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

韩鲁祥; 唐慧昕; 赵振峰; 李珊珊; 王全义; 孔令斌; 毕慧莹; 舒振峰; 段钟平; 陈煜; 洪丰

doi:10.3969/j.issn.1001-5256.2022.05.024

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

DOI: 10.3969/j.issn.1001-5256.2022.05.024

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

基金项目:

国家自然科学基金 (81170395);

国家自然科学基金 (81570556);

中国肝炎防治基金会-天晴肝病研究基金资助课题 (TQGB20210013)

伦理学声明：本研究方案于2021年2月13日经由济宁医学院附属医院伦理委员会审批，批号：2021B169，符合实验室动物管理与使用准则。

利益冲突声明：本研究不存在研究者、伦理委员会成员以及与公开研究成果有关的利益冲突。

作者贡献声明：韩鲁祥、唐慧昕负责课题设计，实验实施，收集数据，论文撰写和修改；赵振峰负责干细胞培养及外泌体鉴定；李珊珊、毕慧莹、舒振峰负责外泌体提取及鉴定；王全义负责病理学检验；孔令斌、段钟平、陈煜负责指导动物实验，统计分析实验数据；洪丰负责指导实验设计，审核实验结果，指导论文撰写与修改。

详细信息

通信作者:
洪丰, fenghong9508@163.com

韩鲁祥、唐慧昕对本文贡献等同, 同为第一作者

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出版历程
- 收稿日期: 2021-09-27
- 录用日期: 2021-10-28
- 出版日期: 2022-05-20

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

1.
Clinical Medical College of Jining Medical University, Jining, Shandong 272013, China
2.
Fourth Department of Liver Disease Center, Beijing YouAn Hospital, Capital Medical University, Beijing 100069, China
3.
Linyi Regional Cell Preparation Center, Linyi, Shandong 276000, China
4.
Department of Gastroenterology, Affiliated Hospital of Jining Medical University, Jining, Shandong 272029, China
5.
Department of Medical Technology, Heze Medical College, Heze, Shandong 274000, China
6.
Shanghai Meifeng Biotechnology Co., Ltd., Shanghai 201203, China

Research funding:

National Natural Science Foundation of China (81170395);

National Natural Science Foundation of China (81570556);

Chinese Foundation for Hepatitis Prevention and Control-TianQing Liver Disease Research Fund Subject (TQGB20210013)

More Information

Corresponding author: HONG Feng, fenghong9508@163.com(ORCID: 0000-0003-4377-7226)

摘要

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 (551.31±17.70)pg/mL、(515.58±7.18)pg/mL vs (556.21±11.15)pg/mL，P值均＜0.001]；且与HLSC-exo 6 h治疗组相比，HLSC-exo 3 h治疗组下降更明显(P＜0.001)。小鼠肝脏大体形态及病理显示HLSC-exo 3 h、6 h治疗组肝细胞坏死程度较ConA 3 h、6 h模型组明显减轻，HLSC-exo 3 h治疗组肝小叶结构基本完整，仅可见散在点状坏死；HLSC-exo 12 h治疗组与ConA 12 h模型组相比，肝细胞坏死无明显改善。结论成体人肝源性干细胞外泌体静脉注射可以减轻ConA诱导的小鼠急性肝损伤，以提前3 h注射保护作用最显著。通过调控细胞因子是HLSC-exo减轻肝损伤的重要机制之一。
- 肝功能衰竭, 急性 /
- 干细胞 /
- 外泌体 /
- 伴刀豆球蛋白A
Abstract: Objective 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 3-hour HLSC-exo treatment group had significantly greater reductions (P < 0.001). In the blank group, the levels of IL-10 and TNF-α were 313.51±10.97 pg/ml and 476.05±7.31 pg/ml, respectively. Compared with the blank control group, the 3-, 6-, and 12-hour ConA model groups had a significant reduction in the level of IL-10 (all P < 0.001); compared with the 3-and 6-hour ConA model groups, the 3-and 6-hour HLSC-exo treatment groups had a significant increase in the level of IL-10(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, both P < 0.001); compared with the 6-hour HLSC-exo treatment group, the 3-hour HLSC-exo treatment group had a significantly greater increase (P < 0.001). Compared with the blank control group, the 3-, 6-, and 12-hour ConA model groups had a significant increase in the level of TNF-α (all P < 0.001); compared with the 3-and 6-hour ConA model groups, the 3-and 6-hour HLSC-exo treatment groups had a significant reduction in the level of TNF-α (478.26±12.99 pg/ml vs 551.31±17.70 pg/ml, 515.58±7.18 pg/ml vs 556.21±11.15 pg/ml, both P < 0.001); compared with the 6-hour HLSC-exo treatment group, the 3-hour HLSC-exo treatment group had a significantly greater reduction (P < 0.001). Compared with the 3-and 6-hour ConA model groups in terms of the gross morphology and histopathology of the liver, the 3-and 6-hour HLSC-exo treatment groups had a significant reduction in the degree of hepatocyte necrosis, and the 3-hour HLSC-exo treatment group had a basically complete lobular structure, with sporadic spotty necrosis; the 12-hour HLSC-exo treatment group had no significant improvement in hepatocyte necrosis compared with the 12-hour ConA model group. Conclusion Intravenous injection of adult HLSC-exo can alleviate acute liver injury induced by ConA in mice, and injection at 3 hours in advance has the most significant protective effect. Regulation of cytokines is one of the important mechanisms for HLSC-exo to alleviate liver injury.
- Liver Failure, Acute /
- Stem Cells /
- Exosomes /
- Concanavalin A

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图 1 外泌体粒径分析结果

Figure 1. Particle size analysis of HLSC-exo

下载: 全尺寸图片幻灯片

图 2 外泌体电镜下显像(×80 000)

Figure 2. Electron microscopic image of HLSC-exo(×80 000)

下载: 全尺寸图片幻灯片

图 3 外泌体表面标志物的表达

Figure 3. Expression of HLSC-exo surface markers

下载: 全尺寸图片幻灯片

图 4 各组肝脏组织HE染色结果(×100)

注：a，空白对照组；b~d，ConA 3 h、6 h、12 h模型组；e~g，HLSC-exo 3 h、6 h、12 h治疗组。

Figure 4. HE staining of liver tissues in each group (×100)

下载: 全尺寸图片幻灯片

表 1 各组小鼠肝功能ALT、AST水平

Table 1. ALT and AST levels of mice in each group

组别	ALT(U/L)	AST(U/L)
空白组	31.81±6.74	69.75±8.30
ConA模型组
3 h	1989.32±347.67¹⁾	2534.27±644.72¹⁾
6 h	2208.33±349.96¹⁾	2437.13±288.13¹⁾
12 h	2223.12±318.48¹⁾	2289.88±274.59¹⁾
HLSC-exo治疗组
3 h	225.58±115.59²⁾³⁾	303.53±126.68²⁾³⁾
6 h	1174.71±203.30⁴⁾	1340.70±262.56⁴⁾
12 h	1990.00±302.59	2293.36±341.96
F值	101.26	79.60
P值	＜0.001	＜0.001
注：与空白组比较，1)P＜0.001；与ConA 3h组比较，2)P＜0.001；与HLSC-exo 6 h组比较，3)P＜0.001；与ConA 6 h组比较，4)P＜0.001。

下载: 导出CSV

表 2 各组小鼠血清IL-10、TNFα水平比较

Table 2. IL-10 and TNFα levels of mice in each group

组别	IL-10(pg/mL)	TNFα(pg/mL)
空白组	313.51±10.97	476.05±7.31
ConA模型组
3 h	266.20±8.15¹⁾	551.31±17.70¹⁾
6 h	264.41±9.12¹⁾	556.21±11.15¹⁾
12 h	260.23±4.56¹⁾	564.42±4.08¹⁾
HLSC-exo治疗组
3 h	331.61±10.46²⁾³⁾	478.26±12.99²⁾³⁾
6 h	288.13±10.74⁴⁾	515.58±7.18⁴⁾
12 h	260.10±7.25	559.85±12.02
F值	81.89	96.24
P值	＜0.001	＜0.001
注：与空白组比较，1)P＜0.001；与ConA 3h组比较，2)P＜0.001；与HLSC-exo 6 h组比较，3)P＜0.001；与ConA 6 h组比较，4)P＜0.001。

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