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中华损伤与修复杂志(电子版) ›› 2025, Vol. 20 ›› Issue (04) : 339 -346. doi: 10.3877/cma.j.issn.1673-9450.2025.04.011

所属专题: 文献

论著

高糖环境下成纤维细胞分泌CXCL11对人脐静脉内皮细胞迁移的影响及其机制
王小双1,2, 阮琼芳3, 金冬梅4, 韩彦5, 曹萍6, 李炳辉7, 褚志刚2,()   
  1. 1 430065 武汉科技大学医学部医学院
    2 430060 武汉大学附属同仁医院暨武汉市第三医院烧伤科
    3 430060 武汉大学附属同仁医院暨武汉市第三医院烧伤研究所
    4 430060 武汉大学附属同仁医院暨武汉市第三医院病理科
    5 430060 武汉大学附属同仁医院暨武汉市第三医院检验科
    6 430060 武汉大学附属同仁医院暨武汉市第三医院内分泌科
    7 430077 武汉,华中科技大学同济医学院附属梨园医院创面修复科
  • 收稿日期:2025-05-01 出版日期:2025-08-01
  • 通信作者: 褚志刚
  • 基金资助:
    武汉市科技局知识创新项目(2023020201010192); 湖北省自然科学基金面上项目(2021CFB532); 湖北省卫健委科研项目(WJ2021M260)

Effect and mechanism of CXCL11 secreted by fibroblasts in high glucose environment on the migration of human umbilical vein endothelial cells

Xiaoshuang Wang1,2, Qiongfang Ruan3, Dongmei Jin4, Yan Han5, Ping Cao6, Binghui Li7, Zhigang Chu,2()   

  1. 1 School of Medicine,Wuhan University of Science and Technology,Wuhan 430065,China
    2 Department of Burns,Tongren Hospital of Wuhan University & Wuhan Third Hospital,Wuhan 430060,China
    3 Institute of Burns,Tongren Hospital of Wuhan University & Wuhan Third Hospital,Wuhan 430060,China
    4 Department of Pathology,Tongren Hospital of Wuhan University & Wuhan Third Hospital,Wuhan 430060,China
    5 Department of Laboratory Medicine,Tongren Hospital of Wuhan University & Wuhan Third Hospital,Wuhan 430060,China
    6 Department of Endocrinology,Tongren Hospital of Wuhan University & Wuhan Third Hospital,Wuhan 430060,China
    7 Department of Wound Repair,Liyuan Hospital Affiliated to Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430077,China
  • Received:2025-05-01 Published:2025-08-01
  • Corresponding author: Zhigang Chu
引用本文:

王小双, 阮琼芳, 金冬梅, 韩彦, 曹萍, 李炳辉, 褚志刚. 高糖环境下成纤维细胞分泌CXCL11对人脐静脉内皮细胞迁移的影响及其机制[J/OL]. 中华损伤与修复杂志(电子版), 2025, 20(04): 339-346.

Xiaoshuang Wang, Qiongfang Ruan, Dongmei Jin, Yan Han, Ping Cao, Binghui Li, Zhigang Chu. Effect and mechanism of CXCL11 secreted by fibroblasts in high glucose environment on the migration of human umbilical vein endothelial cells[J/OL]. Chinese Journal of Injury Repair and Wound Healing(Electronic Edition), 2025, 20(04): 339-346.

目的

研究高糖环境下成纤维细胞分泌CXC趋化因子配体11(CXCL11)对人脐静脉内皮细胞(HUVECs)迁移的作用及其机制。

方法

取华中科技大学同济医学院附属梨园医院创面修复科2021年8月收治的1例糖尿病足溃疡(DFU)患者和该院手外科 2021年9月收治的1例急性足外伤患者创缘皮肤组织,行单细胞转录组测序,分析成纤维细胞亚群中趋化因子配体与血管内皮细胞亚群中趋化因子受体的相互作用。纳入2022年1月至2024年12月武汉市第三医院内分泌科DFU患者和该院烧伤科急性足外伤患者各3例,取其石蜡包埋组织样本,采用免疫组化染色观察创缘皮肤组织中CXCL11及CXC趋化因子受体7(CXCR7)的表达。取正常人包皮成纤维细胞(HFF-1)和经D-葡萄糖处理后的高糖HFF-1细胞,分别为正常组和高糖组,收集2组培养48 h后的上清液作为正常条件培养基(NFs-CM)和高糖条件培养基(GFs-CM)。采用实时荧光定量PCR(RT-qPCR)法检测正常组和高糖组细胞中CXCL11 mRNA表达水平。取HUVECs分为完全培养基组(CPM组)、NFs-CM组和GFs-CM组,行划痕实验计算划痕后12、24、36 h细胞迁移率。采用蛋白质印迹法检测CPM组、NFs-CM组和GFs-CM组HUVECs处理36 h后的CXCR7蛋白表达水平。利用CXCL11中和抗体处理HUVECs,实验分为6组:CPM组、CPM+anti-CXCL11组、NFs-CM组、NFs-CM+anti-CXCL11组、GFs-CM组和GFs-CM+anti-CXCL11组,重复划痕实验。所有实验分别重复3次。对数据行单因素方差分析(ANOVA)、Tukey's检验和t检验。

结果

与急性足外伤患者足背创缘皮肤组织比较,DFU患者创缘皮肤组织成纤维细胞亚群中趋化因子配体CXCL11与血管内皮细胞亚群中趋化因子受体CXCR7的相互作用明显增强,CXCL11及CXCR7蛋白表达水平显著升高(t=25.870、18.150, P<0.001)。与正常组比较,高糖组HFF-1细胞中CXCL11 mRNA表达水平显著升高(t=8.412,P=0.001)。划痕后36 h,GFs-CM组HUVECs细胞迁移率显著低于CPM组和NFs-CM组(P<0.001)。蛋白质印迹分析显示,GFs-CM组CXCR7蛋白表达水平显著高于CPM组和NFs-CM组(P<0.001)。划痕后12、24、36 h,与GFs-CM组比较,GFs-CM+anti-CXCL11组HUVECs细胞迁移明显增强(P<0.05)。

结论

高糖促进HFF-1细胞分泌CXCL11,其细胞培养上清液抑制HUVECs迁移并上调CXCR7蛋白表达;外源性添加CXCL11中和抗体可增强HUVECs迁移能力。

Objective

To investigate the effect of C-X-C motif chemokine ligand 11 (CXCL11) secreted by fibroblasts in high glucose environment on the migration of human umbilical vein endothelial cells (HUVECs) and the potential mechanisms.

Methods

The wound edge skin tissue of a diabetic foot ulcer (DFU) patient admitted to the Department of Wound Repair of Liyuan Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology in August 2021, and an acute foot injury patient treated in the Hand Surgery Department of the same hospital in September 2021 was collected. The single-cell transcriptome sequencing was performed to analyze the interaction between chemokine ligands of fibroblasts subgroup and chemokine receptors of vascular endothelial cells subgroup. Paraffin-embedded tissue samples were collected from DFU patients (n=3) in the Department of Endocrinology and acute foot injury patients (n=3) in the Burns Department at Wuhan Third Hospital between January 2022 and December 2024. Immunohistochemistry staining was used to observe the expression of CXCL11 and C-X-C motif chemokine receptor 7 (CXCR7) in the wound edge skin tissue. Normal human foreskin fibroblasts (HFF-1) and HFF-1 treated with D-glucose were divided into normal group and high glucose group, respectively. After 48 h of culture, the supernatants from normal group and high glucose group were collected as normal fibroblasts-conditioned medium (NFs-CM) and glucose-treated fibroblasts-conditioned medium (GFs-CM), respectively. The mRNA expression levels of CXCL11 in the cells of normal group and high glucose group were detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR). HUVECs were divided into complete medium group (CPM group), NFs-CM group, and GFs-CM group, the scratch test was performed to calculate the cell migration rates at 12, 24, and 36 h after scratching. Western blot was employed to detect the expression levels of CXCR7 protein in HUVECs from CPM group, NFs-CM group, and GFs-CM group, treated for 36 h. After treatment with a neutralizing antibody against CXCL11, the experiment was divided into six groups: CPM group, CPM+anti-CXCL11 group, NFs-CM group, NFs-CM+anti-CXCL11 group, GFs-CM group, and GFs-CM+anti-CXCL11 group. Scratch tests were repeated in each group. All experiments were repeated three times. One-way ANOVA,Tukey's test and t-test were used for statistical analysis.

Results

Compared with the wound edge skin tissue of acute foot injury, the interaction between the chemokine ligand CXCL11 of the fibroblasts subgroup and the chemokine receptor CXCR7 of the vascular endothelial cells subgroup was significantly enhanced in the wound edge skin tissue of DFU, and the expression levels of CXCL11 and CXCR7 protein in tissue from DFU were notably higher (t=25.870, 18.150; P<0.001). Compared with normal group, the mRNA expression level of CXCL11 in HFF-1 from high glucose group was significantly elevated (t=8.412,P=0.001). At 36 h after scratching, the migration rate of HUVECs in GFs-CM group was significantly lower than that in CPM group and NFs-CM group (P<0.001). Western blot analysis demonstrated that CXCR7 protein expression was significantly higher in GFs-CM group than that in CPM group and NFs-CM group (P<0.001). At 12, 24, and 36 h after scratching,compared with GFs-CM group, the migration of HUVECs was significantly enhanced in GFs-CM+anti-CXCL11 group (P<0.001).

Conclusion

High glucose promotes the secretion of CXCL11 by HFF-1, and the stimulation of its cell culture supernatant can inhibit HUVECs migration and upregulate CXCR7 protein expression. Exogenous CXCL11 neutralizing antibody can enhance the migration capacity of HUVECs.

表1 采用RT-qPCR法检测HFF-1细胞中CXCL11 mRNA表达水平相关引物序列及产物大小
图1 基于单细胞转录组测序的DFU创缘皮肤组织成纤维细胞与血管内皮细胞受体-配体相互作用分析 注:AWE为急性足外伤患者足背创缘皮肤组织,CWE为DFU患者创缘皮肤组织;CXCL为趋化因子配体,CXCR为趋化因子受体;ACKR3为非典型趋化因子受体3,又称CXCR7。图中从左至右各列依次为毛细血管内皮细胞与静脉内皮细胞、静脉内皮细胞与静脉内皮细胞、成纤维细胞亚群1与静脉内皮细胞、成纤维细胞亚群3与静脉内皮细胞、成纤维细胞亚群5与静脉内皮细胞、成纤维细胞亚群6与静脉内皮细胞
图2 急性足外伤及DFU患者创缘皮肤组织CXCL11和CXCR7表达免疫组化染色。A、B示急性足外伤患者足背创缘皮肤组织CXCL11和CXCR7表达;C、D示DFU患者创缘皮肤组织CXCL11和CXCR7表达 注:CXCL11和CXCR7阳性表达呈棕黄色。CXCL11为CXC趋化因子配体11,CXCR7为CXC趋化因子受体7
表2 急性足外伤及DFU患者创缘皮肤组织中CXCL11及CXCR7表达阳性面积比(%,x±s
图3 采用划痕实验分析GFs-CM对HUVECs细胞迁移的影响。A-D示CPM组0、12、24、36 h划痕面积;E-H示NFs-CM组0、12、24、36 h划痕面积;I-L示GFs-CM组0、12、24、36 h划痕面积 注:CPM组为使用RPMI-1640完全培养基培养HUVECs;NFs-CM组为使用NFs-CM培养HUVECs;GFs-CM组为使用GFs-CM培养HUVECs。NFs-CM为正常条件培养基,GFs-CM为高糖条件培养基,HUVECs为人脐静脉内皮细胞
表3 CPM组、NFs-CM组和GFs-CM组划痕后各时间点HUVECs细胞迁移率比较(%,x±s
图4 采用蛋白质印迹法检测GFs-CM对HUVECs细胞CXCR7蛋白表达的影响 注:1、2、3分别为CPM组、NFs-CM组、GFs-CM组。CPM组为使用RPMI-1640完全培养基培养HUVECs;NFs-CM组为使用NFs-CM培养HUVECs;GFs-CM组为使用GFs-CM培养HUVECs。CXCR7为CXC趋化因子受体7,GAPDH为3-磷酸甘油醛脱氢酶
图5 采用划痕实验分析CXCL11中和抗体处理后GFs-CM对HUVECs细胞迁移的影响。A-D示CPM组0、12、24、36 h划痕面积;E-H示CPM+anti-CXCL11组0、12、24、36 h划痕面积;I-L示NFs-CM组0、12、24、36 h划痕面积;M-P示NFs-CM+anti-CXCL11组0、12、24、36 h划痕面积;Q-T示GFs-CM组0、12、24、36 h划痕面积;U-X示GFs-CM+anti-CXCL11组0、12、24、36 h划痕面积 注:CPM组为使用RPMI-1640完全培养基培养HUVECs;NFs-CM组为使用NFs-CM培养HUVECs;GFs-CM组为使用GFs-CM培养HUVECs。CPM+anti-CXCL11组、NFs-CM+anti-CXCL11组、GFs-CM+anti-CXCL11组分别为CPM、NFs-CM、GFs-CM经CXCL11中和抗体处理后对应组别。NFs-CM为正常条件培养基,GFs-CM为高糖条件培养基,HUVECs为人脐静脉内皮细胞
表4 CXCL11中和抗体处理后划痕实验各时间点HUVECs细胞迁移率比较(%,x±s
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