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M2型肿瘤相关巨噬细胞参与胃癌细胞转移及免疫逃逸的机制研究
Mechanism of M2 polarization of tumor associated macrophages involved in the metastasis and immune escape of gastric cancer cells
- 局解手术学杂志 2023年32卷第9期 页码：771-777
- 作者机构：
海南医学院第二附属医院胃肠肿瘤外科，海南 海口 571000
- 作者简介：
黄光钺，E-mail：279102562@qq.com
- 基金信息：海南省卫生健康行业科研项目(21A200423)
DOI：10.11659/jjssx.07E022106
中图分类号： R735.2纸质出版日期：2023-09-25，
收稿日期：2022-07-27，
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陈华敏,何志军,涂伟等.M2型肿瘤相关巨噬细胞参与胃癌细胞转移及免疫逃逸的机制研究[J].局解手术学杂志,2023,32(09):771-777.
CHEN Hua-min,HE Zhi-jun,TU Wei,et al.Mechanism of M2 polarization of tumor associated macrophages involved in the metastasis and immune escape of gastric cancer cells[J].JOURNAL OF REGIONAL ANATOMY AND OPERATIVE SURGERY,2023,32(09):771-777.
陈华敏,何志军,涂伟等.M2型肿瘤相关巨噬细胞参与胃癌细胞转移及免疫逃逸的机制研究[J].局解手术学杂志,2023,32(09):771-777. DOI： 10.11659/jjssx.07E022106.
CHEN Hua-min,HE Zhi-jun,TU Wei,et al.Mechanism of M2 polarization of tumor associated macrophages involved in the metastasis and immune escape of gastric cancer cells[J].JOURNAL OF REGIONAL ANATOMY AND OPERATIVE SURGERY,2023,32(09):771-777. DOI： 10.11659/jjssx.07E022106.
摘要
目的
2
探究M2型肿瘤相关巨噬细胞（TAM）通过调控胃癌细胞程序性死亡配体-1（PD-L1）表达影响肿瘤细胞转移和免疫抑制因子分泌的机制。
方法
2
人单核细胞株THP-1经佛波酯（PMA）和白细胞介素-4（IL-4）诱导建立M2型TAM模型，免疫荧光染色检测M2型TAM表面标记物CD206的表达。将人胃癌细胞系MKN-45分为对照组（未经转染的MKN-45细胞）、siPD-L1组（将siPD-L1转染至MKN-45细胞）、M2组（在Transwell小室上室接种M2型TAM，下室接种MKN-45细胞）和siPD-L1+M2组（在Transwell小室上室接种M2型TAM，下室接种转染siPD-L1的MKN-45细胞）。RT-qPCR检测转染效果；免疫荧光染色和流式细胞术检测各组MKN-45细胞PD-L1的表达；划痕实验检测各组MKN-45细胞划痕愈合率；Transwell实验检测各组MKN-45细胞迁移与侵袭；ELISA测定各组MKN-45细胞培养液上清中血管内皮生长因子（VEGF）、转化生长因子-β（TGF-β)和白细胞介素-6（IL-6）水平。
结果
2
THP-1细胞经PMA与IL-4诱导后，细胞贴壁生长，形态多样且伸出伪足，细胞内CD206荧光染色强度明显增加，说明M2型TAM构建成功。转染siPD-L1的MKN-45细胞内PD-L1相对表达量降低，表示转染成功。与对照组比较，M2组的MKN-45细胞内PD-L1荧光强度增强，细胞表面PD-L1表达升高，划痕愈合率、细胞迁移及侵袭数目升高/增加，VEGF、TGF-β和IL-6含量也增加，差异均有统计学意义（
P
<
0.05）。与M2组比较，siPD-L1+M2组的MKN-45细胞内PD-L1荧光强度减弱，细胞表面PD-L1表达降低，划痕愈合率、细胞迁移及侵袭数目降低/减少，VEGF、TGF-β和IL-6水平降低，差异均有统计学意义（
P
<
0.05）。
结论
2
M2型TAM能够促进胃癌细胞迁移与侵袭，并增加免疫抑制因子分泌，从而促进肿瘤细胞转移及免疫逃逸，该作用可能与调控PD-L1表达有关。
Abstract
Objective
2
To explore the mechanism of M2 polarization of tumour associated macrophages （TAM） affecting tumor cell metastasis and immunosuppressive factor secretion by regulating the expression of programmed death ligand-1 （PD-L1） in gastric cancer cells.
Methods
2
The human monocyte line THP-1 was induced by phorbol 12-myristate 13-acetate （PMA） and interleukin-4 （IL-4） to establish the M2 polarization of TAM model， the expression of M2 polarization of TAM surface marker CD206 was detected by immunofluorescence staining. Human gastric cancer cell line MKN-45 was divided into the control group （non-transfected MKN-45 cells）， the siPD-L1 group （siPD-L1 was transfected into MKN-45 cells）， the M2 group （M2 polarization of TAM was inoculated in the upper compartment of Transwell chamber， and MKN-45 cells were inoculated in the lower compartment） and the siPD-L1+M2 group （M2 polarization of TAM was inoculated in the upper compartment of Transwell chamber， and MKN-45 cells transfected with siPD-L1 were inoculated in the lower compartment）. RT-qPCR was used to detect the transfection effect. Cell immunofluorescence staining and flow cytometry were used to detect the expression of PD-L1 on MKN-45 cells in each group. Scratch test was used to detect the scratch healing rate of MKN-45 cells in each group. Transwell assay was used to detect the migration and invasion of MKN-45 cells in each group.ELISA was used to determine the levels of vascular endothelial growth factor （VEGF）， transforming growth factor-β （TGF-β） and interleukin-6 （IL-6） in the supernatant of MKN-45 cells in each group.
Results
2
After THP-1 cells were induced by PMA and IL-4， the cells grew adherently， with diverse shapes and protruding pseudopodia， and the fluorescence staining intensity of CD206 in the cells increased significantly， indicating that the M2 polarization of TAM was successfully constructed. The relative expression of PD-L1 in MKN-45 cells transfected with siPD-L1 decreased， indicating that the transfection was successful. Compared with the control group， the fluorescence intensity of PD-L1 in MKN-45 cells in the M2 group was enhanced， the expression of PD-L1 on the cell surface was increased， the scratch healing rate， the numbers of cell migration and invasion were rised/increased， and the contents of VEGF， TGF-β and IL-6 were also increased， the difference were statistically significant （
P
<
0.05）. Compared with the M2 group， the fluorescence intensity of PD-L1 in MKN-45 cells in the siPD-L1+M2 group was weakened， the expression of PD-L1 on the cell surface was reduced， the scratch healing rate， the numbers of cell migration and invasion were reduced/decreased， and the contents of VEGF， TGF-β and IL-6 were all decreased， the difference were statistically significant （
P
<
0.05）.
Conclusion
2
M2 polarization of TAM can promote the migration and invasion of gastric cancer cells， and increase the secretion of immunosuppressive factors， thereby promoting tumor cell metastasis and immune escape， which may be related to the regulation of PD-L1 expression.
关键词
胃癌M2型肿瘤相关巨噬细胞PD-L1迁移侵袭免疫逃逸
Keywords
gastric cancerM2 polarization of tumor associated macrophagesPD-L1migrationinvasionimmune escape
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