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新規破骨細胞形成促進因子Angiogeninが口腔癌骨浸潤に果たす役割の解明

阐明新型破骨细胞形成促进因子血管生成素在口腔癌骨侵袭中的作用

基本信息

批准号：
20K10137
负责人：
伊原木聰一郎
金额：
$ 2.83万
依托单位：
Okayama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2020
资助国家：
日本
起止时间：
2020-04-01 至 2024-03-31
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-20K10137/
关键词：
Angiogenin 口腔癌骨浸潤

项目摘要

BALB/C系nu/nuヌードマウスの大腿骨骨髄腔内に口腔癌細胞HSC-2を移植し，口腔癌骨浸潤動物モデルを確立した．各種口腔癌細胞株を移植し，浸潤面積をImageJで計測し，骨浸潤を評価した．shRNAを用いて口腔癌細胞のANG発現を抑制し，骨浸潤の抑制効果を調べた．対照群では骨髄内に腫瘍増殖を認め，TRAP染色で腫瘍周囲の骨面に沿って多数の破骨細胞形成と骨吸収窩の形成が認められた．一方shRNA群では腫瘍増殖が抑制され、TRAP染色で破骨細胞形成も抑制されていた．ANGが破骨細胞形成に与える影響の解析を行った．マウス大腿骨から骨髄細胞を回収し，Macrophage colony-stimulating factor（M-CSF）とANGを添加し培養後，TRAP染色を行い，破骨細胞数を計測した．ANGにより破骨細胞形成が促進されることを確認した．野生型およびANG KOマウスから、それぞれ採取した頭蓋骨由来骨芽細胞と脾臓由来破骨細胞前駆細胞の共存培養を行った.破骨細胞前駆細胞におけるANG遺伝子の欠損は，破骨細胞形成を阻害した．破骨細胞の骨吸収能はPit formation assayで評価した．ANG遺伝子の阻害は，破骨細胞の骨吸収能を阻害した．ANGが口腔癌細胞に与える影響の解析を行った．口腔癌細胞のANGおよびPlexinB2発現はELISA法とWestern blot法で調べた．口腔癌細胞をANG 存在下と非存在下で培養し増殖能と浸潤能の解析を行った.口腔癌細胞のANGおよびPlexinB2を発現しており、ANGは口腔癌細胞の増殖能と浸潤能を促進することが明らかとなった．

BALB/C nu/nu cells were transplanted into the femoral bone cavity to establish an animal model of oral cancer bone infiltration. When various oral cancer cell lines are transplanted, the invasion area is measured by ImageJ, and the bone invasion is evaluated.shRNA is used to inhibit the development of ANG in oral cancer cells, and the inhibition effect of bone invasion is modulated. TRAP staining showed that most osteoclasts formed along the bone surface of the tumor. A group of shRNAs inhibited tumor proliferation, TRAP staining inhibited osteoclast formation, and ANG was analyzed for its effect on osteoclast formation. The number of osteoclasts was measured by TRAP staining after culture with Macrophage colony-stimulating factor (M-CSF) and ANG. Wild-type ANG KO cells were cultured in vitro using calvarium-derived bone bud cells and splenic-derived osteoclast-derived precursor cells. The osteoclast precursor cells are damaged by ANG gene, which prevents osteoclast formation. Evaluation of Pit Formation Assay for Osteoclast Bone Absorption. Analysis of the Effects of ANG on Oral Cancer Cells. Expression of ANG and Plexin B2 in oral cancer cells was detected by ELISA and Western blot. Oral cancer cells can be cultured in the presence or absence of ANG and analyzed for proliferation and infiltration. ANG and PlexinB2 in oral cancer cells were found to promote proliferation and infiltration of oral cancer cells.