FGF2对小鼠肠道间质细胞R-spondin 1表达的调控作用

doi:10.3969/j.issn.1674-8115.2025.08.001

上海交通大学学报（医学版） ›› 2025, Vol. 45 ›› Issue (8): 939-948.doi: 10.3969/j.issn.1674-8115.2025.08.001

• 论著 · 基础研究 • 下一篇

FGF2对小鼠肠道间质细胞R-spondin 1表达的调控作用

李景聪¹^,², 赵涵¹^,², 林巧雯¹^,²^,³, 孙宏翔¹^,², 苏冰¹^,²(), 伍宁波¹^,²()

^1.上海交通大学基础医学院免疫学与微生物学系，上海 200025
^2.上海交通大学医学院上海市免疫学研究所，上海 200025
^3.上海交通大学医学院附属瑞金医院消化内科，上海 200025

收稿日期:2025-01-03 接受日期:2025-03-07 出版日期:2025-08-28 发布日期:2025-08-26
通讯作者: 苏冰，教授，博士；电子信箱：bingsu@sjtu.edu.cn
伍宁波，研究员，博士；电子信箱：wuningbo@shsmu.edu.cn。
基金资助:
国家自然科学基金(32170895);上海市科学技术委员会项目(22ZR1480700);上海市科学技术委员会项目(22QA1408000)

Regulatory effect of FGF2 on the expression of R-spondin 1 in mouse intestinal stromal cells

LI Jingcong¹^,², ZHAO Han¹^,², LIN Qiaowen¹^,²^,³, SUN Hongxiang¹^,², SU Bing¹^,²(), WU Ningbo¹^,²()

^1.Department of Immunology and Microbiology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China
^2.Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
^3.Department of Gastroenterology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

Received:2025-01-03 Accepted:2025-03-07 Online:2025-08-28 Published:2025-08-26
Contact: SU bing，E-mail：bingsu@sjtu.edu.cn
WU Ningbo，E-mail：wuningbo@shsmu.edu.cn.
Supported by:
National Natural Science Foundation of China(32170895);Project of Science and Technology Commission of Shanghai Municipality(22ZR1480700)

摘要/Abstract

摘要：

目的·初步探究成纤维细胞生长因子2（fibroblast growth factor 2，FGF2）对小鼠结肠CD34⁺CD81⁺间质细胞R-spondin 1（Rspo1）表达的调控作用及机制。方法·利用流式细胞术分选Rspo1-tdTomato荧光报告基因小鼠结肠中CD45^－CD326^－CD31^－GP38⁺CD81⁺Rspo1-tdTomato⁺细胞，并进行体外培养。运用流式细胞术检测培养14 d后该类细胞表面蛋白标志物分子的表达情况。通过实时荧光定量PCR（quantitative real-time PCR，qPCR）检测该细胞分别在FGF2、FGF9、表皮生长因子（epidermal growth factor，EGF）、血小板衍生生长因子-bb（platelet-derived growth factor-bb，PDGFbb）、胰岛素样生长因子1（insulin-like growth factor 1，IGF1）、肝细胞生长因子（hepatocyte growth factor，HGF）刺激条件下Rspo1的表达情况。通过转录组测序和生物信息学方法分析FGF2调控Rspo1表达的信号通路，并用信号通路抑制剂及qPCR进行初步验证。结果·流式分选得到的小鼠结肠间质细胞经过体外培养14 d后，能够表达CD34、CD81和糖蛋白GP38，而不表达CD45、CD326、CD31等其他细胞谱系标志分子。qPCR结果发现，20 ng/mL的FGF2即可显著抑制该细胞Rspo1的表达，其他生长因子则无显著抑制作用。转录组测序和生物信息学分析结果显示，丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）信号通路可能是FGF2调节Rspo1表达的关键信号通路。qPCR结果显示，丝裂原细胞外激酶1/2（mitogen extracellular kinase 1/2，MEK1/2）抑制剂U0216预处理可逆转FGF2对Rspo1表达的抑制作用。结论·FGF2可能通过MEK1/2-细胞外调节蛋白激酶1/2（extracellular regulated protein kinase 1/2，ERK1/2）通路抑制小鼠结肠CD34⁺CD81⁺间质细胞Rspo1的表达。

关键词: R-spondin 1基因, 成纤维细胞生长因子2, CD34⁺CD81⁺间质细胞, 丝裂原活化蛋白激酶通路

Abstract:

Objective ·To preliminarily investigate the regulatory effect and underlying mechanism of fibroblast growth factor 2 (FGF2) on R-spondin 1 (Rspo1) expression in CD34⁺CD81⁺ stromal cells from the mouse colon. Methods ·Colonic CD45^-CD326^-CD31^-GP38⁺CD81⁺Rspo1-tdTomato⁺ stromal cells were sorted from Rspo1-tdTomato reporter mice by flow cytometry and subsequently cultured in vitro. The expression of surface protein markers was evaluated by flow cytometry after 14 d of culture. qPCR was employed to quantify Rspo1 expression in response to stimulation with FGF2, FGF9, epidermal growth factor (EGF), platelet-derived growth factor-bb (PDGF-bb), insulin-like growth factor 1 (IGF1), or hepatocyte growth factor (HGF). RNA sequencing and bioinformatic analyses were used to identify the signaling pathways underlying FGF2-mediated regulation of Rspo1, followed by preliminary validation with pathway-specific inhibitors and qPCR. Results ·After 14 d of culture, the sorted colonic stromal cells retained expression of CD34, CD81, and glycoprotein GP38, while remaining negative for other lineages markers CD45, CD326, and CD31. qPCR revealed that 20 ng/mL FGF2 significantly suppressed Rspo1 expression, whereas the other tested growth factors exerted no notable effect. RNA sequencing and bioinformatic analysis indicated that mitogen-activated protein kinase (MAPK) signaling pathway played a key role in the regulatory effect of FGF2 on Rspo1. qPCR further demonstrated that pretreatment with U0126, an inhibitor of mitogen extracellular kinase 1/2 (MEK1/2), reversed FGF2-mediated suppression of Rspo1 expression. Conclusion ·FGF2 may inhibit Rspo1 expression in mouse colonic CD34⁺CD81⁺ stromal cells via the MEK1/2-extracellular regulated protein kinase 1/2 (ERK1/2) signaling pathway.

Key words: R-spondin 1 gene, fibroblast growth factor 2 (FGF2), CD34⁺CD81⁺ stromal cell, mitogen-activated protein kinase (MAPK) pathway

中图分类号:

R392.9

李景聪, 赵涵, 林巧雯, 孙宏翔, 苏冰, 伍宁波. FGF2对小鼠肠道间质细胞R-spondin 1表达的调控作用[J]. 上海交通大学学报（医学版）, 2025, 45(8): 939-948.

LI Jingcong, ZHAO Han, LIN Qiaowen, SUN Hongxiang, SU Bing, WU Ningbo. Regulatory effect of FGF2 on the expression of R-spondin 1 in mouse intestinal stromal cells[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(8): 939-948.

图/表 6

表1 PCR引物序列

Tab 1 Primer sequences for PCR

Primer name	Sequence (5'→3')
Hprt-F	TCCTCCTCAGACCGCTTTT
Hprt-R	CCTGGTTCATCATCGCTAATC
Rspo1-F	GGGATCAAGGGCAAGAGACAG
Rspo1-R	CTGGCGGATGTCGTTCCTC

图1 肠道CD34+CD81+ 间质细胞培养体系的构建和流式细胞术的验证Note： A. Sorting and culture strategy of CD34+CD81+ stromal cells. B. Gating strategy for sorting colon stromal cells from Rspo1-tdTomato mice. C. Flow cytometry detection of lineage-related markers in stromal cells. D. Flow cytometry detection of changes in major surface markers of CD34+CD81+ stromal cells. E. The number change of CD34+CD81+ stromal cells before and after culture.

Fig 1 Establishment and flow cytometry validation of CD34+CD81+ intestinal stromal cell culture system

图2 FBS对CD34+CD81+ 间质细胞 Rspo1 表达的抑制作用Note： A. qPCR showing relative expression of Rspo1 in stromal cells before (sorted cells) and after culture (cultured cells) (n=3). B. qPCR showing relative expression of Rspo1 in stromal cells after FBS and other additives were removed (n=3). C. Live-cell fluorescence imaging showing tdTomato changes in stromal cells under basal medium treatment. Scale bar=150 μm. D. qPCR showing relative expression of Rspo1 in stromal cells with FBS treatment (n=3).

Fig 2 Inhibitory effect of FBS on Rspo1 expression in CD34+CD81+ stromal cells

图3 FGF2对CD34+CD81+ 间质细胞 Rspo1 表达的抑制作用Note： A. qPCR showing relative expression of Rspo1 in stromal cells after different cytokine stimulations (n=3). B. qPCR showing relative expression of Rspo1 in stromal cells after treatment with different concentrations of FGF2 (n=3). C. Live-cell fluorescence imaging showing tdTomato changes in stromal cells under FGF2 treatment. Scale bar=150 μm.

Fig 3 Inhibitory effect of FGF2 on the expression of Rspo1 in CD34+CD81+ stromal cells

图4 FGF2通过MAPK通路抑制CD34+CD81+ 间质细胞 Rspo1 表达Note： A. CD34+CD81+ stromal cell stimulation strategy. B. Volcano plot depicting transcript levels of regeneration/wound healing marker genes and homeostatic epithelial growth genes in stromal cells with or without FGF2 treatment (n=3). C/D. Selected GO terms (C) and KEGG pathways (D) for upregulated/downregulated genes in stromal cells with or without FGF2 treatment. E. qPCR showing relative expression of Rspo1 in stromal cells treated with different inhibitors (n=3). Bmp2—bone morphogenetic protein 2; Tgfb1—transforming growth factor β1; Mmp9—matrix metalloproteinase-9; Ptgs2 —prostaglandin-endoperoxide synthase 2; Lif—leukemia inhibitory factor; Grem1—gremlin-1; Fgf18—fibroblast growth factor 18; Serpine1—serpin family E member 1; Twist1—Twist-related protein 1; Sox7—SRY-box transcription factor 7.

Fig 4 FGF2 inhibits the expression of Rspo1 through MAPK signaling pathway in CD34+CD81+ stromal cells

图5 FGF2抑制CD34+CD81+ 间质细胞 Rspo1 表达分子机制示意图Note： FGFR—fibroblast growth factor receptor.

Fig 5 Schematic diagram of the molecular mechanism by which FGF2 inhibits the expression of Rspo1 in CD34+CD81+ stromal cells

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FGF2对小鼠肠道间质细胞R-spondin 1表达的调控作用

Regulatory effect of FGF2 on the expression of R-spondin 1 in mouse intestinal stromal cells

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