生物发光成像对肝损伤小鼠体内骨髓间充质干细胞迁移的动态示踪研究  

The dynamic tracking study on tropism of bone marrow mesenchymal stem cells in mice with liver injury using bioluminescence imaging

作　　者：刘静静[1] 时宇鹏 张勇[1] 汪卫建 宋曼莉 程敬亮[1] Liu Jingjing;Shi Yupeng;Zhang Yong;Wang Weijian;Song Manli;Cheng Jingliang(Department of MRI,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)

机构地区：[1]郑州大学第一附属医院磁共振科,450052

出　　处：《中华放射学杂志》2021年第10期1086-1091,共6页Chinese Journal of Radiology

基　　金：河南省医学科技攻关计划(201702028)。

摘　　要：目的利用生物发光成像活体动态示踪人骨髓间充质干细胞(MSCs)小鼠体内移植后对损伤肝的趋向迁移及其治疗作用。方法通过基因转染将CMV-Luciferase2-mKate2导入MSCs,96 h后运用流式细胞仪对表达远红外荧光蛋白mKate2的MCSs进行纯化筛选,得到基因转染的MSCs-R(MSCs-CMV-Luciferase2-mKate2)用于体外和活体生物发光成像。将小鼠(雄性BALB/c裸鼠)用随机数目表法分为4组,每组9只。(1)肝损伤实验组:以CCl4腹腔注射建立肝损伤模型,24 h后进行脾脏MSCs-R移植;(2)对照实验组:腹腔注射同等体积磷酸缓冲液(PBS),24 h后进行脾脏MSCs-R移植;(3)肝损伤组:建立肝损伤模型,脾脏注射PBS;(4)空白组:腹腔注射PBS。移植后每天对小鼠进行生物发光成像,直至肝区光信号消失,在第14天对肝脏组织进行病理学研究。光信号强度与细胞数量的相关性采用线性回归分析,肝损伤实验组和对照实验组间光信号强度的比较采用独立样本t检验。结果CMV-Luciferase2-mKate2慢病毒感染MSCs 96 h后,经过纯化筛选其蛋白mKate2表达率高于95%。体外实验显示MSCs-R细胞生物发光信号强度与细胞数量呈线性正相关(R2=0.980)。MSCs-R脾内移植后第1天肝损伤实验组和对照实验组均可见干细胞迁移至肝脏,肝损伤实验组的肝区光信号强度明显高于对照实验组(t=15.476,P<0.001)。对照实验组小鼠肝区生物发光信号持续5 d,肝损伤实验组光学信号持续11 d,其他2组无光学信号。组织病理学显示肝损伤实验组小鼠MSCs-R体内移植后肝损伤程度明显减轻。结论生物发光成像可动态示踪脾内移植的MSCs定向迁移并定居在受损肝脏内,肝损伤有助于MSCs定向迁移至损伤组织并发挥其修复肝损伤的作用。Objective To dynamically trace the migration and therapeutic effects of human bone marrow mesenchymal stem cells(MSCs)in mice with liver injury after cell transplantation through in vivo bioluminescent imaging(BLI).Methods The MSCs were transfected with the lentivirus CMV-Luciferase2-mKate2 and mKate2 positive cells were purified and screened by fluorescence-activated cell sorting(FACS)after 96 h.The purified MSCs-R(MSCs-CMV-Luciferase2-mKate2)were used by in vitro and in vivo BLI.The mice(male BALB/c nude mice)were divided into 4 groups with 9 mice per group by random number table method,including(1)Liver injury experimental group:The liver injury model was established by intraperitoneal injection of CCl4,and MSCS-R transplantation through spleen injection was performed 24 h later;(2)Control experimental group:The same volume of phosphate buffer(PBS)was injected intraperitoneally,and MSCS-R transplantation through spleen injection was performed 24 h later;(3)Liver injury group:Liver injury model was established and PBS was injected into the spleen;(4)Blank group:The mice were intraperitoneally injected of PBS.BLI was performed daily after cell transplantation until light signals disappeared in the liver region,and the pathological examination of liver tissue was obtained 14 d after MSCs-R transplantation.Linear regression analyses were performed to determine the correlation between the optical signal intensity and the number of cells,and statistical differences of the optical signal intensity between liver injury experimental group and control experimental group were evaluated using the Student′s t test.Results The MSCs were readily transfected with lentivirus CMV-Luciferase2-mKate2 for 96 h.The transfected MSCs were purified by FACS and more than 95%of MSCs were mKate2 positive.The optical signal intensity of MSCs-R detected by BLI in vitro significantly correlated with cell numbers in vitro(R2=0.980).In both of liver injury experimental group and control experimental group,cell migration to the liver was obs

关 键 词：干细胞 肝损伤 细胞治疗 示踪 生物发光成像 

分 类 号：R575[医药卫生—消化系统]