JP2013255481A - Biomarker for predicting disturbance of circadian rhythm - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for predicting a circadian rhythm sleep disorder or circadian rhythm disturbance, particularly a marker for accurately and easily diagnosing the circadian rhythm sleep disorder.SOLUTION: A marker for diagnosing a circadian rhythm sleep disorder includes HSPA1a protein or an HSPA1a gene. Specifically, a method is provided for diagnosing the circadian rhythm sleep disorder or circadian rhythm disturbance by measuring the increase amount of the expression amount of the HSPA1a or its gene in a subject's blood, and a method is provided for screening a substance for improving the circadian rhythm sleep disorder or circadian rhythm disturbance by measuring the increase control amount of the expression amount of the HSPA1a or its gene in the blood of a circadian rhythm sleep disorder model mouse or a stress sleep disorder mouse dosed with a test substance.

Description

  The present invention relates to a method for predicting circadian rhythm disturbance such as circadian rhythm sleep disorder and a marker therefor. Specifically, the present invention relates to a sleep disorder detection means, a sleep disorder detection method and detection kit, and a sleep disorder therapeutic agent screening method.

At present, one out of every five Japanese people is said to have some problems with sleep, and in particular elderly people, one in three people are said to have trouble with sleep.
Sleep disorders include sleep disordered breathing caused by respiratory disorders such as sleep apnea, but circadian rhythm sleep disorder (circadian rhythm sleep disorder) that is related to the onset of the circadian clock. ), It was difficult to identify and predict the cause and difficult to make an objective diagnosis. For circadian rhythm sleep disorder, exogenous acute syndrome caused by jet lag, night shift, shift work, etc., sleep phase regression syndrome (DSPS), sleep phase advance syndrome (ASPS), non-24-hour sleep It can be classified into intrinsic chronic syndromes such as arousal disorder and irregular sleep-wake pattern.
As a cause of circadian rhythm sleep disorders, some familial genetic causes have been reported in advanced sleep phase syndrome, but most of them are night shifts, shift work, jet lag, irregular diet, mental It is thought that lifestyle habits such as social stress are involved.
Diagnosis of sleep disorders is centered on interviews by the person and family, and definitive diagnosis also requires electroencephalogram measurement, and the development of a simple tool for objectively diagnosing sleep disorders is awaited. ing.
In the past, transthyretin, insulin-like growth factor, prostaglandin D synthase, HSP70, and BACE1 that have been observed to increase gene expression in experimental animals that have fallen asleep have been reported as possible sleep disorder markers. (Patent Document 1). However, the increase in the expression level of these genes is a phenomenon observed as a result of a sleep deprivation experiment conducted by a forced technique in rats, which are experimental animals, and therefore humans who develop as a result of chronic mental stress. It is difficult to extrapolate to other sleep disorders and human circadian rhythm sleep disorders. Furthermore, the induction of these gene expressions is a phenomenon observed in the brain, and its expression in blood has not been confirmed, and the possibility of clinical application in humans is unknown. .
The center of the circadian clock in mammals exists in the suprachiasmatic nucleus of the hypothalamus in the brain, and when this nerve nucleus is electrically destroyed, almost all circadian rhythms such as behavioral rhythm, sleep / wake rhythm, etc. disappear. To do. On the other hand, it is clear that circadian rhythmic biological clocks exist not only in the brain but also in almost whole body tissues such as heart, liver, kidney, fat, and peripheral blood leukocytes (non-patented). Reference 2). The body clocks present in these peripheral tissues are called peripheral clocks to distinguish them from the central clocks present in the suprachiasmatic nucleus of the hypothalamus.
As a result of exhaustive gene expression analysis using DNA microarrays in various tissues, it was found that several to several tens of% of genes were expressed daily in each tissue. It is considered to be involved in circadian rhythm formation of specific physiological functions (Non-patent Document 3). This also indicates that when comparing gene expression levels, it is necessary to take into account the body time, that is, the sampling time from the individual.
In order to examine the body time, a method using the expression level of a gene (Non-patent Document 4), a method using the amount of a metabolite in blood (Non-Patent Document 5), and the like can be considered.
Thus, although research on the circadian rhythmic biological clock has progressed, it has not yet been predicted that circadian rhythm is disturbed, and it is still difficult to accurately and easily diagnose circadian rhythm sleep disorders. Met.

JP 2007-75071 A

Lavie L, Dyugovskaya L, Golan-Shany O, Lavie P., (2010) J Sleep Res. 19: 139-47. Peripheral Clock, Time Biology Encyclopedia, pp.158-159, Asakura Shoten, 2008 Gachon, F. et al .: Chromosoma, 113, 103-112, 2004. Ueda HR. Et al., Proc Natl Acad Sci U S A.2004; 101: 11227-32. Minami Y. et al., Proc Natl Acad Sci U S A. 2009; 106: 9890-5. Oishi K., (2009) Neuro Endocrinol Lett. 30: 458-61 Miyazaki K, Itoh N, Ohyama S, Ohkura N, Oishi K (2011) Neurosci Res, 71S: e172-e173. Doi: 10.1016 / j.neures.2011.07.746 Maret S, Dorsaz S, Gurcel L, Pradervand S, Petit B, Pfister C, Hagenbuchle O, O'Hara BF, Franken P, Tafti M, (2007) Proc NatlAcad Sci U S A. 104: 20090-5

  The present invention provides a method for predicting disturbance of biological rhythm caused by disturbance of a biological clock such as circadian rhythm sleep disorder, and in particular, a marker for accurately and easily diagnosing circadian rhythm sleep disorder Is intended to provide.

Therefore, the present inventors eliminated the circadian rhythm of behavior by breeding under a 6-hour light-dark cycle (light period 3 hours: dark period 3 hours), which was previously developed by the present inventors. Circadian rhythm sleep disorder model mice (Non-patent Document 6) were used, and changes in gene expression levels in the liver tissues were comprehensively searched using a DNA microarray.
As a result, it was observed that the expression level of the HSPA1a gene in the HSP70 family was significantly increased in the liver compared to normal mice. HSPA1a is a type of heat shock protein (HSP) that is not directly related to the circadian clock gene. Conventionally, there is a report (Non-Patent Document 1) that the peripheral blood mononuclear cells of patients with sleep disordered breathing such as sleep apnea are increased, but the body clock is involved in the onset of sleep disordered breathing. The circadian rhythm sleep disorder is not common in the onset mechanism. It is also the first time that an increase in the expression level of the HSPA1a gene has been observed in relation to the circadian clock.
By the way, the present inventors previously created a stress sleep disorder mouse by using a normal breeding cage and continuing a breeding method that restricts the mouse from getting off the rotating wheel by physical shielding for two weeks. (Non-Patent Document 7). This stress sleep disorder mouse can be said to be an excellent stress sleep disorder model animal that exhibits a rhythm disorder that can be extrapolated to a general sleep disorder. In addition, because of the extreme decrease in activity during the active period (nighttime) shown by this mouse, it is now positioned as a model animal for chronic fatigue syndrome.
Therefore, the present inventors quantitatively determined the expression level of the HSPA1a gene in the cerebrum, hypothalamus, adipose tissue around the testis, heart, liver, and peripheral whole blood (including leukocytes) of the stress sleep disorder model mouse. When examined by the method, it was confirmed that all but the adipose tissue were not only significantly increased compared to the control mice but also increased with an expression rhythm corresponding to the light-dark cycle (FIG. 4). ). The fact that sleep disorders related to the circadian clock can be observed as an increase in gene expression in other organs other than the cerebrum, especially an increase in gene expression in peripheral blood, is extremely important for simple diagnosis of circadian rhythm sleep disorders. Meaningful.
By obtaining the above knowledge, the present invention relating to a circadian rhythm sleep disorder diagnostic marker comprising HSPA1a and its gene was completed.

That is, the present invention includes the following inventions.
[1] A circadian rhythm sleep disorder marker comprising HSPA1a or a gene thereof.
[2] A method for determining circadian rhythm sleep disorder or disturbance of circadian rhythm, characterized by using HSPA1a or a gene thereof as a circadian rhythm sleep disorder marker, comprising the following (1) to (3): A method comprising steps;
(1) a step of measuring the expression level of HSPA1a protein in a sample derived from a test mammal or the gene transcription level thereof,
(2) a step of comparing the expression level of HSPA1a protein in a sample derived from a healthy mammal or a gene transcription level thereof measured in advance,
(3) When the measured value in step (1) exceeds the measured value in step (2) with a significant difference, circadian rhythm sleep disorder or circadian rhythm disturbance in the test mammal A step of determining.
[3] The method according to [2], wherein the sample is white blood cells or blood containing white blood cells.
[4] A diagnostic agent for circadian rhythm sleep disorder, comprising an anti-HSPA1a antibody capable of quantitatively measuring the expression level of HSPA1a or a primer set for amplification of HSPA1a gene capable of quantitatively measuring the transcription level of HSPA1a gene.
[5] An anti-HSPA1a antibody capable of quantitatively measuring HSPA1a expression level or a primer set for amplification of HSPA1a gene capable of quantitatively measuring HSPA1a gene transcription level, or circadian rhythm sleep disorder or circadian Rhythm disturbance kit.
[6] A screening method for a substance for improving circadian rhythm sleep disorder or circadian rhythm disturbance, characterized by using HSPA1a or a gene thereof as a circadian rhythm sleep disorder marker, comprising the following (1) to (4): A method comprising the step of
(1) a step of preparing a circadian rhythm sleep disorder model mouse or a stress sleep disorder model mouse;
(2) dividing one of the model mice into two groups, administering a test substance to one, not administering to the other, and rearing both for a certain period, and collecting samples from both;
(3) measuring the expression level of HSPA1a protein or the transcription level of the gene in both samples and comparing the two,
(4) When the measured value of the group to which the test substance is administered is significantly lower than the measured value of the group to which the test substance is not administered, the test substance is used to improve circadian rhythm sleep disorder or circadian rhythm disturbance The process of evaluating that it is.
[7] The method according to [6], wherein the sample is white blood cells or blood containing white blood cells.

  HSPA1a which is a circadian rhythm sleep disorder diagnostic marker found in the present invention and its gene can be observed as an increase in the expression level in peripheral blood, the increase in the expression level in the cerebrum or other organs, Whether or not it is a circadian rhythm sleep disorder such as a stress sleep disorder can be determined only by measuring the amount of HSPA1a present in the blood of a subject suspected of having a sleep disorder. If the amount of HSPA1a present in the subject increases, it can be predicted and confirmed that the patient has circadian rhythm sleep disorder even if the patient does not complain of sleep disorder. It is also useful for diagnosis of chronic fatigue syndrome.

Daily behavior pattern of mice (drinking behavior of control group mice). In the figure, the vertical axis represents the date, and the horizontal axis represents the time. Daily behavior pattern of mice (drinking behavior of circadian rhythm sleep disorder mice). MRNA expression level of HSP70 (HSPA1a) in the liver (calculated as a ratio to β-actin expression level). The increase of the expression level in a circadian rhythm sleep disorder mouse | mouth is represented by making a control group 100%. Daily behavior pattern of mice (rolling behavior of control mice). Daily behavior pattern of mice (spinning behavior of stress sleep disorder mice). MRNA expression level in each organ in mice with stress sleep disorder. The notation of `` 10:00 '' on the left side of each organ indicates that it was measured at the beginning of the light period (10:00), and `` 22:00 '' indicates the beginning of the dark period (22 : 00). MRNA expression level of sleep deprivation stress response gene in cerebrum of stress sleep disorder mouse. “10:00” on the left side indicates that it was measured at the beginning of the light period (10:00), and “22:00” indicates that it was measured at the beginning of the dark period (22:00). Indicates that it was measured.

1. About HSP70 (HSPA1a) gene
HSPA1a is a member of the subfamily of “HSP70” attached to a protein having a molecular weight of 70 kDa among heat shock proteins (HSP) conserved in various species from humans to bacteria. Its amino acid sequence and base sequence are known, and various mutants are known for mammals. For example, typical amino acid sequences and base sequences of human HSPA1A can be obtained from NCBI Reference Sequences NP_005336.3 and NM_005345.5, respectively, and mouse Hspa1a can be obtained from NP_034609.2 and NM_010479.2, respectively. As a general notation method, a human gene (or protein) is represented as “HSPA1A” and a mouse gene (or protein) as “Hspa1a”. Rather, it means a gene (or protein) derived from another mammal and containing each allelic variant.
It is known that HSP is generally induced by various stresses on cells such as bacterial infection, inflammation, active oxygen, ultraviolet rays, and heavy metals. The expression of other HSPs, including other HSP70s, was not observed in the two model mice this time, and a significant increase in HSPA1a alone was confirmed. HSPA1a belongs to the HspA family that is involved in the control of king during the fall of proteins through biological membranes, and is constantly expressed in many organs such as the digestive tract and skin, and has anti-cell death and anti-inflammatory effects. It is known that it has an effect of protecting cells from various stresses. In addition, as described above, there is a report (Non-patent Document 1) that HSPA1a is increased in peripheral blood mononuclear cells of patients with sleep respiratory disorder such as sleep apnea. In sleep apnea, oxidative stress is caused by hypoxia, so it can be easily imagined that the expression of HSPA1a is induced by oxidative stress. The observed increase in HSPA1a gene expression in circadian rhythm sleep disorders is very surprising given that heat shock proteins have never been associated with a circadian clock.
Further, in the present invention, an increase in the expression level of HSPA1a is not only observed in the hypothalamus, heart, liver organs and peripheral whole blood (including leukocytes) but also in the early light period, in addition to the cerebrum. The circadian rhythm of expression that the amount of increase in the early dark period is significantly higher than the amount is synchronized not only in the cerebrum but also in the hypothalamus, heart and liver organs and peripheral whole blood including white blood cells Has been observed.

2. Circadian rhythm sleep disorder model mouse As previously reported by the present inventors (Non-patent Document 6), a mouse usually raised in a 24-hour cycle of 12 hours light period and 12 hours dark period is 3 hours light period. By rearing under a 6-hour light-dark cycle with a dark period of 3 hours, the circadian rhythm of behavior cannot synchronize with the light-dark cycle, and the circadian rhythm of behavior disappears. The disappearance of the behavior rhythm can be clearly determined, for example, by measuring the drinking behavior or the rotating behavior of the rotating basket using a chronobiology kit (Stanford Software Systems, CA). Since the mouse has also lost its circadian rhythm, it can be said that the mouse is a model mouse for circadian rhythm sleep disorder.

3. Stress-induced sleep disorder model mouse The present inventors still used a normal breeding cage, and the breeding method provided with a behavior restriction by physical shielding that the bottom of the cage is filled with water and the mouse cannot get off the rotating wheel For 2 weeks, a stress sleep disorder model mouse was created (Non-patent Document 7). This stress sleep disorder mouse exhibits a rhythm disorder that can be extrapolated to a general sleep disorder. In addition, the total amount of activity (especially the amount of activity during the dark period) decreases, and disturbances in behavioral rhythms, in which activity is observed during the light period and dark period, are observed. It is especially characterized by overactivity in the first half of the light period. Further, in conjunction with this, a decrease in sleep amount in the first half of the light period and an increase in sleep amount in the active period (dark period) are observed (Non-patent Document 7). For the determination of this behavioral rhythm, as in the case of the circadian rhythm sleep disorder model mouse, a method for measuring the momentum such as drinking behavior by using the Chronobiology Kit (Stanford Software Systems, CA) or rotating behavior of a rotating basket. Can be used. In addition, since there is an extreme decrease in activity during the active period (nighttime), it is positioned as a model animal for chronic fatigue syndrome, which is currently a major social problem. A specific method for producing such a stress sleep disorder mouse is described in detail in the application specification (Japanese Patent Application No. 2012-46806) concerning the circadian rhythm improving agent by the present inventors.

4). Method of using marker for diagnosing circadian rhythm sleep disorder in the present invention Expression level of HSPA1a in leukocytes or blood containing leukocytes collected from mammals such as patients suspected of having circadian rhythm sleep disorder, typically peripheral blood Measure with HSPA1a antibody. If the expression level of a healthy mammal (for example, a healthy person) is set in advance as a reference value, it can be diagnosed as circadian rhythm sleep disorder when the reference value is exceeded. HSPA1a antibody can be obtained by appropriately immunizing experimental animals such as mice, but is commercially available from Uscn Life Science Inc., Merck Millipore, and the like.
In particular, more accurate diagnosis is possible by collecting blood during the sleep start time zone.
Alternatively, quantitative PCR analysis or the like may be applied to the blood of the test mammal to similarly measure the increase in the expression level of HSPA1a at the gene transcription level. HSPA1a gene amplification primers are also commercially available from Invitrogen Corporation.
The human clock gene is also present in hair follicle cells, skin, and oral cells, and it is known that the circadian rhythm is inscribed. HSPA1a gene also varies in the expression level in these cells. Is predicted. Therefore, it is possible to monitor using follicular cells, skin cells, and oral cells instead of the blood sample of the test mammal. In the case of rodents, which are experimental animals, a technique of monitoring while collecting the tail is also possible.
The mammals that are subject to the diagnosis of circadian rhythm sleep disorders are typically humans, but they are pets such as dogs and cats, livestock animals such as cows, horses and pigs, mice and rats, etc. Any mammal such as a laboratory animal may be used.

5. 1. Screening method for circadian rhythm sleep disorder improving agent in the present invention Or 3. Circadian rhythm sleep disorder model mice or stress sleep disorder mice described in 1), these mice are divided into two groups, and a test substance is administered to one group of mice. Administration forms such as intravenous injection and application are also possible, but it is typically preferable to add a test substance in the feed or in the water supply. Samples such as blood from mice in the group to which the test substance was administered and samples from the control mice in the remaining groups were collected, and the respective HSPA1a expression levels or gene transcription levels were compared. When it decreases, the used test substance is selected as a circadian rhythm sleep disorder improving agent candidate. In this case, the expression level of HSPA1a or the gene transcription level is the same as that described in 4. It can be measured using the HSPA1a antibody or HSPA1a gene amplification primer described in 1.
Here, the mouse-derived sample is preferably simple and sensitive peripheral blood or leukocytes, but the cerebrum, hypothalamus, heart, and liver are removed and ground to extract protein or DNA, and HSPA1a expression in the extract is obtained. It is also possible to measure the quantity or its gene transcription level. Hair follicle cells, skin cells, oral cells, or tails may be collected and monitored.

EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not particularly limited to these examples.
Technical terms used in the present invention have meanings commonly understood by those skilled in the art unless otherwise defined.
Moreover, the description content of the prior art literature or the patent application specification cited in the present invention is incorporated as the description of this specification.

(Example 1) Observation of increased expression of HSPA1a gene in circadian rhythm sleep disorder model mice
(1-1) Production of circadian rhythm sleep disorder model mouse A circadian rhythm sleep disorder model mouse was produced according to the method described in Non-Patent Document 5. Specifically, it is as follows.
For acclimation of 13 mice of ICR strain (3-week-old male, Japan SLC Co., Ltd.) under a light / dark cycle of 12 hours light period, 12 hours dark period (lights on at 8:00, lights off at 20:00) Preliminary breeding was performed. After acclimation, the mice were divided into 2 groups (6 control groups, 7 experimental groups), the control group remained 12 hours light and 12 hours dark, and the experimental group 3 hours light and 3 hours dark Breeding in the light and dark 6-hour cycle (8: 00 lights, 11:00 lights off, 14:00 lights on, 17:00 lights off, 20:00 lights on, 23:00 lights off, 2:00 lights on, 5:00 lights off) went. As the feed, AIN-93M (manufactured by Oriental Yeast Co., Ltd.) was used.
Throughout the period, the drinking behavior of mice was measured with a chronobiology kit (Stanford Software Systems, CA). Since mice are nocturnal, drinking behavior is often observed at 12 hours at night when kept under normal light-dark cycle (12 hours light period, 12 hours dark period). However, it is described in Non-Patent Document 6 because the body clock that controls the circadian rhythm of behavior cannot synchronize with the light-dark cycle under a light-dark cycle of 6 hours period of light period 3 hours and dark period 3 hours. As a result, a clear behavioral rhythm disappeared (FIG. 1B).

(1-2) Remarkable induction of HSPA1a gene expression After rearing for 8 weeks under a 6-hour light-dark cycle of 3 hours in the light period and 3 hours in the dark period, total RNA is extracted from the liver of the sacrificed mouse. The expression level of HSPA1a mRNA was examined by quantitative PCR. In FIG. 2, the expression level of HSPA1a mRNA is quantified as a ratio to the expression level of β-actin mRNA, and then shown as a ratio with the control group as 100%. That is, it was found that the expression of the HSPA1a gene in the liver was remarkably induced by breeding under a light / dark 6 hour cycle.

(Example 2) Increased expression of HSPA1a gene expression using stress sleep disorder model mice
(2-1) Method for producing stress sleep disorder model mouse The stress sleep disorder mouse was produced according to the method described in the above-mentioned application specification (Japanese Patent Application No. 2012-46806) by the present inventors. Specifically, it is as follows.
Prepare 20 mice of Slc: B6C3F1 strain (4-week-old male, Japan SLC Co., Ltd.) under a light-dark cycle of 12 hours light period, 12 hours dark period (lights on at 8:00, lights off at 20:00) Raised. Mice were individually housed in a rotating cage (SW-15s, Merquest Co., Ltd.) throughout the period. The amount of activity of the mouse is represented by dots in the rotation of the rotating cage using a chronobiology kit (Stanford Systems, CA). CE-2 (manufactured by Clea Japan Co., Ltd.) was used as the feed.
FIG. 3 is a diagram showing an activity rhythm of a mouse. Ten mice out of 20 were kept under a 12-hour light-dark cycle (lighting on at 8:00, light-off at 20:00) for a period of more than one week (acclimation) for 12 weeks or longer. Stress-induced sleep disturbance was continuously induced for one week by filling the bottom with water and restricting the mouse from getting off the rotating wheel (stress breeding period). The other 10 mice continued to be non-stressed as described above as control mice.
This stress-induced sleep disorder model mouse exhibits a rhythm disorder that can be extrapolated to a general human sleep disorder (FIG. 3B). For example, the activity amount in the daytime (light period), which is the original inactive period, of the nocturnal mouse increases, and the activity amount in the night (dark period), which is the active period, decreases. In particular, it is characterized by overactivity in the first half of the light period. Further, in conjunction with this, it is also characteristic that a decrease in sleep amount in the first half of the light period and an increase in sleep amount in the active period (dark period) are observed (Non-patent Document 7).
FIG. 3 is a diagram showing a daily behavior pattern of a mouse. In FIG. 3, the vertical axis represents the date, and the horizontal axis represents the time. The dots indicate that the mouse's rolling behavior was observed. In addition, (FIG. 3A) shows the behavior pattern of the mouse | mouth which is not stressed, (FIG. 3B) shows the behavior pattern of the mouse | mouth which stressed. From FIG. 3, it can be seen that in the light period (8-20 o'clock: sleeping hours), there is almost no rotating behavior observed in the light period (no dots). On the other hand, it can be seen that in the dark period (20 to 8 o'clock: activity time zone), it is actively spinning around (many dots). In contrast, in the case of a stress sleep disorder model mouse, an increase in dots in the light period and a decrease in dots in the dark period are observed, indicating that the circadian rhythm of behavior is disturbed and sleep disorders occur. In addition, it can be seen that the activity amount of the model mouse is decreased as a whole, and particularly, an extreme decrease in the activity amount in the dark period (active period) is characteristic, and it can be said that this is a chronic fatigue syndrome model.

(2-2) Measurement of the expression level of HSPA1a mRNA in each organ of stress-induced sleep disorder mice After the stress sleep disorder was loaded for 2 weeks, the early period of light (10:00) and the early period of dark (22:00) ), The total RNA was extracted from the cerebrum, hypothalamus, adipose tissue around the testis, heart, liver, and peripheral whole blood (including leukocytes), and the expression level of HSPA1a mRNA was examined by quantitative PCR. It was. The primer sequences used are as follows.

Hspa1a-F: 5′-ATGGCCAAGAACACGGCGATC-3 ′ (SEQ ID NO: 1)
Hspa1a-R: 5′-ACCTGGAAGGGCCAGTGCTTC-3 ′ (SEQ ID NO: 2)

In FIG. 4, the expression level of HSPA1a mRNA is quantified as a ratio to the expression level of β-actin mRNA, and is shown as a ratio with the control group being 100%.
As shown in FIG. 4, the expression level of the HSPA1a gene in the liver, heart, cerebrum, hypothalamus, and whole blood, excluding adipose tissue, was low in the control group at the beginning of the light period (10:00) and dark. There is a daily rhythm that rises at the beginning of the season (22:00).
Moreover, the expression level of the HSPA1a gene in stress sleep disorder model mice was higher than that of the control group except for adipose tissue. In particular, the expression level of HSPA1a gene in whole blood including leukocytes was remarkably high at the beginning of the light period (10:00). That is, it is considered that abnormalities in biological rhythms such as sleep disorders can be estimated by examining the expression level of the HSPA1a gene in whole blood including blood cells, taking blood collection time into consideration.

(Example 3) Measurement of gene expression level of sleep deprivation stress response gene in the brain of stress sleep disorder mice For mice of the same Slc: B6C3F1 strain as in Example 2 (4 weeks old male, Japan SLC, Inc.) According to the method described in Example 2, stress sleep disorder mice were prepared (10 mice and 10 control mice were used). After 2 weeks of stressful sleep disorder, mice were killed in the early light phase (10:00) and early dark phase (22:00), and total RNA was extracted from the cerebrum and quantitative PCR was used. The expression levels of Ttr (transthyretin) (patent document 1), Homer1a (non-patent document 8), and Egr2 (NGFI-B) (non-patent document 8), which are known as sleeplessness stress response genes, were examined. Each of these genes is a gene group in which an increase in gene expression level was observed in the brain of experimental animals subjected to forced sleep deprivation.
The primer sequences used are as follows.

Ttr-F: 5'-CATGAATTCGCGGATGTG-3 '(SEQ ID NO: 3)
Ttr-R: 5′-GATGGTGTAGTGGCGATGG-3 ′ (SEQ ID NO: 4)
Homer1a-F: 5'-AGCTCATGTCTTCCAGATTGACC-3 '(SEQ ID NO: 5)
Homer1a-R: 5'-GTCATGTTTGGTGTGATGGTGCT-3 '(SEQ ID NO: 6)
Egr2-F: 5'-TGACCAGATGAACGGAGTGGC-3 '(SEQ ID NO: 7)
Egr2-R: 5'-GTGAAGGTCTGGTTTCTAGGTGC-3 '(SEQ ID NO: 8)

In FIG. 5, the mRNA expression level of each gene is quantified as a ratio to the expression level of β-actin mRNA, and then shown as a ratio with the control group as 100%.
As shown in FIG. 5, the expression levels of Ttr (transthyretin), Homer1a, and Egr2 genes known as sleep deprivation stress response genes are significantly different between control mice and stress sleep disorder model mice. There was no difference.
This indicates that the gene expression responsiveness in this stress-induced sleep disorder model mouse is clearly different from the gene expression responsiveness due to forced sleep deprivation stress. However, this proved to be a very accurate model animal for human sleep disorders that develop as a result of chronic mental stress.
At the same time, by examining the expression level of HSPA1a or HSPA1a gene, it is possible to estimate biological rhythm abnormalities such as chronic sleep disorders different from forced sleepless stress. That is, it was demonstrated that the HSPA1a or HSPA1a gene of the present invention is an excellent circadian rhythm sleep disorder marker capable of determining circadian rhythm sleep disorder or circadian rhythm disturbance.

Claims (7)

  A circadian rhythm sleep disorder marker comprising HSPA1a or its gene. A method for determining circadian rhythm sleep disorder or circadian rhythm disturbance, characterized by using HSPA1a or a gene thereof as a circadian rhythm sleep disorder marker, comprising the following steps (1) to (3): Method;
(1) a step of measuring the expression level of HSPA1a protein in a sample derived from a test mammal or the gene transcription level thereof,
(2) a step of comparing the expression level of HSPA1a protein in a sample derived from a healthy mammal or a gene transcription level thereof measured in advance,
(3) When the measured value in step (1) exceeds the measured value in step (2) with a significant difference, circadian rhythm sleep disorder or circadian rhythm disturbance in the test mammal A step of determining.   The method according to claim 2, wherein the sample is white blood cells or blood containing white blood cells.   A diagnostic agent for circadian rhythm sleep disorder, comprising an anti-HSPA1a antibody capable of quantitatively measuring the expression level of HSPA1a or a primer set for amplification of HSPA1a gene capable of quantitatively measuring the transcription level of HSPA1a gene.   Circadian rhythm sleep disorder or disturbance of circadian rhythm, characterized by including anti-HSPA1a antibody that can quantitatively measure HSPA1a expression level or primer set for HSPA1a gene amplification that can quantitatively measure HSPA1a gene transcription level Evaluation kit. A method for screening a substance for improving circadian rhythm sleep disorder or circadian rhythm disturbance, characterized by using HSPA1a or a gene thereof as a circadian rhythm sleep disorder marker, comprising the following steps (1) to (4): A method comprising:
(1) a step of preparing a circadian rhythm sleep disorder model mouse or a stress sleep disorder model mouse;
(2) dividing one of the model mice into two groups, administering a test substance to one, not administering to the other, and rearing both for a certain period, and collecting samples from both;
(3) measuring the expression level of HSPA1a protein or the transcription level of the gene in both samples and comparing the two,
(4) When the measured value of the group to which the test substance is administered is significantly lower than the measured value of the group to which the test substance is not administered, the test substance is used to improve circadian rhythm sleep disorder or circadian rhythm disturbance The process of evaluating that it is.   The method according to claim 6, wherein the sample is white blood cells or blood containing white blood cells.

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