CXCL2-CXCR2轴在血管内皮细胞趋化循环纤维细胞过程中的作用研究

doi:10.3969/j.issn.1005-4979.2022.05.004

摘要/Abstract

摘要：

目的：探讨人CXC趋化因子配体2[chemokine (C-X-C motif) ligand 2，CXCL2]及其受体CXC趋化因子受体2 [chemokine (C-X-C) receptor 2，CXCR2]，即CXCL2-CXCR2轴，在血管内皮细胞（vascular endothelial cells, VECs）趋化循环纤维细胞（circulating fibrocytes, CFs）中的作用。方法：将人CFs与人脐静脉内皮细胞（human umbilical vein endothelial cells，HUVECs）在Transwell小室内共培养；用基因芯片技术检测共培养后上调的趋化因子，并筛选出CXCL2、CC趋化因子配体23[chemokine (C-C motif) ligand 23，CCL23]、CXC趋化因子配体16[chemokine (C-X-C motif) ligand 16，CXCL16] 3种趋化因子；然后利用实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）、酶联免疫吸附试验（enzyme-linked immunosorbent assay，ELISA）、蛋白质印迹法（Western blotting）进一步筛选共培养后上调的趋化因子及其受体；最后采用趋化因子及趋化因子受体阻断实验明确在VECs趋化循环纤维细胞过程中起作用的趋化因子及其受体。结果：HUVECs与CFs共培养后，CCL23的mRNA表达水平上调；CXCL2的mRNA表达水平及蛋白表达水平均发生了显著上调。同时，CXCR2的mRNA表达水平及蛋白表达水平也发生了显著上调。趋化因子阻断实验表明，在2种细胞的Transwell共培养体系内分别添加CXCL2及CXCR2的多克隆抗体后，发生迁移的人CFs数量明显减少。结论：CXCL2-CXCR2轴可能在VECs趋化CFs的过程中起一定的正向调节作用。

关键词: 颌面部创伤, 血管新生, 血管内皮细胞, 循环纤维细胞, 趋化

Abstract:

Objective: To investigate the role of human chemokine(C-X-C motif) ligand 2(CXCL2) and its receptor chemokine(C-X-C) receptor 2(CXCR2) in chemotaxis of circulating fibrocytes(CFs) by vascular endothelial cells (VECs). Methods: Human CFs and human umbilical vein endothelial cells (HUVECs) were co-cultured in the Transwell plate. The up-regulated chemokines after co-culture were detected by gene chip technology, and three chemokines CXCL2, chemokine (C-C motif) ligand 23 (CCL23), chemokine (C-X-C motif) ligand 16 (CXCL16) were screened out, and then real-time quantitative polymerase chain reaction (RT-qPCR), enzyme-linked immunosorbent assay (ELISA） and Western blotting were utilized to further screen the up-regulated chemokines and their receptors after co-culture. Finally, the chemokine and chemokine receptor blocking experiments were used to confirm the chemokine-receptor axis, which might play a role in the chemotaxis of CFs by VECs. Results: After HUVECs were co-cultured with CFs, the mRNA expression level of CCL23 was up-regulated; the mRNA expression level and protein expression level of CXCL2 were significantly up-regulated. At the same time, the mRNA expression level and protein expression level of CXCR2 were significantly up-regulated. Chemokines blocking experiments showed that after adding CXCL2 and CXCR2 polyclonal antibodies in the Transwell co-culture system of the two cells, the number of migrating human CFs was significantly reduced. Conclusion: The CXCL2-CXCR2 axis may play a positive regulatory role in the chemotaxis of CFs by VECs.

Key words: maxillofacial trauma, angiogenesis, vascular endothelial cells, circulating fibrocytes, chemotaxis

中图分类号:

R782

庞楠, 王艳, 李学拥. CXCL2-CXCR2轴在血管内皮细胞趋化循环纤维细胞过程中的作用研究[J]. 《口腔颌面外科杂志》, 2022, 32(5): 284-291.

PANG Nan, WANG Yan, LI Xueyong. The role of CXCL2-CXCR2 axis in chemotaxis of circulating fibrocytes by vascular endothelial cells[J]. 《Journal of Oral and Maxillofacial Surgery》, 2022, 32(5): 284-291.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2022/V32/I5/284

图/表 11

表1 RT-qPCR引物序列信息

Table 1 Coding sequence of primers involved in RT-qPCR

基因	正向引物（5′-3′）	反向引物（5′-3′）
CXCL16	5′-TACACGAGGTTCCAGCTCCT-3′	5′-CACAATCCCCGAGTAAGCAT-3′
CXCL2	5′-GGGAATTCACCTCAAGAACATC-3′	5′-GACAAGCTTTCTGCCCATTC-3′
CCL23	5′-TTTGAAACGAACAGCGAGTG-3′	5′-TGTGTCCAGCTTCAGCATTC-3′
CXCR2	5′-GTGAACCAGAATCCCTGGAA-3′	5′-GCCGACCCTGCTGTATAAGA-3′
GAPDH	5′-TGGATGGCCCCTCCGGGAAA-3′	5′-AGTGGGGACACGGAAGGCCA-3′

图1 CFs体外培养2周时的形态（×200）

Figure 1 Morphology of CFs cultured for 2 weeks in vitro（×200）

图2 HUVECs体外培养3 d时的形态（×200）

Figure 2 Morphology of HUVECs cultured for 3 days in vitro（×200）

图3 CFs体外培养14 d后行的免疫荧光染色（×200）

Figure 3 Immunofluorescent staining of CFs cultured for 14 days in vitro (×200) A. DAPI; B. CD45; C. COL-Ⅰ; D. CD45/COL-Ⅰ

图4 基因芯片检测CFs与HUVECs共培养前后6种趋化因子的表达水平 *表示P<0.05，**表示P<0.01，与HUVECs单独培养组比较。

Figure 4 Expression levels of the six chemokines in HUVECs before and after co-culture with CFs by gene microarray

图5 RT-qPCR检测CFs与HUVECs共培养前后3种趋化因子mRNA的表达水平 *表示P<0.05，与HUVECs单独培养组比较。

Figure 5 Relative mRNA expression levels of three chemokines in HUVECs before and after co-culture with CFs by RT-qPCR

图6 HUVECs与CFs共培养前后培养液中3种趋化因子的ELISA检测 A. CCL23的表达；B. CXCL2的表达；C. CXCL16的表达。**表示P<0.01，与HUVECs单独培养组比较。

Figure 6 Concentrations of the three chemokines in the medium of HUVECs before and after co-culture with CFs measured by ELISA

图7 RT-qPCR检测HUVECs与CFs共培养前后CFs中CXCR2的 mRNA表达 **表示P<0.01，与CFs单独培养组比较。

Figure 7 Expression of CXCR2 mRNA in CFs before and after co-culture with HUVECs measured by RT-qPCR

图8 Western blotting检测HUVECs与CFs共培养前后CFs中CXCR2的表达 A. CXCR2蛋白表达的电泳图，a代表CFs单独培养组，b代表CFs与HUVECs共培养组；B. CXCR2的蛋白表达水平，**表示P<0.01，与CFs单独培养组比较。

Figure 8 Expression of CXCR2 in CFs before and after co-culture with HUVECs measured by Western blotting

图9 CXCL2阻断实验 A. 结晶紫染色显示迁移的CFs（×200）；B. CXCL2多抗组与对照组中迁移的CFs细胞数，**表示P<0.01，与对照组比较。

Figure 9 CXCL2 blocking experiment

图10 CXCR2阻断实验 A. 结晶紫染色显示迁移的CFs（×200）；B. CXCR2多抗组与对照组中迁移的CFs细胞数，*表示P<0.05，与对照组比较。

Figure 10 CXCR2 blocking experiment

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