WO2011114732A1 - Circadian rhythm measurement method - Google Patents

Circadian rhythm measurement method Download PDF

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WO2011114732A1
WO2011114732A1 PCT/JP2011/001565 JP2011001565W WO2011114732A1 WO 2011114732 A1 WO2011114732 A1 WO 2011114732A1 JP 2011001565 W JP2011001565 W JP 2011001565W WO 2011114732 A1 WO2011114732 A1 WO 2011114732A1
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expression level
gene
circadian rhythm
saliva
clock
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合津陽子
土師信一郎
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株式会社 資生堂
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

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  • the present invention relates to a novel method for measuring a circadian rhythm of a living body. More specifically, the present invention relates to a method for measuring circadian rhythm based on the expression level of a clock gene in saliva, a method for detecting modulation of circadian rhythm, and a method for screening a substance that modulates circadian rhythm.
  • circadian rhythms biological diurnal fluctuations
  • the biological clock causes biological diurnal fluctuations called circadian rhythms, including the sleep / wake cycle of organisms, body temperature / blood pressure, hormone secretion, metabolism, and psychosomatic activity, feeding, etc. It is thought that it controls the diurnal variation of various biological phenomena (activities).
  • disturbance of circadian rhythm has been pointed out as a cause of various psychosomatic symptoms or diseases such as sleep disorders, lifestyle-related diseases, and even neuropsychiatric disorders such as depression. The development of methods is highly desired.
  • the circadian rhythm control center (central clock) is present in the suprachiasmatic nucleus (suprachiasmatic nucleus: ⁇ SCN) of the hypothalamus, but there is also a “peripheral clock” that vibrates uniquely in peripheral tissues. Rhythm is thought to be expressed and controlled by synchronizing and integrating the vibration of the peripheral clock in each tissue and cell under the influence of the central clock.
  • a group of genes called “clock genes” are involved in the control of biological rhythms, and function as “internal clocks” by autonomously changing their expression periodically (vibrating).
  • the clock gene controls various biological phenomena (physiological states) as described above by controlling the expression of other various gene groups (see, for example, Non-Patent Document 1).
  • the molecular mechanism of biological rhythm is in individual cells.
  • fibroblasts isolated from mice with behavioral rhythm abnormalities also have rhythm abnormalities. Therefore, cultured cells collected from individuals can be used to measure the rhythm of the individual.
  • measurement of circadian rhythm using cells induced from biopsied skin is also being studied in humans. For example, an attempt has been made to evaluate circadian rhythm by measuring the amplitude of clock genes in cultured cells obtained from isolated skin (Non-patent Document 2).
  • a method of determining the degree of biological rhythm disorder from blood has been proposed (Patent Document 1).
  • Non-patent Document 3 melatonin
  • Patent Document 2 information related to biological rhythm is acquired based on the temporal change in the expression level of clock genes in hair follicle cells of living organisms.
  • the present invention has been made in view of the above circumstances, and an object thereof is to provide a novel method capable of measuring circadian rhythm easily with a small burden.
  • the present inventor can detect the expression of clock genes Period, Bmal and Clock in saliva, and further find that these clock genes are expressed with a circadian rhythm in saliva to complete the present invention. It came. Although it is known that various genes are expressed in saliva, it has not been known at all that clock genes are expressed in saliva. As clock genes in mammals, Period gene (Period1, Period2, Period3), Bmal gene (Bmal1, Bmal2), Clock gene, Cryptochrome gene, albumin site D-binding protein (Dbp) gene, E4BP4 gene, Npas2 gene, Rev- Many erb genes are known, but in saliva, remarkable rhythmicity was observed in the expression of Bmal gene, Period gene and Clock gene.
  • the method for measuring circadian rhythm of the present invention measures the expression level of one or more genes selected from the group consisting of Period, Bmal and Clock in saliva collected from an organism individual, The circadian rhythm of an individual organism is measured based on the expression level. Since these clock genes are expressed in saliva with a circadian rhythm, it is possible to measure an individual's circadian rhythm using the expression level as an index, and to detect a circadian rhythm modulated by various factors. .
  • modulation of circadian rhythm means to bring about some change in the phase, period, etc. of circadian rhythm, and normal circadian rhythm is disturbed ( In addition to the improvement of circadian rhythms that are not good.
  • the expression level of genes in saliva collected from individuals over time can be measured, and the circadian rhythm can be measured based on changes over time in the expression levels of those genes in saliva. .
  • the expression level of genes in saliva collected from an organism individual between 8-12 am (expression amount in the morning) and the expression level of genes in saliva collected from an organism individual between 8:00 pm and 12:00 (The modulation of circadian rhythm may be detected based on the comparison with the expression level in the afternoon.
  • Period, Bmal, and Clock each have their own expression patterns, and changes in their expression patterns, that is, modulation of circadian rhythm, can be detected by comparing expression levels in the morning and afternoon.
  • the gene is a Period gene
  • the expression level in the morning of the gene in saliva is higher than the expression level in the afternoon in a person with a normal life pattern who is active during the day and goes to sleep at night. Therefore, the morning expression level of the Period gene in saliva is lower than the expression level in the afternoon, suggesting a circadian rhythm disorder.
  • the gene when the gene is a Bmal gene, the expression level in the morning of the gene in saliva is normally lower than the expression level in the afternoon. Therefore, the morning expression level of the Bmal gene in saliva is higher than the expression level in the afternoon, suggesting a circadian rhythm disorder.
  • the screening method of the present invention is characterized by screening a substance that modulates circadian rhythm using the above method.
  • the method for measuring circadian rhythm of the present invention uses saliva as a measurement sample, the sample can be collected non-invasively and easily, the burden on the subject can be reduced, and more detailed by repeated sample collection. It is possible to provide accurate circadian rhythm information.
  • the circadian rhythm governs various metabolic functions of the living body, and the circadian rhythm is disturbed by various disorders such as sleep disorders, lifestyle-related diseases, and even neuropsychiatric disorders such as depression. Since it becomes a psychosomatic symptom or disease onset factor, by measuring the circadian rhythm by the method of the present invention, the modulation of the circadian rhythm can be detected easily and quickly, and various circadian rhythm disturbances Information useful for diagnosing symptoms can be provided.
  • the graph which shows the time-dependent change of the expression level of Period, Bmal, and Clock in saliva A graph showing the modulation of circadian rhythm by comparing the expression levels of Period and Bmal in saliva in the morning and afternoon.
  • the expression level of one or more genes selected from the Period gene, Bmal gene and Clock gene in saliva collected from an individual organism is measured.
  • Period1, 2 and 3 three types of Period genes, Period1, 2 and 3, are known, and two types of Bmal genes, Bmal1 and Bmal2, are known.
  • Bmal1 and Bmal2 are known.
  • the circadian rhythm can be measured more accurately by using one or more of the genes in the saliva of the Period gene and Bmal gene.
  • the expression level of the above gene in saliva can be measured according to a known method.
  • the RNA amount may be measured using RT-PCR, Northern blotting, or the like, or the protein may be measured using ELISA, Western blotting, or the like.
  • RNA amount of each gene in saliva can be measured by RT-PCR using commercially available PCR primers.
  • PCR primers for Bmal1, Clock and Period1 for example, Perfect Real Time Primer (Takara Bio) is commercially available, and quantitative RT-PCR kits (BrilliantII SYBR Green QRT-PCR Kit, Stratagene), and real-time PCR devices (Stratagene, Mx3000P) etc. can be used to quantify the expression level of each gene.
  • the method for collecting saliva is not particularly limited as long as the object of the present invention can be achieved.
  • commercially available saliva such as RNeasy Protect Saliva (Qiagen) or Oragene-RNA (DNA genotek)
  • saliva can be collected easily, and RNA can be stabilized by suppressing the action of RNase.
  • RNA can be extracted from saliva according to a known method such as a solvent extraction method or a column adsorption / desorption method, or a commercially available extraction kit may be used.
  • the gene expression level in saliva collected over time from the same individual organism is measured, and the phase of the circadian rhythm and the shift in the cycle are detected based on the change in the expression level over time. Can do. Further, the modulation of circadian rhythm may be detected by comparing the gene expression level in saliva collected at one or more predetermined time points with, for example, the gene expression level in a control sample.
  • the circadian rhythm modulation may be detected by comparing gene expression levels in saliva collected at one or more times each in the morning and afternoon. For example, by comparing gene expression levels in saliva collected at one or more time points between 8-12 am and 8-12 pm, more preferably between 9-11 am and 9-11 pm Can detect circadian rhythm modulation.
  • the expression level of the Period gene normally peaks around 9 am, and the expression level between 8-12 am is higher than the expression level between 8-12 pm. Therefore, in the Period gene, when the morning expression level is lower than the afternoon expression level, it is suggested that the circadian rhythm is modulated.
  • the expression level of the Bmal gene normally peaks around 1 am, and the expression level between 8-12 am is lower than the expression level between 8-12 pm. Therefore, in the Bmal gene, when the morning expression level is higher than the afternoon expression level, it is suggested that the circadian rhythm is modulated.
  • a substance that modulates the circadian rhythm can be screened.
  • the circadian rhythm is measured based on the expression of the Period, Bmal and / or Clock genes in the saliva according to the above methods, and the comparison is made before and after the administration.
  • the influence (modulation effect) of the test substance on the circadian rhythm can be evaluated.
  • the individual organism in the present invention is exemplified by animals that secrete saliva, humans, mice, rats, monkeys, etc., but the main purpose is particularly measurement in humans.
  • Example 1 Examination of circadian rhythm measurement in saliva samples was investigated the temporal change in the expression level of clock genes in saliva samples and examined whether circadian rhythms could be detected (measured) using saliva samples.
  • RNA extraction kit RNeasy Protect Saliva, Qiagen
  • a commercial PCR primer for Bmal1, Clock and Period1 Perfect Real Time Primer, Takara Bio
  • a quantitative RT-PCR kit Batagene
  • the RNA amount (expression level) of Bmal1, Clock and Period1 was measured.
  • a real-time PCR apparatus (Stratagene, Mx3000P) was used.
  • the expression level of ⁇ -Actin a housekeeping gene, was quantified and used as an internal standard.
  • the expression level is shown as the ratio of the expression level of each clock gene to the expression level of ⁇ -Actin (relative expression level).
  • Period1, Bmal1 and Clock have peak expression at around 9am, 1am and 5am, respectively, and are expressed in saliva with circadian rhythms in their own patterns. It was. In the suprachiasmatic nucleus of the brain, which is the center of the body clock, Period1 is known to have a circadian rhythm with peak expression in the daytime and Bmal and Clock at night, but peripheral secretions It was found for the first time that the same circadian rhythm can be detected in the saliva sample.
  • Example 2 Examination of detection of circadian rhythm modulation
  • the circadian rhythm can be measured by measuring the expression levels of Period1, Bmal1 and Clock in saliva.
  • the expression level of the Period gene normally peaks around 9 am, and the expression level between 8 and 12 am is higher than the expression level between 8 and 12 pm.
  • the expression level of the Bmal gene normally peaks at around 1 am, and the expression level between 8-12 am is lower than the expression level between 8-12 pm. Therefore, in the Period gene, when the morning expression level is lower than the afternoon expression level, it is suggested that the circadian rhythm is modulated, and in the Bmal gene, when the morning expression level is higher than the afternoon expression level, It is suggested that circadian rhythm is modulated.

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Abstract

Disclosed is a method of measuring circadian rhythms in a simple, non-intrusive manner by measuring the expression of the Period gene, the Bmal gene and/or the Clock gene in saliva collected from a living organism, and then measuring the circadian rhythm of the living organism on the basis of the expression of said gene(s) in the saliva.

Description

概日リズムの測定方法Circadian rhythm measurement method
　本発明は、生体の概日リズムを測定する新規方法に関するものである。より詳しくは、唾液中の時計遺伝子の発現量に基づき、概日リズムを測定する方法、概日リズムの変調を検出する方法、ならびに概日リズムを変調させる物質をスクリーニングする方法に関する。 The present invention relates to a novel method for measuring a circadian rhythm of a living body. More specifically, the present invention relates to a method for measuring circadian rhythm based on the expression level of a clock gene in saliva, a method for detecting modulation of circadian rhythm, and a method for screening a substance that modulates circadian rhythm.
　地球上の殆ど全ての生物は体内に約２４時間周期で自律振動する「体内時計」を持っている。体内時計は、概日リズム（サーカディアンリズム）という生物学的な日周変動を引き起こし、生物の睡眠・覚醒サイクルをはじめ、体温・血圧、ホルモン分泌、代謝、さらには、心身の活動、摂食などの、様々な生体現象（活動）の日周変動を支配すると考えられている。近年、概日リズムの乱れが、睡眠障害や、生活習慣病、さらにはうつ病等の精神神経疾患など、様々な心身の症状または疾患の発症要因として指摘されており、概日リズムを測定する方法の開発が強く望まれている。 殆 ど Almost all living organisms on the earth have a “body clock” that vibrates autonomously every 24 hours. The biological clock causes biological diurnal fluctuations called circadian rhythms, including the sleep / wake cycle of organisms, body temperature / blood pressure, hormone secretion, metabolism, and psychosomatic activity, feeding, etc. It is thought that it controls the diurnal variation of various biological phenomena (activities). In recent years, disturbance of circadian rhythm has been pointed out as a cause of various psychosomatic symptoms or diseases such as sleep disorders, lifestyle-related diseases, and even neuropsychiatric disorders such as depression. The development of methods is highly desired.
　概日リズムの制御中枢（中枢時計）は視床下部の視交叉上核（suprachiasmatic nucleus : SCN）に存在しているが、末梢組織にも独自に振動する「末梢時計」が存在しており、生体リズムは、各組織や細胞中の末梢時計が独自に振動しながらも、中枢時計の影響を受けてその振動を同調・統合することによって、発現・制御されていると考えられている。生体リズムの制御には、「時計遺伝子（クロックジーン）」と呼ばれる遺伝子群が関与しており、その発現を自律的に周期変動（振動）させることによって、「体内時計」として機能している。時計遺伝子は、他の種々の遺伝子群の発現を制御することで、上記のような様々な生体現象（生理状態）を支配している（例えば、非特許文献１を参照）。 The circadian rhythm control center (central clock) is present in the suprachiasmatic nucleus (suprachiasmatic nucleus: 、 SCN) of the hypothalamus, but there is also a “peripheral clock” that vibrates uniquely in peripheral tissues. Rhythm is thought to be expressed and controlled by synchronizing and integrating the vibration of the peripheral clock in each tissue and cell under the influence of the central clock. A group of genes called “clock genes” are involved in the control of biological rhythms, and function as “internal clocks” by autonomously changing their expression periodically (vibrating). The clock gene controls various biological phenomena (physiological states) as described above by controlling the expression of other various gene groups (see, for example, Non-Patent Document 1).
　生体リズムの分子機構は個々の細胞内にあり、例えば、行動リズム異常マウスから分離した線維芽細胞にもリズム異常が認められることから、個体から採取した培養細胞を、その個体のリズム測定に応用する試みがされている。また、ヒトでもバイオプシした皮膚より誘導した細胞を用いて概日リズムを測定することが検討されている。例えば、分離した皮膚から得られる培養細胞の時計遺伝子の振幅を測定することで、概日リズムを評価する試みがなされている（非特許文献２）。あるいは、血液より生体リズム障害の度合いを判定する方法なども提案されている（特許文献１）。 The molecular mechanism of biological rhythm is in individual cells. For example, fibroblasts isolated from mice with behavioral rhythm abnormalities also have rhythm abnormalities. Therefore, cultured cells collected from individuals can be used to measure the rhythm of the individual. An attempt has been made. In addition, measurement of circadian rhythm using cells induced from biopsied skin is also being studied in humans. For example, an attempt has been made to evaluate circadian rhythm by measuring the amplitude of clock genes in cultured cells obtained from isolated skin (Non-patent Document 2). Alternatively, a method of determining the degree of biological rhythm disorder from blood has been proposed (Patent Document 1).
　しかしながら、皮膚のバイオプシや血液の採取は被験者への負担が大きく、長期間繰り返し試料を採取して測定を行うことが困難である。マウスの場合には、髄液をマイクロダイアリシスにより連続抽出して、メラトニンなどの生体リズムに関連するホルモンをモニターする試みもされているが(非特許文献３)、ヒトへの応用は難しい。 However, collection of skin biopsies and blood is a burden on the subject, and it is difficult to perform measurement by repeatedly collecting samples for a long period of time. In the case of mice, attempts have been made to continuously extract cerebrospinal fluid by microdialysis and monitor hormones related to biological rhythm such as melatonin (Non-patent Document 3), but application to humans is difficult.
　さらに、生物個体の毛包細胞中の時計遺伝子の発現量の経時変化に基づき、生体リズムに関わる情報を取得することも報告されている（特許文献２）が、より非侵襲的でかつ簡便な方法が求められている。 Furthermore, it has been reported that information related to biological rhythm is acquired based on the temporal change in the expression level of clock genes in hair follicle cells of living organisms (Patent Document 2), but it is more noninvasive and simpler. There is a need for a method.
特開２００５－８０６０３号公報JP 2005-80603 A 特開２００９－２７９５２号公報JP 2009-27952 A
　本発明は、上記のような事情に鑑み、少ない負担でかつ簡便に概日リズムを測定できる新規方法を提供することを目的とするものである。 The present invention has been made in view of the above circumstances, and an object thereof is to provide a novel method capable of measuring circadian rhythm easily with a small burden.
　本発明者は、唾液において、時計遺伝子であるPeriod、BmalおよびClockの発現を検出でき、さらに、それら時計遺伝子が唾液中において概日リズムをもって発現されていることを見出し、本発明を完成するに至った。唾液中に様々な遺伝子が発現されていることは知られているが、時計遺伝子が唾液中に発現されていることは全く知られていなかった。哺乳動物における時計遺伝子として、Period遺伝子（Period1, Period2, Period3）、Bmal遺伝子（Bmal1, Bmal2）、Clock遺伝子、Cryptochrome遺伝子、albumin site D-binding protein (Dbp)遺伝子、E4BP4遺伝子、Npas2遺伝子、Rev-erb遺伝子など多数知られているが、唾液中においては、Bmal遺伝子、Period遺伝子およびClock遺伝子の発現に顕著なリズム性が認められた。 The present inventor can detect the expression of clock genes Period, Bmal and Clock in saliva, and further find that these clock genes are expressed with a circadian rhythm in saliva to complete the present invention. It came. Although it is known that various genes are expressed in saliva, it has not been known at all that clock genes are expressed in saliva. As clock genes in mammals, Period gene (Period1, Period2, Period3), Bmal gene (Bmal1, Bmal2), Clock gene, Cryptochrome gene, albumin site D-binding protein (Dbp) gene, E4BP4 gene, Npas2 gene, Rev- Many erb genes are known, but in saliva, remarkable rhythmicity was observed in the expression of Bmal gene, Period gene and Clock gene.
　本発明の概日リズムを測定する方法は、生物個体から採取された唾液中のPeriod、BmalおよびClockより成る群から選択される１以上の遺伝子の発現量を測定し、唾液中のそれら遺伝子の発現量に基づき、生物個体の概日リズムを測定することを特徴とする。それら時計遺伝子は、唾液中に概日リズムをもって発現されるため、その発現量を指標として個体の概日リズムを測定できると共に、様々な要因で変調した概日リズムを検出することを可能にする。 The method for measuring circadian rhythm of the present invention measures the expression level of one or more genes selected from the group consisting of Period, Bmal and Clock in saliva collected from an organism individual, The circadian rhythm of an individual organism is measured based on the expression level. Since these clock genes are expressed in saliva with a circadian rhythm, it is possible to measure an individual's circadian rhythm using the expression level as an index, and to detect a circadian rhythm modulated by various factors. .
　本発明において、「概日リズムの変調」または「概日リズムを変調させる」とは、概日リズムの位相、周期などに何らかの変化をもたらすことを意味し、正常な概日リズムが乱れること（不調）のみならず、不調である概日リズムの改善も含む。 In the present invention, “modulation of circadian rhythm” or “modulate circadian rhythm” means to bring about some change in the phase, period, etc. of circadian rhythm, and normal circadian rhythm is disturbed ( In addition to the improvement of circadian rhythms that are not good.
　本発明の方法において、例えば、経時的に個体から採取された唾液中の遺伝子の発現量を測定し、唾液中のそれら遺伝子の発現量の経時変化に基づき、概日リズムを測定することができる。 In the method of the present invention, for example, the expression level of genes in saliva collected from individuals over time can be measured, and the circadian rhythm can be measured based on changes over time in the expression levels of those genes in saliva. .
　あるいは、午前８～１２時の間に生物個体から採取された唾液中の遺伝子の発現量（午前の発現量）と、午後８時～１２時の間に生物個体から採取された唾液中の遺伝子の発現量（午後の発現量）との比較に基づき、概日リズムの変調を検出してもよい。Period、BmalおよびClockはそれぞれ独自の発現パターンを有しており、午前と午後の発現量の比較によりその発現パターンの変化、すなわち概日リズムの変調を検出できる。
　例えば、遺伝子がPeriod遺伝子である場合、日中活動し夜間に就寝する通常の生活パターンの人では、唾液中の遺伝子の午前の発現量が午後の発現量より高い。従って、唾液中のPeriod遺伝子の午前の発現量が午後の発現量より低いことが、概日リズムの不調を示唆する。 Alternatively, the expression level of genes in saliva collected from an organism individual between 8-12 am (expression amount in the morning) and the expression level of genes in saliva collected from an organism individual between 8:00 pm and 12:00 ( The modulation of circadian rhythm may be detected based on the comparison with the expression level in the afternoon. Period, Bmal, and Clock each have their own expression patterns, and changes in their expression patterns, that is, modulation of circadian rhythm, can be detected by comparing expression levels in the morning and afternoon.
For example, when the gene is a Period gene, the expression level in the morning of the gene in saliva is higher than the expression level in the afternoon in a person with a normal life pattern who is active during the day and goes to sleep at night. Therefore, the morning expression level of the Period gene in saliva is lower than the expression level in the afternoon, suggesting a circadian rhythm disorder.
　また、遺伝子がBmal遺伝子である場合には、正常では唾液中の遺伝子の午前の発現量が午後の発現量より低い。従って、唾液中のBmal遺伝子の午前の発現量が午後の発現量より高いことが、概日リズムの不調を示唆する。 In addition, when the gene is a Bmal gene, the expression level in the morning of the gene in saliva is normally lower than the expression level in the afternoon. Therefore, the morning expression level of the Bmal gene in saliva is higher than the expression level in the afternoon, suggesting a circadian rhythm disorder.
　本発明のスクリーニング方法は、上記方法を用いて、概日リズムを変調させる物質をスクリーニングすることを特徴とする。 The screening method of the present invention is characterized by screening a substance that modulates circadian rhythm using the above method.
　本発明の概日リズムを測定する方法は、測定試料として唾液を用いるため、非侵襲的かつ簡便に試料を採取することができ、被験者の負担を軽減できると共に、繰り返しの試料採取によって、より詳細かつ正確な概日リズムの情報をもたらすことを可能にする。 Since the method for measuring circadian rhythm of the present invention uses saliva as a measurement sample, the sample can be collected non-invasively and easily, the burden on the subject can be reduced, and more detailed by repeated sample collection. It is possible to provide accurate circadian rhythm information.
　上述したように、概日リズムは、生体の様々な代謝機能を司っており、概日リズムの乱れが、睡眠障害や、生活習慣病、さらにはうつ病等の精神神経疾患など、様々な心身の症状または疾患の発症要因となるため、本発明の方法により概日リズムを測定することにより、概日リズムの変調を簡便かつ迅速に検出できると共に、概日リズムの乱れに起因する様々な症状の診断に有用な情報を提供することができる。 As described above, the circadian rhythm governs various metabolic functions of the living body, and the circadian rhythm is disturbed by various disorders such as sleep disorders, lifestyle-related diseases, and even neuropsychiatric disorders such as depression. Since it becomes a psychosomatic symptom or disease onset factor, by measuring the circadian rhythm by the method of the present invention, the modulation of the circadian rhythm can be detected easily and quickly, and various circadian rhythm disturbances Information useful for diagnosing symptoms can be provided.
　さらに、本発明の方法を用いて、概日リズムを変調させる物質をスクリーニングすることにより、簡便かつ効率的に概日リズム不調に対する薬剤候補を特定することができる。 Furthermore, by using the method of the present invention to screen for substances that modulate circadian rhythm, drug candidates for circadian rhythm disorders can be identified easily and efficiently.
唾液中のPeriod、BmalおよびClockの発現量の経時変化を示すグラフ。The graph which shows the time-dependent change of the expression level of Period, Bmal, and Clock in saliva. 唾液中のPeriodおよびBmalの午前と午後の発現量の比較により概日リズムの変調を示すグラフ。A graph showing the modulation of circadian rhythm by comparing the expression levels of Period and Bmal in saliva in the morning and afternoon.
　本発明の方法では、生物個体から採取された唾液中の、Period遺伝子、Bmal遺伝子およびClock遺伝子から選択される１種または２種以上の遺伝子の発現量を測定する。ヒトでは、Period遺伝子としてPeriod1, 2および3の３種類の存在が知られており、またBmal遺伝子としてBmal1および Bmal2の２種類の存在が知られているが、いずれも同じ遺伝子群で同様の発現リズム（日内変動）を有している。特に、Period遺伝子およびBmal遺伝子の唾液中の発現量の日内変動は顕著であり、それら遺伝子の１種以上を用いることにより、より正確に概日リズムを測定することができる。 In the method of the present invention, the expression level of one or more genes selected from the Period gene, Bmal gene and Clock gene in saliva collected from an individual organism is measured. In humans, three types of Period genes, Period1, 2 and 3, are known, and two types of Bmal genes, Bmal1 and Bmal2, are known. Has a rhythm (daily fluctuation). In particular, the circadian rhythm can be measured more accurately by using one or more of the genes in the saliva of the Period gene and Bmal gene.
　上記遺伝子の唾液中の発現量は公知の方法に従い測定することができる。例えば、ＲＴ－ＰＣＲやノーザンブロット法等を用いてＲＮＡ量を測定してもよいし、あるいはＥＬＩＳＡやウェスタンブロット法等を用いてタンパク質を測定してもよい。 The expression level of the above gene in saliva can be measured according to a known method. For example, the RNA amount may be measured using RT-PCR, Northern blotting, or the like, or the protein may be measured using ELISA, Western blotting, or the like.
　限定はされないが、例えば、唾液中の各遺伝子のＲＮＡ量を、市販されているＰＣＲプライマーを用いてＲＴ－ＰＣＲによって測定することができる。Bmal1、ClockおよびPeriod1用ＰＣＲプライマーとして、例えばPerfect Real Time Primer（タカラバイオ社）等が市販されており、また定量ＲＴ－ＰＣＲキット（BrilliantII SYBR Green QRT-PCR Kit、Stratagene社）、ならびにリアルタイムＰＣＲ装置（Stratagene社、Mx3000P）等を用いて各遺伝子の発現量を定量することができる。 Although not limited, for example, the RNA amount of each gene in saliva can be measured by RT-PCR using commercially available PCR primers. As PCR primers for Bmal1, Clock and Period1, for example, Perfect Real Time Primer (Takara Bio) is commercially available, and quantitative RT-PCR kits (BrilliantII SYBR Green QRT-PCR Kit, Stratagene), and real-time PCR devices (Stratagene, Mx3000P) etc. can be used to quantify the expression level of each gene.
　唾液の採取方法は、本発明の目的を達成できる限り特に限定されないが、例えばＲＮＡの発現量を測定する場合、RNeasy Protect Saliva （Qiagen社）やOragene-RNA (DNA genotek社)などの市販の唾液採取キットを用いることによって、容易に唾液を採取できると共に、ＲＮＡ分解酵素の働きを抑制してＲＮＡを安定化することができる。また、唾液からのＲＮＡの抽出は、例えば、溶媒抽出法やカラム吸脱着法など周知の方法に従い行うことができ、あるいは市販の抽出キットを使用してもよい。 The method for collecting saliva is not particularly limited as long as the object of the present invention can be achieved. For example, when measuring the expression level of RNA, commercially available saliva such as RNeasy Protect Saliva (Qiagen) or Oragene-RNA (DNA genotek) By using the collection kit, saliva can be collected easily, and RNA can be stabilized by suppressing the action of RNase. In addition, RNA can be extracted from saliva according to a known method such as a solvent extraction method or a column adsorption / desorption method, or a commercially available extraction kit may be used.
　本発明の方法において、例えば、同一の生物個体から経時的に採取した唾液中の遺伝子発現量を測定して、発現量の経時変化に基づき、概日リズムの位相や周期のずれを検出することができる。また、所定の１または２以上の時点で採取された唾液中の遺伝子発現量を、例えば対照試料における遺伝子発現量と比較して、概日リズムの変調を検出してもよい。 In the method of the present invention, for example, the gene expression level in saliva collected over time from the same individual organism is measured, and the phase of the circadian rhythm and the shift in the cycle are detected based on the change in the expression level over time. Can do. Further, the modulation of circadian rhythm may be detected by comparing the gene expression level in saliva collected at one or more predetermined time points with, for example, the gene expression level in a control sample.
　あるいは、午前と午後のそれぞれ１以上の時点で採取された唾液中の遺伝子発現量を比較することにより、概日リズムの変調を検出してもよい。例えば、午前８～１２時と午後８～１２時の間、より好ましくは午前９～１１時と午後９～１１時の間の、それぞれ１以上の時点で採取された唾液中の遺伝子発現量を比較することにより、概日リズムの変調を検出することができる。 Alternatively, the circadian rhythm modulation may be detected by comparing gene expression levels in saliva collected at one or more times each in the morning and afternoon. For example, by comparing gene expression levels in saliva collected at one or more time points between 8-12 am and 8-12 pm, more preferably between 9-11 am and 9-11 pm Can detect circadian rhythm modulation.
　Period遺伝子の発現量は、正常では、午前９時頃にピークとなり、午前８～１２時の間の発現量が午後８～１２時の間の発現量より高い。従って、Period遺伝子では、午前の発現量が午後の発現量より低くなると、概日リズムが変調されていることが示唆される。 The expression level of the Period gene normally peaks around 9 am, and the expression level between 8-12 am is higher than the expression level between 8-12 pm. Therefore, in the Period gene, when the morning expression level is lower than the afternoon expression level, it is suggested that the circadian rhythm is modulated.
　Bmal遺伝子の発現量は、正常では、午前１時頃にピークとなり、午前８～１２時の間の発現量が午後８～１２時の間の発現量より低い。従って、Bmal遺伝子では、午前の発現量が午後の発現量より高くなると、概日リズムが変調されていることが示唆される。 The expression level of the Bmal gene normally peaks around 1 am, and the expression level between 8-12 am is lower than the expression level between 8-12 pm. Therefore, in the Bmal gene, when the morning expression level is higher than the afternoon expression level, it is suggested that the circadian rhythm is modulated.
　本発明の測定方法を用いて、概日リズムを変調させる物質をスクリーングすることができる。
　例えば、被験物質を投与する前および後に採取した唾液を用いて、それぞれ上記方法に従い、唾液中のPeriod、Bmal および／またはClock遺伝子の発現に基づき概日リズムを測定し、投与前後での比較により、被験物質による概日リズムに対する影響（変調効果）を評価することができる。 Using the measurement method of the present invention, a substance that modulates the circadian rhythm can be screened.
For example, using the saliva collected before and after administration of the test substance, the circadian rhythm is measured based on the expression of the Period, Bmal and / or Clock genes in the saliva according to the above methods, and the comparison is made before and after the administration. The influence (modulation effect) of the test substance on the circadian rhythm can be evaluated.
　なお、本発明における生物個体とは、唾液を分泌する動物類、ヒト、マウス、ラット、サル等が例示されるが、特にヒトにおける測定を主な目的とする。 The individual organism in the present invention is exemplified by animals that secrete saliva, humans, mice, rats, monkeys, etc., but the main purpose is particularly measurement in humans.
　以下、実施例を挙げて本発明を具体的に説明するが、本発明は下記の実施例に限定されるものではない。 Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to the following examples.
実施例１
唾液サンプルでの概日リズム測定の検討
　唾液サンプルにおける時計遺伝子の発現量の経時変化を調べて、唾液サンプルを用いて概日リズムを検出（測定）することが可能であるか検討した。 Example 1
Examination of circadian rhythm measurement in saliva samples We investigated the temporal change in the expression level of clock genes in saliva samples and examined whether circadian rhythms could be detected (measured) using saliva samples.
　被験者において、夜９時から翌日の夜９時までの２４時間にわたり４時間間隔で、それぞれ約２ｍＬの唾液を採取した。 In the subject, about 2 mL of saliva was collected at intervals of 4 hours over 24 hours from 9:00 pm to 9:00 pm the next day.
　ＲＮＡ抽出キット（RNeasy Protect Saliva、Qiagen社）を用い、添付のマニュアルに従って、採取した唾液からＲＮＡを抽出した。Bmal1、ClockおよびPeriod1用の市販のＰＣＲプライマー（Perfect Real Time Primer、タカラバイオ社）と、定量ＲＴ－ＰＣＲキット（BrilliantII SYBR Green QRT-PCR Kit、Stratagene社）を用いて、定量ＲＴ－ＰＣＲ法によりBmal1、ClockおよびPeriod1のＲＮＡ量（発現量）を測定した。測定にはリアルタイムＰＣＲ装置（Stratagene社、Mx3000P）を用いた。同時に、ハウスキーピング遺伝子であるβ-Actinの発現量を定量し、内部標準として用いた。 RNA was extracted from the collected saliva using an RNA extraction kit (RNeasy Protect Saliva, Qiagen) according to the attached manual. By using a commercial PCR primer for Bmal1, Clock and Period1 (Perfect Real Time Primer, Takara Bio) and a quantitative RT-PCR kit (Brilliant II SYBR Green GRT-PCR Kit, Stratagene) The RNA amount (expression level) of Bmal1, Clock and Period1 was measured. For the measurement, a real-time PCR apparatus (Stratagene, Mx3000P) was used. At the same time, the expression level of β-Actin, a housekeeping gene, was quantified and used as an internal standard.
　結果を図１に示す。発現量は、β-Actinの発現量に対する各時計遺伝子の発現量の比（相対的発現量）として示す。Period1、Bmal1およびClockはそれぞれ、午前９時頃、深夜１時頃および早朝５時頃に発現ピークを示し、それぞれ独自のパターンで概日リズムを刻んで唾液中に発現されていることが認められた。体内時計の中枢である脳の視交叉上核において、Period1は昼間に、BmalとClockは夜間に発現量がピークとなる概日リズムを刻んでいることが知られているが、末梢の分泌物である唾液サンプルにおいても、同様の概日リズムを検出できることを初めて見出した。 The results are shown in FIG. The expression level is shown as the ratio of the expression level of each clock gene to the expression level of β-Actin (relative expression level). Period1, Bmal1 and Clock have peak expression at around 9am, 1am and 5am, respectively, and are expressed in saliva with circadian rhythms in their own patterns. It was. In the suprachiasmatic nucleus of the brain, which is the center of the body clock, Period1 is known to have a circadian rhythm with peak expression in the daytime and Bmal and Clock at night, but peripheral secretions It was found for the first time that the same circadian rhythm can be detected in the saliva sample.
実施例２
概日リズムの変調の検出の検討
　上記実施例において、唾液中のPeriod1、Bmal1およびClockの発現量を測定して概日リズムを測定できることを確認できたので、次に、この測定系を用いて概日リズムの変調を検出できるか検討した。尚、以下の実験では、唾液中での発現量の日内変動がより大きいPeriod1およびBmal1を用いて評価した。 Example 2
Examination of detection of circadian rhythm modulation In the above example, it was confirmed that the circadian rhythm can be measured by measuring the expression levels of Period1, Bmal1 and Clock in saliva. We examined whether the circadian rhythm modulation could be detected. In the following experiments, evaluation was performed using Period1 and Bmal1, which have larger daily fluctuations in the expression level in saliva.
　１８名の女性に、２晩連続で、就寝中の深夜２時に一時的に起床して算数や図形問題に回答するという作業（所要時間5～10分）を行なわせ、実験的に概日リズムを乱した。 Eighteen women were woken up for 2 consecutive nights at 2 o'clock in the middle of bed at night to temporarily answer mathematical and graphic problems (required time: 5-10 minutes). Disturbed.
　試験開始前、睡眠中断１日目と２日目、および１週間後の計４回、夜１０時と朝１０時に唾液を採取し、実施例１と同様の方法によりPeriod1とBmal1の発現量を測定し、概日リズムを評価した。唾液の採取と保存には専用キット（Oragen-RNA、DNA Genotek社）を用いた。 Before the start of the test, saliva was collected 4 times, 10:00 and 10:00 in the morning, 1st and 2nd day after sleep interruption, and the expression levels of Period1 and Bmal1 were determined in the same manner as in Example 1. Measure and evaluate circadian rhythm. A special kit (Oragen-RNA, DNA-Genotek) was used for saliva collection and storage.
　図１から明らかであるように、Period遺伝子の発現量は、正常では、午前９時頃にピークとなり、午前８～１２時の間の発現量が午後８～１２時の間の発現量より高い。一方、Bmal遺伝子の発現量は、正常では、午前１時頃にピークとなり、午前８～１２時の間の発現量が午後８～１２時の間の発現量より低い。従って、Period遺伝子では、午前の発現量が午後の発現量より低くなると、概日リズムが変調されていることが示唆され、またBmal遺伝子では、午前の発現量が午後の発現量より高くなると、概日リズムが変調されていることが示唆される。 As is apparent from FIG. 1, the expression level of the Period gene normally peaks around 9 am, and the expression level between 8 and 12 am is higher than the expression level between 8 and 12 pm. On the other hand, the expression level of the Bmal gene normally peaks at around 1 am, and the expression level between 8-12 am is lower than the expression level between 8-12 pm. Therefore, in the Period gene, when the morning expression level is lower than the afternoon expression level, it is suggested that the circadian rhythm is modulated, and in the Bmal gene, when the morning expression level is higher than the afternoon expression level, It is suggested that circadian rhythm is modulated.
　結果を図２および下記表１に示す。
Figure JPOXMLDOC01-appb-T000001
 　試験開始前および睡眠中断１日目は、Period１の午前１０時の発現量は午後１０時の発現量よりも高く、またBmal1の午前１０時の発現量は午後１０時の発現量よりも低く、いずれも正常な概日リズムを示した。一方、睡眠中断２日目には、Period１の午前１０時の発現量は午後１０時の発現量よりも低くなり、またBmal1の午前１０時の発現量は午後１０時の発現量よりも高くなり、概日リズムが乱れていることが明確に示された。さらに、1週間後にはいずれも正常な発現パターンに戻っており、概日リズムが回復したことが示された。 The results are shown in FIG. 2 and Table 1 below.
Figure JPOXMLDOC01-appb-T000001
 Before the start of the study and on the first day of sleep interruption, the expression level of Period 1 at 10am is higher than the expression level at 10pm, and the expression level of Bmal1 at 10am is lower than the expression level at 10pm. All showed normal circadian rhythm. On the other hand, on the second day of sleep interruption, the expression level of Period 1 at 10am is lower than the expression level at 10pm, and the expression level of Bmal1 at 10am is higher than the expression level at 10pm. It was clearly shown that the circadian rhythm was disturbed. Furthermore, after 1 week, all returned to normal expression patterns, indicating that circadian rhythm was restored.
　以上の結果から、唾液中のPeriod、BmalおよびClockの発現量に基づき、概日リズムを測定し、さらに概日リズムの変調を検出できることが確認できた。 From the above results, it was confirmed that circadian rhythm was measured based on the expression levels of Period, Bmal, and Clock in saliva, and that the modulation of circadian rhythm could be detected.

Claims (6)

  1. 生物個体から採取された唾液中のPeriod、BmalおよびClockより成る群から選択される１以上の遺伝子の発現量を測定し、唾液中の該遺伝子の発現量に基づき、前記生物個体の概日リズムを測定する方法。 The expression level of one or more genes selected from the group consisting of Period, Bmal and Clock in saliva collected from an individual is measured, and the circadian rhythm of the individual is measured based on the expression level of the gene in saliva How to measure.
  2. 経時的に前記生物個体から採取された唾液中の前記遺伝子の発現量を測定し、唾液中の前記遺伝子の発現量の経時変化に基づき概日リズムを測定する請求項１記載の方法。 The method according to claim 1, wherein the expression level of the gene in saliva collected from the individual organism over time is measured, and the circadian rhythm is measured based on the temporal change in the expression level of the gene in saliva.
  3. 午前８～１２時の間に前記生物個体から採取された唾液中の遺伝子の発現量と、午後８時～１２時の間に前記生物個体から採取された唾液中の遺伝子の発現量との比較に基づき、概日リズムの変調を検出することを含む請求項１または２記載の方法。 Based on the comparison of the expression level of the gene in saliva collected from the organism individual between 8-12 am and the expression level of the gene in saliva collected from 8-8 pm 3. A method according to claim 1 or 2, comprising detecting a modulation of a daily rhythm.
  4. 前記遺伝子がPeriod遺伝子を含み、唾液中の該遺伝子の前記午前の発現量が前記午後の発現量より低いことが、概日リズムの変調を示唆する請求項３記載の方法。 The method according to claim 3, wherein the gene includes a Period gene, and that the expression level of the gene in saliva is lower than the expression level in the afternoon suggests modulation of circadian rhythm.
  5. 前記遺伝子がBmal遺伝子を含み、唾液中の該遺伝子の前記午前の発現量が前記午後の発現量より高いことが、概日リズムの変調を示唆する請求項３または４記載の方法。 The method according to claim 3 or 4, wherein the gene comprises a Bmal gene, and that the morning expression level of the gene in saliva is higher than the afternoon expression level, suggests modulation of circadian rhythm.
  6. 請求項１から５いずれか１項記載の方法を用いて、概日リズムを変調させる物質をスクリーニングする方法。 A method for screening a substance that modulates circadian rhythm using the method according to any one of claims 1 to 5.
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WO2022043528A1 (en) * 2020-08-27 2022-03-03 Moreira Borralho Relogio Angela Method of assessing the circadian rhythm of a subject and/or assessing and predicting the athletic performance of said subject
EP4202059A1 (en) * 2021-12-22 2023-06-28 Charité - Universitätsmedizin Berlin Method for determining a circadian rhythm type of a human subject
WO2023118425A1 (en) 2021-12-22 2023-06-29 Charité - Universitätsmedizin Berlin Method for determining a circadian rhythm of a human subject

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