歯周病と非アルコール性脂肪肝炎における新規バイオマーカーの検討

牙周病和非酒精性脂肪性肝炎新生物标志物的检查

• 批准号: 20K18519
• 负责人: 大野 祐
• 金额: $ 2.5万
• 依托单位: Aichi Gakuin University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Early-Career Scientists
• 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-20K18519/
• 关键词: 歯周炎; NASH; ANGPTL2; 慢性炎症; 歯周病; ペリオドンタルメディスン; 肝臓

1.実験的歯周炎モデルマウスの作成12週齢の NASHモデルマウス（C57BL/6J-NASH）を用い、上顎第二大臼歯に 5-0絹糸を留置することより炎症を惹起させ、実験的歯周炎モデルとする。また炎症状態長期化のため片側２週間絹糸の留置を行い、計４週間歯周炎状態を維持する。なお擬似処置を行ったNASHモデルマウスを対照群とする。実験的歯周炎惹起後、４週間ごとに体重測定、μ-CTを用いた歯槽骨吸収の解析ならびに尾静脈から採血を行う。16週齢にて屠殺後、歯周組織、肝臓を採取し以下の実験に用いる。凍結包埋にて歯肉における連続切片を作製し、組織染色を用いて炎症性細胞浸潤および ANGPTL2の局在を病理組織学的に評価する。この結果より、歯周炎の惹起による歯周組織局所におけるANGPTL2タンパク産生量を可視化して評価する。パラフィン包埋にて肝臓の組織切片を作成し、NASHの病態確認および肝臓でのANGPTL2の局在を確認するため、肝細胞の線維化、風船化、炎症性細胞浸潤などの病理組織学的評価を行う。次に、肝臓組織の一部を gentle MACSを用いてホモジナイズし、NucleoSpin RNA II systemを用い total RNA抽出を行う。その後、Reva-tra ACEを用いて、cDNAに逆転写を行う。そして、①ANGPTL2の遺伝子発現量を Real-time quantitative PCR (qPCR)法で測定する。②高発現しているその他の遺伝子を網羅的に解析するため、PCR Array Kit (RT2 Profiler PCR Arrray)を用いて、NASH病態における肝臓組織の遺伝子発現を網羅的に解析する。これらの結果より、肝臓局所における ANGPTL2の遺伝子発現の変化を確認するとともに、歯周炎がNASHの病態形成に影響を与える因子を同定する。

1. The periodontitis (C57BL/6J-NASH) was made into a 12-inch NASH-induced periodontitis (C57BL/6J-NASH), and the second acetabular was placed in a 5-0 series. The symptoms of chronic inflammation were prolonged, and the symptoms of inflammation were maintained during the period of 2 years and 4 years. It seems that the line is NASH and the group is full of photos. After the onset of periodontitis, the body weight was measured and μ-CT was analyzed by bone resorption in 4 years. 16. After being slaughtered, the tissue and liver tissue will be collected and the following samples will be used. The tissue staining was performed by impregnating the tissue with inflammatory cells, and the ANGPTL2 was examined by histopathology. The results showed that the growth rate of ANGPTL2 caused by periodontitis could be reduced by the local government. The tissue sections of the embedded liver were confirmed by NASH, and the pathological changes of the liver were confirmed by NASH. The pathological changes of the liver were confirmed by the pathology of the liver, the maintenance of the liver cells, the infiltration of the liver cells, and the pathology of the inflammatory cells. The secondary and liver tissues use gentle MACS to extract data from each other, and NucleoSpin RNA II system uses total RNA to extract data. After that, Reva-tra ACE writes the line in reverse with the words "cDNA" and "write". The Real-time quantitative PCR (qPCR) method is used to measure the temperature of the sample. The measurement is based on the 1ANGPTL2. 2. High-level users can detect the resolution of other sub-networks, PCR Array Kit (RT2 Profiler PCR Arrray) usage, and NASH pathological diagnosis of the liver organization subnet. the resolution of the network. The results showed that the symptoms of ANGPTL2 infection were confirmed and that the symptoms of periodontitis and NASH were the same as those of other factors.