作　　者：冯佳 谢芳莘 杨嘉洁 黄雨 李晓红 左凤琼[4] 刘少先 张勇刚 陈勇军 马峰[1] FENG Jia;XIE Fangxin;YANG Jiajie;HUANG Yu;LI Xiaohong;ZUO Fengqiong;LIU Shaoxian;ZHANG Yonggang;CHEN Yongjun;MA Feng(Institute of Blood Transfusion,Chinese Academy of Medical Sciences&Peking Union Medical College(CAMS&PUMC),Chengdu 610052,China;Kangjing Biotechnology Co Ltd;Shanghai University of Science and Technology;School of Basic Medical Science,Sichuan University)

机构地区：[1]中国医学科学院北京协和医学院输血研究所,四川成都610052 [2]康景生物科技有限公司 [3]上海科技大学 [4]四川大学基础医学院

出　　处：《中国输血杂志》2020年第5期441-445,共5页Chinese Journal of Blood Transfusion

基　　金：中国医学科学院医学与健康创新工程(2016I1M-1-018,2019-I2M-006)。

摘　　要：目的建立并验证组织金属蛋白酶抑制因子2(TIMP2)基因修饰间充质干细胞(MSCs)治疗阿尔兹海默症(AD)有效性的实验动物模型。方法 1)TIMP2基因修饰MSCs的获得:利用腺相关病毒(AAV)基因表达载体和人脐带来源的MSCs(hUB-MSCs)构建TIMP2基因修饰的MSCs(命名为hUB-MSCs-T),采用ELISA检测hUB-MSCs-T分泌TIMP2蛋白含量,通过流式检测hUB-MSCs-T表面分子,以及通过脂肪、成骨、软骨细胞分化试验鉴定对hUB-MSCs-T的分化能力。2)hUB-MSCs-T对AD实验小鼠的影响:将30只7月龄雄性AD小鼠随机均分为3组,PBS组:小鼠尾静脉注射0.006 7 mol/L PBS缓冲液0.2 mL/只;hUB-MSCs组:注射1×10^6个/mL悬浮hUB-MSCs0.2 mL/只;hUB-MSCs-T组:注射1×10^6个/mL悬浮hUB-MSCs-T0.2 mL/只;均为1次/周,连续8周。治疗后第1—4周,采用旷场试验、新物体识别试验和巴恩斯迷宫试验评价小鼠的记忆和认知能力;治疗后第5周解剖小鼠3只/组,取海马区域组织做测序分析。结果 hUB-MSCs-T、hUB-MSCs和PBS 3组AD小鼠在巴恩斯迷宫试验d1第1次训练的逃避潜伏期(s)分别为76±93 vs 148±83 vs 150±89 (P<0.05),第4次训练的逃避潜伏期(s)分别为52±46 vs 87±87 vs 96±78(P<0.05);在巴恩斯迷宫试验d12的长时记忆试验中,进入目标洞次数分别为17±9.6 vs 12.0±5.3 vs 10.0±7.7(P<0.05)。结论 hUB-MSCs-T在一定程度上改善了AD小鼠的记忆和认知能力。Objective To investigate the TIMP2 modified human umbilical cord mesenchymal stem cells(hUB-MSCs-T) and its influence on Alzheimer′s disease(AD) mice. Methods hUB-MSCs-T were obtained by the use of an AAV gene expression vector and human umbilical mesenchymal stem cells(hUB-MSCs). ELISA was used to detect the protein content of TIMP2 secreted by hUB-MSCs-T, the surface molecules of hUB-MSCs-T were detected by flow cytometry, and the differentiation ability of hUB-MSCs-T was identified byadipogenesis, osteogenesis and chondrogenesis. Thirty seven-month-old male AD mice were randomly allocated into PBS group, hUB-MSCs group, and hUB-MSCs-T group. The mice were injected with 0.2 mLPBS(0.006 7 mol/L) via tail vein in the PBS group, with 0.2 mL hUB-MSCs suspension of 1 × 10^6 cells/mL in the hUB-MSCs group,with 0.2 mL hUB-MSCs-T suspension of 1 × 10^6 cells/mL in the hUB-MSCs-T group;once a week for eight consecutive weeks. From the 1 st to 4 th week after treatment, the memory and cognitive ability of mice were evaluated by open field experiment, new object recognition experiment and Barnes maze experiment;at the 5 th week after treatment, nine mice were dissected and the hippocampal tissues were taken for transcriptome sequencing analysis Results The escape latency of the 1 st training on the first day of Barnes maze experiment in hUB-MSCs-T group was 76±93 vs 148±83 vs 150±89 as compared to hUB-MSCs group and PBS group(P<0.05). The escape latency of the 4 th training on the first day of Barnes maze experiment in hUB-MSCs-T group was 52±46 vs 87±87 vs 96±78 as compared to hUB-MSCs group and PBS group(P<0.05). In the long-term memory test on the 12 th day of Barnes maze experiment, the number of times that mice entered the target hole in hUB-MSCs-T group was 17±9.6 vs 12.0±5.3 vs 10.0±7.7 as compared to hUB-MSCs group and PBS group(P<0.05). Conclusion hUB-MSCs-T could improve the memory ability and cognitive ability of AD mice to some extent.

关 键 词：阿尔兹海默症 人脐带间充质干细胞 组织金属蛋白酶抑制因子2 腺相关病毒 AD动物模型 小鼠 

分 类 号：R457.7[医药卫生—治疗学] R741[医药卫生—临床医学]