肿瘤相关血管内皮细胞对单核细胞趋化的影响

doi:10.3969/j.issn.1005-4979.2020.01.003

《口腔颌面外科杂志》 ›› 2020, Vol. 30 ›› Issue (1): 9-15. doi: 10.3969/j.issn.1005-4979.2020.01.003

肿瘤相关血管内皮细胞对单核细胞趋化的影响

陈雨蒙¹^,²(), 乔春燕¹^,², 刘欣辰¹^,³, 孟琳¹^,², 任春霞¹^,⁴, 郑梦丹¹^,², 郑适泽¹^,², 孙宏晨¹^,²()

¹ 吉林省牙发育及颌骨重塑与再生重点实验室吉林长春 130021
² 吉林大学口腔医院病理科，吉林长春 130021
³ 牙体牙髓科，吉林长春 130021
⁴ 牙周科，吉林长春 130021

收稿日期:2019-11-28 修回日期:2019-12-09 出版日期:2020-02-28 发布日期:2020-02-21
通讯作者: 孙宏晨，教授. E-mail: hcsun@mail.jlu.edu.cn
作者简介:
陈雨蒙（1995—），女，黑龙江省双鸭山市人，硕士研究生. E-mail: 892808247@qq.com
基金资助:
国家自然科学基金(81920108012); 吉林省科技厅优秀青年人才基金(20280520058H)

Effect of Tumor Associated Vascular Endothelial Cells on Monocyte Chemotaxis

CHEN Yumeng¹^,²(), QIAO Chunyan¹^,², LIU Xinchen¹^,³, MENG Lin¹^,², REN Chunxia¹^,⁴, ZHENG Mengdan¹^,², ZHENG Shize¹^,², SUN Hongchen¹^,²()

¹ Key Laboratory of Tooth Development and Jaw Remodeling and Regeneration, Changchun 130021
² Department of Pathology, School and Hospital of Stomatology, Jilin University,Changchun 130021, Jilin Province, China
³ Department of Conservative Dentistry, School and Hospital of Stomatology, Jilin University,Changchun 130021, Jilin Province, China
⁴ Department of Periodontology, School and Hospital of Stomatology, Jilin University,Changchun 130021, Jilin Province, China

Received:2019-11-28 Revised:2019-12-09 Online:2020-02-28 Published:2020-02-21

摘要/Abstract

摘要：

目的：探究头颈部鳞状细胞癌（head and neck squamous cell carcinoma，HNSCC）中肿瘤相关巨噬细胞（tumor-associated macrophage, TAM）的来源和极化机制。方法：用HNSCC的条件培养基将血管内皮细胞（vascular endothe-lial cells, VEC）诱导为肿瘤相关VEC，使用显微镜观察肿瘤相关VEC的形态，以RT-qPCR检测单核细胞趋化因子在肿瘤相关内皮细胞的表达，Transwell趋化实验检测肿瘤相关内皮细胞对单核细胞的趋化能力。进一步使用佛波酯（phorbol 12-myristate 13-acetate，PMA）将人单核细胞（THP-1）诱导为贴壁的巨噬细胞，与肿瘤相关内皮细胞共培养后，qRT-PCR检测巨噬细胞极化相关基因的表达。结果：HNSCC相关VEC形态呈多角形，细胞间连接疏松，排列紊乱，并分泌多种单核细胞趋化因子将单核细胞趋化至肿瘤微环境，同时将其极化为白细胞介素-10（IL-10）、精氨酸酶1（arginase 1, Arg-1）高表达的M2型TAM。结论：在HNSCC中，肿瘤相关VEC招募单核细胞并将其极化为M2型TAM。

关键词: 头颈部鳞状细胞癌, 血管内皮细胞, 单核细胞, 巨噬细胞

Abstract:

Objective: To investigate the origin and polarization mechanism of TAM（tumor-associated macrophage ） in squamous cell carcinoma of head and neck. Methods: The vascular endothelial cells were induced into tumor related vascular endothelial cells through the conditioned medium of head and neck squamous cell carcinoma cells. The morphology of tumor related vascular endothelial cells was observed by microscope, the expression of monocyte chemokine in tumor related endothelial cells was detected by qRT-PCR, and the chemotaxis ability of tumor related endothelial cells to monocytes was detected by transwell chemotaxis test. Furthermore, （phorbol 12-myristate 13-acetate, PMA） was used to induce THP-1 monocytes into adherent macrophages. After co-culture with tumor related endothelial cells, qRT-PCR was used to detect the expression of macrophage polarization related genes. Results: The endothelial cells associated with head and neck squamous cell carcinoma were polyhedral in shape, with loose intercellular connections and disordered arrangement, and secreted a variety of monocyte chemokines to recruit the monocytes to the tumor microenvironment, at the same time, they were polarized into M2 TAM with high expression of IL-10 and Arg-1. Conclusion: In squamous cell carcinoma of head and neck, tumor associated vascular endothelial cells recruit monocytes and polarize them to M2 TAM.

Key words: head and neck squamous cell carcinoma, vascular endothelial cell, monocyte, macrophage

中图分类号:

R739.8

陈雨蒙, 乔春燕, 刘欣辰, 孟琳, 任春霞, 郑梦丹, 郑适泽, 孙宏晨. 肿瘤相关血管内皮细胞对单核细胞趋化的影响[J]. 《口腔颌面外科杂志》, 2020, 30(1): 9-15.

CHEN Yumeng, QIAO Chunyan, LIU Xinchen, MENG Lin, REN Chunxia, ZHENG Mengdan, ZHENG Shize, SUN Hongchen. Effect of Tumor Associated Vascular Endothelial Cells on Monocyte Chemotaxis[J]. 《Journal of Oral and Maxillofacial Surgery》, 2020, 30(1): 9-15.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2020/V30/I1/9

图/表 6

表1 RT-qPCR引物序列

Table 1 Primer sequences used for RT-qPCR

基因名称	上游引物序列 (5'→3′)	下游引物序列(5'→3′)
CCL2	CTTCTGTGCCTGCTGCTCATA	CTTTGGGACACTTGCTGCTG
CCL5	CTCGCTGTCATCCTCATTG	ATCCGGCGGTTCTTTCG
CCL8	AACATGAAGGTCTCCGCTGG	GCAGCAGGTGATTGGGGTAG
CSF	GATGGAGACCTCGTGCCAAATTA	TGTTATCTCTGAAGCGCATGGTG
CXCL12	GAGCCAACGTCAAGCATCTCAA	TTAGCTTCGGGTCAATGCACAC
CXCL16	TCCCACAGCCAGGACATCAG	TCAGGTATTAGAGTCAGGTGCCACA
TNF-α	AGCTGGTGGTGCCATCAGAGG	TGGTAGGAGACGGCGATGCG
IL-1β	GCCAGTGAAATGATGGCTTATT	AGGAGCACTTCATCTGTTTAGG
IL-6	CACTGGTCTTTTGGAGTTTGAG	GGACTTTTGTACTCATCTGCAC
IL-10	GTTGTTAAAGGAGTCCTTGCTG	TTCACAGGGAAGAAATCGATGA
Arg-1	TGGACAGACTAGGAATTGGCA	CCAGTCCGTCAACATCAAAACT
β-actin	GGAGATTACTGCCCTGGCTCCTA	GACTCATCGTACTCCTGCTTGCTG

图1 VEC的形态学表现（×20）注：A. 正常VEC；B. Cal27相关VEC。

Figure 1 Morphology of vascular endothelial cells（×20）

图2 VEC胞经不同细胞诱导后的Tanswell实验（×4）注：A. 正常VEC；B. Cal27相关VEC；C. Fadu相关VEC；D. 定量分析及统计学分析。

Figure 2 Transwell experiment of vascular endothelial cells induced by different tumor cells（×4）

图3 RT-qPCR检测单核细胞趋化因子

Figure 3 Detection of monocyte chemokines by RT-qPCR

图4 RT-qPCR检测巨噬细胞极化表型

Figure 4 Detection of macrophage polarization phenotype by RT-qPCR

图5 实验模式图

Figure 5 Experimental model diagram

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