成体人肝源性干细胞外泌体对刀豆蛋白A诱导的小鼠急性肝损伤的作用  

Effect of exosomes from adult human liver-derived stem cells on concanavalin A-induced acute liver injury in mice

作　　者：韩鲁祥 唐慧昕 赵振峰 李珊珊[2] 王全义[4] 孔令斌[4] 毕慧莹 舒振峰 段钟平[2] 陈煜[2] 洪丰[4] HAN Luxiang;TANG Huixin;ZHAO Zhenfeng;LI Shanshan;WANG Quanyi;KONG Lingbin;BI Huiying;SHU Zhenfeng;DUAN Zhongping;CHEN Yu;HONG feng(Clinical Medical College of Jining Medical University,Jining,Shandong 272013,China;Fourth Department of Liver Disease Center,Beijing YouAn Hospital,Capital Medical University,Beijing 100069,China;Linyi Regional Cell Preparation Center,Linyi,Shandong 276000,China;Department of Gastroenterology,Affiliated Hospital of Jining Medical University,Jining,Shandong 272029,China;Department of Medical Technology,Heze Medical College,Heze,Shandong 274000,China;Shanghai Meifeng Biotechnology Co.,Ltd.,Shanghai 201203,China)

机构地区：[1]济宁医学院临床医学院,山东济宁272013 [2]首都医科大学附属北京佑安医院肝病中心四科,北京100069 [3]临沂区域细胞制备中心,山东临沂276000 [4]济宁医学院附属医院消化内科,山东济宁272029 [5]菏泽医学专科学校医学技术系,山东菏泽274000 [6]上海美峰生物技术有限公司,上海201203

出　　处：《临床肝胆病杂志》2022年第5期1101-1105,共5页Journal of Clinical Hepatology

基　　金：国家自然科学基金(81170395,81570556);中国肝炎防治基金会-天晴肝病研究基金资助课题(TQGB20210013)。

摘　　要：目的探讨成体人肝源性干细胞外泌体(HLSC-exo)不同时间静脉注射对刀豆蛋白A(ConA)诱导的小鼠急性肝损伤的保护作用。方法差速离心法提取HLSC-exo,Western blot检测其标志蛋白CD9、CD63的表达,纳米颗粒跟踪分析粒径分布。将56只C57BL/6雄性小鼠随机分为3组,空白对照组、ConA模型组和HLSC-exo治疗组。根据给予磷酸盐缓冲液或HLSC-exo后再注射ConA间隔时间不同,分为3 h、6 h、12 h亚组;检测血清中ALT、AST、TNFα、IL-10水平;比较各组小鼠肝脏大体形态及病理组织变化。计量资料多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。结果HLSC-exo是直径为90~110 nm的囊泡体,电镜下可见清晰的“茶托样”结构,其特异性标志蛋白CD9和CD63均有明显的表达;空白对照组小鼠ALT、AST水平分别为(31.81±6.74)U/L、(69.75±8.30)U/L。相较于空白对照组,ConA 3 h、6 h、12 h模型组小鼠ALT、AST水平明显升高(P值均<0.001);HLSC-exo 3 h、6 h治疗组ALT、AST水平较ConA 3 h、6 h模型组明显下降[(225.58±115.59)U/L vs(1989.32±347.67)U/L、(1174.71±203.30)U/L vs(2208.33±349.96)U/L,(303.53±126.68)U/L vs(2534.27±644.72)U/L、(1340.70±262.56)U/L vs(2437.13±288.13)U/L,P值均<0.001];与HLSC-exo 6 h治疗组相比,HLSC-exo 3 h治疗组下降更明显(P<0.001)。空白对照组小鼠IL-10、TNFα水平分别为(313.51±10.97)pg/mL,(476.05±7.31)pg/mL。相较于空白对照组,ConA3 h、6 h、12 h模型组IL-10水平均明显下降(P值均<0.001);HLSC-exo 3 h、6 h治疗组IL-10水平相较于ConA 3 h、6 h模型组明显升高[(331.61±10.46)pg/mL vs(266.20±8.15)pg/mL、(288.13±10.74)pg/mL vs(264.41±9.12)pg/mL,P值均<0.001];且与HLSC-exo 6 h治疗组相比,HLSC-exo 3 h治疗组升高更明显(P<0.001)。相较于空白对照组,ConA 3 h、6 h、12 h模型组TNFα水平较正常组明显升高(P值均<0.001);HLSC-exo 3 h、6 h治疗组TNFα水平相较于ConA 3 h、6 h模型组明显下降[(478.26±12.99)pg/mL vs(551Objective To investigate the protective effect of adult human liver-derived stem cell exosomes(HLSC-exo)intravenously injected at different time points against acute liver injury induced by concanavalin A(ConA)in mice.Methods HLSC-exo was extracted by differential centrifugation.Western blot was used to measure the expression of the marker proteins CD9 and CD63,and nanoparticle tracking analysis was used to investigate particle size distribution.A total of 56 male C57BL/6 mice were randomly divided into blank control group,ConA model group,and HLSC-exo treatment group.The ConA model group and the HLSC-exo treatment group were further divided into 3-,6-,and 12-hour subgroups according to the interval between phosphate buffer or HLSC-exo injection and ConA injection.The serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),tumor necrosis factor-α(TNF-α),and interleukin-10(IL-10)were measured,and the gross morphology and histopathology of the liver were compared between groups.A one-way analysis of variance was used for comparison of continuous data between multiple groups,and the least significant difference t-test was used for further comparison between two groups.Results HLSC-exo was a membranous vesicle with a diameter of 90-110 nm,with a clear saucer-like structure under an electron microscope and marked expression of its specific marker proteins CD9 and CD63.In the blank control group,the levels of ALT and AST were 31.81±6.74 U/L and 69.75±8.30 U/L,respectively.Compared with the blank control group,the 3-,6-,and 12-hour ConA model groups had significant increases in the levels of ALT and AST(all P<0.001);compared with the 3-and 6-hour ConA model groups,the 3-and 6-hour HLSC-exo treatment groups had significant reductions in the levels of ALT and AST(225.58±115.59 U/L vs 1989.32±347.67 U/L,1174.71±203.30 U/L vs 2208.33±349.96 U/L,303.53±126.68 U/L vs 2534.27±644.72 U/L,1340.70±262.56 U/L vs 2437.13±288.13 U/L,all P<0.001);compared with the 6-hour HLSC-exo treatment group,the

关 键 词：肝功能衰竭 急性 干细胞 外泌体 伴刀豆球蛋白A 

分 类 号：R28[医药卫生—中药学]