文献类型：期刊文献

中文题名：人脐带间充质干细胞外泌体对创伤性脑损伤后神经功能的保护作用

英文题名：Protective effect of human umbilical cord mesenchymal stem cell exosomes on nerve function after traumatic brain injury

作者：张震文[1,2,3];严菁兴[1];彭晓明[1];唐珂[1];李晓红[3,4]

第一作者：张震文

机构：[1]甘肃中医药大学附属医院脑病科,兰州730000;[2]甘肃中医药大学中西医结合学院,兰州730000;[3]中国人民武装警察部队特色医学中心脑科中心,天津300072;[4]天津大学医学工程与转化医学研究院,300072

第一机构：甘肃中医药大学第二附属医院

年份：2020

卷号：29

期号：12

起止页码：1061

中文期刊名：中华行为医学与脑科学杂志

外文期刊名：Chinese Journal of Behavioral Medicine and Brain Science

收录：CSTPCD;;北大核心:【北大核心2017】；CSCD:【CSCD_E2019_2020】；

基金：国家自然科学基金项目(81771350)。

语种：中文

中文关键词：外泌体;人脐带间充质干细胞;神经功能恢复;创伤性脑损伤

外文关键词：Exosomes;Human umbilical cord mesenchymal stem cells;Neurological recovery;Traumatic brain injury

摘要：目的探讨人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,HUCMSCs)外泌体对创伤性脑损伤(traumatic brain injury,TBI)后神经保护的作用及可能机制。方法分离、培养HUMSCs并提取外泌体,使用透射电镜和蛋白免疫印迹方法进行外泌体鉴定;将60只健康雄性SPF级SD大鼠按照随机数字表法分为3组,分别为假手术组(n=20)、TBI对照组(n=20)、外泌体治疗组(n=20),采用电子脑皮质损伤撞击仪建立大鼠TBI模型,采用改良神经功能缺损评分(modified neurological severity score,mNSS)和Morris水迷宫实验(Morris water maze,MWM)评估大鼠的神经功能运动和空间记忆功能,使用免疫荧光法检测胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)和小胶质细胞特异性蛋白离子钙结合衔接分子1(ionized calcium binding adaptor molecule-1,IBA1)活化表达水平。TUNEL检测大脑皮质损伤区细胞凋亡的变化。所有的数据采用SPSS 22.0软件进行统计分析,两组间比较采用t检验,多组均数的比较采用单因素方差分析,双因素方差分析进行神经行为学统计。结果相差显微镜下,HUCMSCs的外泌体直径为40~120 nm,呈圆形,荧光显微镜下显示标记的外泌体CD9、CD63蛋白表达阳性。与TBI组相比,外泌体治疗组mNSS评分[术后28 d(3.23±0.72)分]较TBI组[术后28 d(5.14±0.96)分]明显降低(t=3.559,P<0.01);水迷宫实验结果显示,术后第26天外泌体治疗组的逃避潜伏期[(21.25±4.72)s]较TBI组[(33.06±8.81)s]变短(t=3.737,P<0.01),外泌体治疗组穿越平台次数[(3.56±0.48)次]与TBI组[(2.06±0.36)次]相比明显增加(t=7.906,P<0.01),经过外泌体治疗后,大鼠的运动功能和空间记忆功能明显改善;免疫荧光显示,TBI组与假手术组相比GFAP和IBA1表达量明显增高,外泌体治疗组与TBI组相比明显降低;TUNEL染色结果显示TBI组大脑皮质TUNEL阳性细胞数较假手术组明显增多(P<0.01),给予外泌体治疗后,TUNEL阳性细胞的数量较TBI组明显减少(P<0.01)。结论 HUCMSCs外泌体在脑外伤后可促进神经功能恢复,抑制胶质细胞过度活化,保护神经元细胞,并减少细胞凋亡。
Objective To investigate the neuroprotective effect and possible mechanism of human umbilical cord mesenchymal stem cells(HUCMSCs)exosomes after traumatic brain injury(TBI).Methods HUCMSCs were isolated and cultured,and the exosomes were extracted.The exosomes were identified by transmission electron microscopy(TEM)and Western blot.Sixty healthy male SPF SD rats were randomly divided into three groups:sham operation group(n=20),TBI control group(n=20)and exosomes treatment group(n=20).The modified neurological severity score(mNSS)and Morris water maze(MWM)were used to evaluate the neural function,motor function and spatial memory function of the rats.The expression levels of glial fibrillary acidic protein(GFAP)and microglia specific protein of ionized calcium binding adaptor molecule 1(IBA1)were detected by immunofluorescence.The TUNEL staining was used to detect the changes of apoptosis in the cerebral cortex.All the data were analyzed by SPSS 22.0 software.The comparison between the two groups was conducted by t-tesi,and the comparison of multiple groups was conducted by oneway ANOVA and two-way ANOVA for neurobehavioral statistics.Results The diameter of exosomes of HUCMSCs ranged from 40 nm to 120 nm under phase contrast microscope?and the expression of CD9 and CD63 in exosomes was observed under fluorescence microscope.Compared with TBI group,the mNSS score of exosomes treatment group(3.23±0.72)was significantly lower than that of the TBI group(5.14±0.96)(t=3.559,P<0.05)28 days after operation.The results of water maze test showed that 26 days after operation the escape latency of exosomes treatment group((21.25±4.72)s)was shorter than that of TBI group((33.06±8.81)s)(3.737,P<0.01),and the number of platform crossing in exosomes treatment group(3.56±0.48)was significantly increased compared with that in TBI group(2.06±0.36)(t=1.906,P<0.01).After exosomes treatment,the motor function and spatial memory function of rats were significantly increased.The expression levels of GFAP and IBA1 in TBI group were significantly increased compared with sham group,while in exosomes treatment group were significantly lower than TBI group.The results of TUNEL staining showed that the number of TUNEL positive cells in TBI group were significantly higher than those in the sham group(P<0.01),and the number of TUNEL positive cells in exosomes treatment group were significantly lower than those in TBI group(P<0.01).Conclusion HUCMSCs exosomes can promote the recovery of neural function,inhibit the excessive activation of glial cells,protect neurons and reduce apoptosis after traumatic brain injury.