人脐带间充质干细胞（hUC-MSC）对肝纤维化小鼠模型的治疗作用及其机制分析

刘平箕; 姚黎超; 胡雪; 王铮; 熊芷玉; 江应安

doi:10.12449/JCH240315

人脐带间充质干细胞（hUC-MSC）对肝纤维化小鼠模型的治疗作用及其机制分析

DOI: 10.12449/JCH240315

武汉大学人民医院感染科，武汉 430060

基金项目:

武汉大学教育发展基金会-抗衰老研究中心专款 (2002330);

中央高校基本科研业务费专项资金 (2042022kf1115)

伦理学声明：本研究方案于2022年9月1日经由武汉大学人民医院伦理委员会审批，批号：20220901A，符合实验室动物管理与使用准则。

利益冲突声明：本文不存在任何利益冲突。

作者贡献声明：刘平箕、姚黎超负责设计论文框架，起草论文；刘平箕、胡雪负责实验操作，研究过程的实施；王铮、熊芷玉负责数据收集，统计学分析，绘制图表；江应安负责论文修改，拟定写作思路，指导撰写文章并最后定稿。

详细信息

通信作者:
江应安， jiangya_cn@aliyun.com （ORCID： 0000-0003-4912-7494）

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出版历程
- 收稿日期: 2023-06-09
- 录用日期: 2023-08-17
- 出版日期: 2024-03-20

Effect of human umbilical cord mesenchymal stem cells in treatment of mice with liver fibrosis and its mechanism

Department of Infectious Diseases，Renmin Hospital of Wuhan University，Wuhan 430060，China

Research funding:

The Grant From the Anti-Aging Research Center, Wuhan University Education & Development Foundation (2002330);

The Fundamental Research Funds for the Central Universities (2042022kf1115)

More Information

Corresponding author: JIANG Yingan， jiangya_cn@aliyun.com （ORCID： 0000-0003-4912-7494）

摘要

摘要: 目的探讨人脐带间充质干细胞（hUC-MSC）对肝纤维化小鼠的治疗作用及其机制。方法选取18只SPF级6周龄C57BL/6小鼠，使用随机数字表法随机分为3组，分别为对照组、CCl₄模型组（CCl₄组）和hUC-MSC治疗组（MSC组），每组6只。CCl₄组和MSC组腹腔注射CCl₄溶液构建小鼠肝纤维化模型，对照组同时注射相同剂量玉米油，MSC组在注射CCl₄的过程经尾静脉注射hUC-MSC。于第8周末采取小鼠血清，处死小鼠，取小鼠肝脏固定。使用酶联免疫吸附法测定炎症因子水平，自动生化检测仪检测肝功能相关指标，HE染色、Masson染色、天狼星红染色及α-SMA免疫荧光用于评估肝纤维化情况；TGF-β刺激的肝星状细胞（HSC）分别在有无几丁质酶3样蛋白1（CHI3L1）添加的培养基中与hUC-MSC共培养，Western Blot试验检测蛋白表达水平。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Dunnett-t检验。结果 Masson染色、天狼星红染色提示CCl₄组小鼠纤维化较对照组明显（P值均<0.05），MSC组小鼠纤维化较CCl₄组减轻（P值均<0.05）。CCl₄组IL-1β、IL-6、AST、ALT和ALP水平较对照组明显升高（P值均<0.05），MSC组IL-6、AST、ALT及ALP水平较CCl₄组明显降低（P值均<0.05）。CCl₄组CHI3L1和α-SMA的表达均高于对照组和MSC组（P值均<0.05）。细胞培养实验显示，MSC+HSC组Bax表达高于HSC组和MSC+CHI3L1组（P值均<0.05），提示CHI3L1逆转了MSC对活化的HSC的促凋亡作用。结论 hUC-MSC治疗可以改善小鼠肝纤维化，其作用机制可能与抑制CHI3L1从而促进HSC的凋亡相关。
- 肝纤维化 /
- 间充质干细胞 /
- 壳多糖酶3样蛋白质1 /
- 小鼠，近交C57BL
Abstract: Objective To investigate the effect of human umbilical cord mesenchymal stem cells （hUCMSCs） in the treatment of mice with liver fibrosis and its mechanism. Methods A total of 18 specific pathogen-free C57BL/6 mice， aged 6 weeks， were selected and divided into control group （n=6）， carbon tetrachloride （CCl₄） model group （CCl₄ group， n=6）， and hUCMSCs treatment group （MSC group， n=6） using a random number table. The mice in the CCl₄ group and the MSC group were given intraperitoneal injection of CCl₄ solution to establish a mouse model of liver fibrosis， while those in the control group were injected with the same dose of corn oil， and the mice in the MSC group were injected with hUCMSCs via the caudal vein during the injection of CCl₄. At the end of week 8， mouse serum was collected， and the mice were sacrificed to collect and fix the liver. Enzyme-linked immunosorbent assay was used to measure the levels of inflammatory factors； an automatic biochemical detector was used to measure liver function parameters； HE staining， Masson staining， Sirius Red staining， and α-SMA immunofluorescence assay were used to evaluate liver fibrosis. Hepatic stellate cells （HSCs） stimulated by TGF-β were co-cultured with hUCMSCs in the medium with or without chitinase-3 like-protein-1 （CHI3L1）， and Western blot was used to measure the expression levels of proteins. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the Dunnett’s t-test was used for further comparison between two groups. Results Masson staining and Sirius Red staining showed that the CCl₄ group had a significantly higher degree of fibrosis than the control group （both P<0.05）， and the MSC group had significant alleviation of fibrosis compared with the CCl₄ group （both P<0.05）. Compared with the control group， the CCl₄ group had significant increases in the levels of interleukin-1β， interleukin-6 （IL-6）， aspartate aminotransferase （AST）， alanine aminotransferase （ALT）， and alkaline phosphatase （ALP）（all P<0.05）， and compared with the CCl₄ group， the MSC group had significant reductions in the levels of IL-6， AST， ALT， and ALP （all P<0.05）. The CCl₄ group had significantly higher expression levels of CHI3L1 and α-SMA than the control group and the MSC group （all P<0.05）. The cell culture experiment showed that the MSC+HSC group had a significantly higher expression level of Bax than the HSC group and the MSC+CHI3L1 group （both P<0.05）， suggesting that CHI3L1 reversed the pro-apoptotic effect of MSC on activated HSCs. Conclusion This study shows that hUCMSCs can improve liver fibrosis in mice， possibly by inhibiting CHI3L1 to promote the apoptosis of HSCs.
- Hepatic Fibrosis /
- Mesenchymal Stem Cells /
- Chitinase-3-Like Protein 1 /
- Mice， Inbred C57BL

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图 1 各组小鼠肝脏组织病理变化

注： a，HE染色（×100）；b，Masson染色（×100）；c，天狼星红染色（×100）；d，各组小鼠Masson染色面积、天狼星红染色面积比较。

Figure 1. Pathological changes in liver tissue of mice in each group

下载: 全尺寸图片幻灯片

图 2 各组小鼠肝脏组织中α-SMA的表达（×200）

Figure 2. Expression of α-SMA in liver tissue of mice in each group （×200）

下载: 全尺寸图片幻灯片

图 3 CHI3L1和Bax在各组小鼠中的表达水平比较

注： a，肝组织CHI3L1表达水平；b，HSC中Bax表达水平；c，肝组织中Bax表达水平。

Figure 3. Protein expression of CHI3L1 and Bax in each group

下载: 全尺寸图片幻灯片

表 1 各组小鼠血清炎症因子表达水平比较

Table 1. Serum inflammatory factors expression levels in mice of each group

组别	动物数（只）	TNF-α（pg/mL）	IL-1β（pg/mL）	IL-6（pg/mL）	IL-10（pg/mL）
对照组	4	16.39±4.18	12.08±6.67	5.79±1.21	147.15±55.14
CCl₄组	4	14.80±3.00	21.88±4.11^1）	32.31±8.15^1）	204.67±117.45
MSC组	3	19.66±9.44	11.65±3.09	6.27±0.42^2）	144.54±27.82
F值		0.46	5.00	37.15	0.67
P值		>0.05	<0.05	<0.001	>0.05
注：与对照组比较，1） P<0.05；与CCl₄组比较，2） P<0.05。对照组TNF-α和CCl₄组IL-6各有1例样本未测出，CCl₄组TNF-α按Chauvenet准则剔除1例偏离值。

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表 2 各组小鼠血清ALT、AST、ALP水平比较

Table 2. Serum ALT， AST， and ALP levels in mice of each group

组别	动物数（只）	ALT（U/L）	AST（U/L）	ALP（U/L）
对照组	4	43.60±2.97	120.00±6.56	7.58±4.66
CCl₄组	4	126.00±15.51^1）	182.00±20.65^1）	119.00±9.51^1）
MSC组	3	49.00±6.52^2）	146.00±12.65^2）	85.20±9.04^2）
F值		78.62	16.77	32.46
P值		<0.001	<0.01	<0.001
注：与对照组比较，1） P<0.05；与CCl₄组比较，2） P<0.05。

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