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Significance of neutrophil extracellular traps in the formation of pre-metastatic niches by exosomes

中性粒细胞胞外陷阱在外泌体形成转移前生态位中的意义

基本信息

批准号：
21K08602
负责人：
大西康晴
金额：
$ 2.66万
依托单位：
Jichi Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2021
资助国家：
日本
起止时间：
2021-04-01 至 2024-03-31
项目状态：
已结题

项目摘要

マウス胃癌細胞YTN16をC57/BL6マウスの腹腔内に接種することを繰り返し、腹膜播種高転移株YTN16Pを作成した。これらの細胞株の培養上清から超遠心法を用いてエクソソームを分離、抽出し、カゼイン腹腔内投与後に腹腔内に滲出した好中球を分離し、それに添加して細胞膜非透過性核酸検出試薬SYTOXで染色してNETs誘導性を測定したところ、YTN16P由来のエクソソーム添加群でNETsの産生が有意に上昇することが確認できた。また、マウス大腿骨から採取した骨髄細胞に添加、4～24時間培養し、同様の方法でNETsを作成したところ、TN16P由来のエクソソーム添加群でNETsの産生が若干上昇する傾向が認められた。YTN16と亜株YTN16pL6マウスの腹腔内に接種し作成した腹膜播種巣から組織切片を作成し、抗 Gr1抗体で好中球を NETs をシトルリン化ヒストン３ (Cit H3) に対する抗体で免疫染色を行ったところ、YTN16PにてGr-1(+)好中球とCit H3(+)のNETsの密度が多く、CD8(+)T細胞は少ない傾向を認めた。また、ヒト大腸癌患者267例において53名(19.9%)が手術時メトホルミンを服用していたが、メトホルミン内服群におけるCD66b(+)好中球の密度は非内服群よりも有意に低く(100.0（34-322）/0.25mm2 vs 169.0（36-553）/0.25mm2 p <0.001）、CD66b(+)CitH3(+)のNETｓの密度もメトホルミン内服群は非内服群と比較して有意に低かった(20.0（0-104）/0.25mm2 v vs 36.0（8-272）/0.25mm2 v, p <0.001）。

YTN16 gastric cancer cells were inoculated intraperitoneally with C57/BL6 inoculation and inoculated with highly inoculated YTN16P in the peritoneum. The supernatant of the cell line was cultured, and the supernatant was isolated, extracted, extracted, injected into the abdominal cavity and then injected into the abdominal cavity to separate the cells, the cell membrane non-permeable nucleic acid was detected by SYTOX staining, and the NETs was assayed by the method of cell membrane non-permeable nucleic acid. The reason for the YTN16P is to add a group of NETs students who are interested in confirming that they are not. The thigh bone samples were collected and the cells were added in 4-24 hours. The same method was used in NETs to make the bone cells. The TN16P method was used to add a group of NETs cells. The tissue sections of YTN16 strain YTN16pL6 strain were inoculated in the abdominal cavity, and the tissue sections of peritoneal inoculation were made. Anti-Gr1 antibodies were used to detect NETs antibody, Cit H3, Gr-1 (+), Cit H3 (+), NETs (+), Cit H3 (+) and CD8 (+). A total of 53 patients (19.9%) were diagnosed with severe acute respiratory syndrome. 53 patients (19.9%) were treated with CD66b (+). The density was low (100.0