人源脐带间充质干细胞移植通过Chi3l1抑制M1型巨噬细胞极化减轻1型糖尿病小鼠的炎症反应

doi:10.12122/j.issn.1673-4254.2025.12.21

摘要/Abstract

摘要：

目的探究Chi3l1调控人源脐带间充质干细胞（hUC-MSCs）治疗1型糖尿病（T1DM）中的重要作用。方法通过慢病毒载体构建稳定低表达Chi3l1的hUC-MSCs（sh-Chi3l1-MSCs），经流式细胞术及成脂、成骨诱导分化鉴定其干细胞特性；采用链脲佐菌素诱导T1DM小鼠模型，分别给予sh-NC-MSCs和sh-Chi3l1-MSCs治疗，监测小鼠临床表现、血糖、体质量变化，苏木精-伊红染色（HE）评价胰腺组织病理变化，免疫组化与免疫荧光检测胰岛内胰岛素含量和巨噬细胞浸润；体外共培养实验诱导骨髓巨噬细胞极化，流式细胞术检测M1和M2比例，采用qPCR检测巨噬细胞标志分子iNOS、Arg-1和炎症因子TNF-α、IL-6、IL-10、IL-13、IL-1β转录水平表达。结果 sh-Chi3l1-MSCs仍保持MSCs的特性，但对T1DM小鼠治疗效果变差；免疫荧光结果显示sh-Chi3l1-MSCs治疗组小鼠胰腺组织中巨噬细胞含量要高于sh-NC-MSCs治疗组（P<0.05）；体外巨噬细胞诱导分化实验结果表明，sh-Chi3l1-MSCs抑制M1型巨噬细胞极化能力降低，同时促进M2型产生的能力也降低。qPCR结果表明， sh-Chi3l1-MSCs组中巨噬细胞M1型标志分子iNOS及相关的炎症因子TNF-α、IL-6和IL-1β表达升高（P<0.05），而M2型巨噬细胞标志分子Arg-1及分泌的细胞因子IL-13和IL-10降低（P<0.05）。结论 hUC-MSCs通过Chi3l1抑制M1型巨噬细胞产生，从而减轻T1DM模型小鼠的炎症反应。

关键词: 人源脐带间充质干细胞, Chi3l1, MI型巨噬细胞极化, 1型糖尿病

Abstract:

Objective To explore the role of Chi3l1 in human umbilical cord mesenchymal stem cell (hUC-MSCs) therapy of type 1 diabetes. Methods hUC-MSCs with stable Chi3l1 knockdown (sh-Chi3l1-MSCs) were constructed using a lentiviral vector and characterized by flow cytometry and adipogenic and osteogenic induction. In adult C57BL/6J mouse models of streptozotocin-induced T1DM, the therapeutic effects of sh-NC-MSCs and sh-Chi3l1-MSCs grafting were evaluated by observing changes in clinical manifestations, blood glucose, body weight and pancreatic tissue pathologies. Insulin content and macrophage infiltration in the islets were detected using immunohistochemistry and immunofluorescence staining. The effects of these two stem cells on induced polarization of co-cultured mouse bone marrow macrophages were assessed using flow cytometry by detecting the mRNA expressions of iNOS, Arg-1, TNF-α, IL-6, IL-10, IL-13, and IL-1β using qPCR. Results The constructed sh-Chi3l1-MSCs retained the characteristics of MSCs but showed reduced therapeutic efficacy in T1DM mice. Immunofluorescence staining showed that the number of macrophages in the pancreatic tissue of the mice treated with sh-Chi3l1-MSCs was higher than that in MSCs treatment group. In the co-culture experiments, sh-Chi3l1-MSCs exhibited a lowered capacity to suppress M1 polarization of the macrophages and a reduced efficacy to promote differentiation of M2-type macrophage subset. Analysis with qPCR showed that the expressions of M1 macrophage marker iNOS and the inflammatory factors TNF-α, IL-6, and IL-1β increased, while the expressions of M2 macrophage marker Arg-1 and the cytokines IL-13 and IL-10 were decreased significantly in sh-Chi3l1-MSCs group. Conclusion In T1DM mouse models, hUC-MSCs mitigate inflammatory responses by suppressing the production of pro-inflammatory M1-type macrophages via Chi3l1.

Key words: human umbilical cord mesenchymal stem cells, Chi3l1, M1-type macrophage polarization, type 1 diabetes mellitus

刘新新, 徐迎芮, 盛红娜, 刘昊. 人源脐带间充质干细胞移植通过Chi3l1抑制M1型巨噬细胞极化减轻1型糖尿病小鼠的炎症反应[J]. 南方医科大学学报, 2025, 45(12): 2738-2746.

Xinxin LIU, Yingrui XU, Hongna SHENG, Hao LIU. Human umbilical cord mesenchymal stem cell grafting alleviates inflammatory response in type 1 diabetic mice by suppressing M1 macrophage polarization through Chi3l1[J]. Journal of Southern Medical University, 2025, 45(12): 2738-2746.

图/表 5

表1 qPCR引物序列

Tab.1 Primer sequences for qPCR

Primer	Sequence (5'→3')
GAPDH	Forword:	ACACTCCAGCTGGGGCTGTACTGACTTGATG
	Reverse:	CTCAACTGGTGTCGTGGAGTCG GCAATTCAGTTGAGCATCAGAT
Chi3l1	Forword:	AAGCAACGATCACATCGACAC
	Reverse:	TCAGGTTGGGGTTCCTGTTCT
iNOS	Forword:	CCAACAATACAAGATGACCCT
	Reverse:	TTCTGGAACATTCTGTGCTG
Arg-1	Forword:	GAACTGAAAGGAAAGTTCCCA
	Reverse:	AATGTACACGATGTCTTTGGC
TNF-α	Forword:	GGCAGGTCTACTTTFFAGTCATTGC
	Reverse:	ACATTCGAGGCTCCAGTGAATTCGG
IL-6	Forword:	TACCACTTCACAAGTCGGA
	Reverse:	AATTGCCATTGCACAACTC
IL-10	Forword:	TTAATAAGCTCCAAGACCAAGG
	Reverse:	CATCATGTATGCTTCTATGCAG
IL-13	Forword:	TCCCTCAAGTTCTTTGTTCG
	Reverse:	CGCACCTTTCTGGTTACAC
IL-1β	Forword:	CCTCAAAGGAAAGAATCTATACCTG
	Reverse:	CTTGGGATCCACACTCTCC

图1 敲低Chi3l1的MSCs的鉴定

Fig.1 Identification of MSCs with stable Chi3l1 knockdown. A: Morphology of sh-NC-MSCs. B: Oil Red O staining of sh-NC-MSCs after adipogenic induction. C: Alizarin Red S staining of sh-NC-MSCs after osteogenic induction. D: Morphology of sh-Chi3l1-MSCs. E: Oil Red O staining of sh-Chi3l1-MSCs after adipogenic induction. F: Alizarin Red S staining of sh-Chi3l1-MSCs after osteogenic induction. G: Detection of surface markers on sh-Chi3l1-MSCs by flow cytometry. H: Chi3l1 mRNA expression level in sh-Chi3l1-MSCs. I: Protein level of Chi3l1 in sh-Chi3l1-MSCs detected by Western blotting. **P<0.01 vs sh-NC-MSCs.

图2 Chi3l1敲低影响MSCs治疗T1DM小鼠的疗效

Fig.2 Chi3l1 knockdown attenuates therapeutic efficacy of MSCs in T1DM mice. A: Blood glucose levels of the mice in each group over the course of treatment. B: Body weight changes of the mice in each group (*P<0.05, **P<0.01, ***P<0.001 vs T1DM group; #P<0.05, ##P<0.01 vs sh-NC-MSCs group). C: Pancreatic pathological changes in each group (HE staining, original magnification: ×100). D: Insulin content in the pancreas of the mice in each group detected by immunohistochemical staining (×100).

图3 小鼠胰腺组织内胰岛素含量及巨噬细胞数量变化

Fig.3 Changes in insulin content and macrophage infiltration in pancreatic tissues of mice. A: Insulin content (green) and macrophage infiltration (red) in pancreatic tissues of mice from each group detected by immunofluorescence staining (×200). B. Statistical analysis of insulin content within individual islets of mice in each group. C: Quantitative analysis of the mean immunofluorescence intensity of macrophages in pancreatic tissues across the groups. **P<0.01,***P<0.001 vs NC group; #P<0.05, ###P<0.001 vs T1DM group; ▲P<0.05 vs sh-NC-MSCs group.

图4 Chi3l1影响hUC-MSCs调控巨噬细胞极化

Fig.4 Chi3l1 influences hUC-MSCs-mediated regulation of macrophage polarization. A: Frequency of M1-type macrophages analyzed using flow cytometry. B: Frequency of M2-type macrophages analyzed using flow cytometry. C: Expression of iNOS mRNA detected with qPCR. D: Expressions of mRNAs for inflammatory cytokines detected by qPCR. E: Expression of Arg-1 mRNA detected with qPCR. F: Expressions of mRNAs for anti-inflammatory cytokines detected by qPCR. *P<0.05, **P<0.01 vs Mφ group; #P<0.05 vs sh-NC-MSCs group.

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