個体における概日時計制御機構の解析

个体生物钟控制机制分析

• 批准号: 12J00639
• 负责人: 田宮 寛之
• 金额: $ 1.92万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for JSPS Fellows
• 财政年份: 2012
• 资助国家: 日本
• 起止时间: 2012-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12J00639/
• 关键词: 概日時計; 12灯用LED照度制御装置; ES分化誘導系; Per1とPer2の協調機構; 睡眠相前進症候群(FASPS); Per2ノックアウトレスキュー系; 短周期親和性PER1,長周期親和性PER2蛋白; 病的破綻; プロモーター; ES細胞; 分化誘導; 同調性

Per1とPer2は概日振動に必須の遺伝子であるが,両遺伝子の協調性に関しては未解明な部分が多い.本研究では新しい実験手法の開発とそれに基づく解析を行った.Per1,Per2各々のヘテロ,ホモノックアウトマウスを恒常明条件下で飼育した所,周期長はPer1とPer2の量比に依存しており,周期性消失はPer1,Per2の片方がないと起こりにくい事が示された.次に,位相応答が起きないような緩徐な光変化に対する個々のマウスの応答性を確認する為,12灯用LED照度制御装置を開発し,1分毎に任意のパターンの光をマウスに照射した際の概日リズム応答を検出可能な系を構築した.この装置を用いて22時間から27時間周期の緩徐に変化する光を照射した所,WTと比較してPer1(-/-)は長周期側に,Per2(-/-)は短周期側に,同調可能な周期の幅が広い事が示された.次にPer1とPer2の協調性を細胞レベルで検出する為,細胞レベルのPer2ノックアウトレスキュー系の構築を目指した.まず,ES細胞は概日リズムを持っていないが分化誘導をすると概日振動が発現することに着目し,ES細胞から10周期分の概日振動を検出し,小さな誤差で周期を測定可能な実験プロトコールを確立した.また,Per2 ノックアウトES細胞のRosa26にPer2遺伝子1コピーを正確に導入し,分化誘導することで,約24時間の振動を検出した.更に,Per2が原因遺伝子である睡眠相前進症候群(FASPS)の変異体を導入すると,個体同様に周期短縮が見られ,FASPS変異体の周期短縮を世界で初めて細胞レベルで再現する事に成功した.また,mut6変異体を導入するとFASPS同様の周期短縮がみられた.更にPer2 promoter下にPer1の翻訳領域を導入したところ,周期短縮が見られ,蛋白レベルの違いが周期親和性の違いを規定する事が示された.総じて,短周期親和性のPER1蛋白と長周期親和性のPER2蛋白は協調し,非生理的な環境条件下において概日周期長を24時間に限定する役割を持っている反面,低柔軟性のため病的破綻しうることを示した.

Per1 and Per2 are the essential components of the vibration, and the coordination of the components is not explained. In this study, the development of a new method for the detection of per1 and per2 was studied. Under constant light conditions, the period length of the breeding was dependent on the ratio of per1 and per2, and the periodic disappearance of per1 and per2 was shown. Second, the phase response is to start, slow down the light conversion, and confirm the responsiveness of the light conversion. For example, 12 LED illumination control devices for lamps are started, and 1 minute for any light conversion, the general light conversion, and detection of possible systems are constructed. The device uses 22 time periods and 27 time periods to slow down the change of light irradiation,WT comparison, Per1(-/-) on the long period side,Per2(-/-) on the short period side, the coherence of the possible period of amplitude is shown. Second, the coordination of Per1 and Per2 is the key to the construction of cell cycle system. The ES cell cycle was detected in 10 minutes, and the error in determining the cycle was small. In addition, the Per2 gene was correctly introduced into the Rosa26 ES cells and induced to differentiate. The vibration was detected in about 24 hours. In addition,Per2 is the cause of progressive sleep phase syndrome (FASPS) and its variant is introduced into individuals with the same cycle shortening, and FASPS variant cycle shortening is successfully reproduced in the world's first cells. FASPS cycle shortening is the same as the introduction of mut6. In addition, under the Per2 promoter, the Per1 domain is introduced, and cycle shortening is observed. The PER1 protein with short cycle affinity and the PER2 protein with long cycle affinity are coordinated under non-physiological environmental conditions. The PER1 protein with long cycle affinity and the PER2 protein with long cycle affinity are coordinated under non-physiological environmental conditions.