WO2021085425A1 - Method for predicting vascular aging, method for predicting disease risk, biomarker for predicting vascular aging, biomarker for diseases, measurement kit, and diagnostic device - Google Patents

Method for predicting vascular aging, method for predicting disease risk, biomarker for predicting vascular aging, biomarker for diseases, measurement kit, and diagnostic device Download PDF

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WO2021085425A1
WO2021085425A1 PCT/JP2020/040285 JP2020040285W WO2021085425A1 WO 2021085425 A1 WO2021085425 A1 WO 2021085425A1 JP 2020040285 W JP2020040285 W JP 2020040285W WO 2021085425 A1 WO2021085425 A1 WO 2021085425A1
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biomarker
aging
vascular aging
disease
cmpk2
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義知 本田
章代 川本
一也 ▲高▼橋
秀郎 志水
衛 上田
雄一 二宮
堯彬 川添
吉宏 ▲濱▼田
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義知 本田
株式会社Ｙ．Ｏｃｅａｎ
章代 川本
一也 ▲高▼橋
秀郎 志水
衛 上田
雄一 二宮
堯彬 川添
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

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  • the present invention relates to a biomarker contained in a biological sample collected from a subject, and by measuring the expression level of the biomarker, the degree of progression of vascular aging of the subject is evaluated, and the subject's arteriosclerosis and brain Related to methods and biomarkers for determining the presence or absence of vascular disease, cardiovascular disease, and other age-related diseases.
  • vascular aging Since the degree of aging associated with human aging is not necessarily proportional to the calendar age and varies greatly among individuals, a scale capable of accurately expressing the degree of aging is required. Human cells and tissues are subject to changes with aging, but it is known that structural and functional changes are particularly likely to appear in blood vessels with aging. Structural changes that appear as vascular aging include thickening and calcification of the intima of arteries and an increase in the collagen / elastin ratio, and functional changes such as decreased vascular extensibility, arteriosclerosis, and occlusion of arteries. It becomes a factor that causes it.
  • vascular aging is expressed not only as aging but also as "vascular age” because aging is accelerated not only by aging but also by diabetes, hypertension, hyperlipidemia, obesity, etc. that cause metabolic syndrome.
  • vascular age By evaluating the degree of vascular aging, it is possible to predict the occurrence of serious and irreversible tissue disorders such as cerebral infarction, myocardial infarction, and obese arteriosclerosis, and take measures to prevent them. Be expected.
  • FMD Flow-Mediated Dilatation
  • Patent Document 1 a system for detecting aging of peripheral blood vessels by measuring volume pulse waves of the fundus artery and the fundus vein is disclosed (see Patent Document 1).
  • a method of detecting a voluminous pulse wave a method of applying an ultrasonic echo to a blood vessel and obtaining the transmission speed of blood flow from the Doppler effect observed in the reflected wave, or in the blood
  • the volume pulse wave is measured by irradiating blood cells such as floating red blood cells with a laser, detecting the light that bounces off the blood cells, and measuring the time difference between them. What is a simple method for measuring blood vessel aging? It's hard to say.
  • a method of measuring a change in forearm volume while venous perfusion of the upper arm is stopped by using a volume pulse wave called plethysmography can be mentioned.
  • this measurement method it has been mainly used for research purposes such as injecting vasoactive substances such as acetylcholine and bradykinin directly into blood vessels and evaluating their effects, but repeated measurement is not possible.
  • it is not used in actual clinical examinations because it requires skill in operation.
  • RH-PAT reactive hyperemic index using peripheral arterial volume pulse wave
  • various biochemistrys that are present in blood and can be used as biomarkers that reflect the condition of the vascular endothelium of the subject.
  • a method for measuring a substance can also be mentioned.
  • the vasodilatory action depends on the action of NO (nitric oxide) produced by the action of eNOS existing in vascular endothelial cells, but when the vascular endothelial function is impaired due to various causes, the function of eNOS decreases. It is known that vasodilation is inhibited.
  • PTX3 the acute phase reaction protein known as PTX3 (Pentraxin 3) has been used as an inflammation marker for a long time and has a strong inverse correlation with the previous FMD, so it can be used as one of the vascular endothelial function tests. is there.
  • PTX3 does not necessarily reflect vascular aging, and is widely used as a biomarker for the entire circulatory system.
  • biomarkers related to vascular endothelial function are known.
  • adhesion molecules ICAM and VCAM are known to correlate with FMD, and von Willebrand factor and the like are also mentioned. These are factors related to thrombus coagulation due to damage to the vascular endothelium and are not directly linked to vascular aging.
  • Patent Document 2 a predictor of vascular aging and a method for examining early lesions caused by vascular aging are known (see Patent Document 2), and in such an examination method, a specific human apolypoprotein B100 in a blood sample is specified. Glutathione formation of thiol groups is used as an index, and by measuring this concentration, it is used as a biomarker for vascular aging.
  • the test method of Patent Document 2 is a method of detecting B100 contained in the serum of a subject by reacting a specific antibody that specifically binds to a domain in which a glutathioneized thiol group is present in apolypoprotein B100.
  • the present invention can be used for diagnosing vascular aging in a subject, and by measuring the biomarker contained in the biological sample collected from the subject and the expression level of the biomarker, the subject.
  • CMPK2 contained in biological samples collected from subjects. It was found that it can be discriminated by measuring the expression level of any gene of RNF213 or both genes.
  • the present invention is characterized in that, as a biomarker for predicting vascular aging, the expression level of either or both genes of CMPK2 and RNF213 contained in a biological sample collected from a subject is measured by such measurement. , Predict vascular aging.
  • CMPK2 and RNF213 contained in a biological sample collected from a subject as biomarkers for any of arteriosclerosis, cerebrovascular disease such as cerebral infarction, and cardiovascular disease. It is characterized by measuring the expression level of either or both genes, and such measurement predicts the presence or absence of those diseases.
  • saliva, blood, urine and the like can be used as a biological sample.
  • the biomarker for predicting vascular aging of the present invention comprises one or both genes of CMPK2 and RNF213 contained in a biological sample.
  • the biomarker for diseases of the present invention comprises one or both genes of CMPK2 and RNF213 contained in a biological sample.
  • saliva, blood, urine and the like can be used as a biological sample.
  • the measurement kit of the present invention contains a reagent for measuring the expression level of either or both genes of CMPK2 and RNF213 in a biological sample collected from a subject.
  • the diagnostic apparatus of the present invention measures the expression level of either or both genes of CMPK2 and RNF213 in a biological sample obtained from a subject, and diagnoses a disease related to a blood vessel of the subject from whom the biological sample was collected. ..
  • the present invention can be used for diagnosing vascular aging in a subject, and the progress of vascular aging in the subject is measured by measuring the biomarker contained in the biological sample collected from the subject and the expression level of the biomarker. It has the effect of being able to evaluate the degree of the disease and determine the presence or absence of arteriosclerosis, cerebrovascular disease, cardiovascular disease, and other age-related diseases of the subject.
  • Biochemical changes that occur along with structural changes in blood vessels due to aging of blood vessels include decreased expression of the endothelial NO synthase eNOS, which contributes to vasodilation, and angiotensin II, endothelin, and thrombo, which are vasoconstrictor factors produced in the body. It is known that the production of xanthin A2 and the like is conversely increased. Furthermore, looking at vascular senescence at the level of cells that make up blood vessels, cells that have undergone cellular senescence stop dividing, and at the same time, the expression of various inflammatory cytokines is enhanced, resulting in infiltration of inflammatory cells such as macrophages. It is known that tissue remodeling of the blood vessel wall progresses.
  • Vascular aging further accelerates the aging process due to the increased production and secretion of inflammatory cytokines and the progress of structural remodeling due to infiltration of macrophages.
  • inflammatory cytokines cause abnormalities in telomeres that control cell division and accelerate cell aging (Dayeon Shin, et al., J. Clin. Med. 2019, 8). , 711; doi: 10.3390 / jcm8050711).
  • Macrophages have TLRs (Toll-like receptors), RLRs, NLRs, and other PRRs (pattern recognition receptors) on the surface that are not only PAMPs (pathogen-associated molecular patterns) derived from microorganisms, but also cytoplasm and nuclear substances of autologous cells. When is damaged, it is also activated by DAMPs (damage-associated molecular patterns) as a self-tissue-derived molecular pattern, and as a result of inflammation-induced cell death, IL-1 ⁇ is released from the cells. It is known to provoke an inflammatory response in the surrounding area.
  • PAMPs pathogen-associated molecular patterns
  • DAMPs damage-associated molecular patterns
  • IL-1 ⁇ is released from the cells. It is known to provoke an inflammatory response in the surrounding area.
  • telomeres of vascular cells are shortened, and when cell division is no longer possible, cell death occurs and cell components begin to decompose due to apoptosis.
  • the PRR on the surface of macrophages in the blood recognizes the DAMPs produced at this time, and activates the transcription factor NF- ⁇ B pathway, thereby initiating an inflammatory reaction.
  • interleukin IL-1 ⁇ plays a major role in the inflammatory response as an inflammatory cytokine, and its production is enhanced by the activation of caspase 1 by the NLRP3 inflammasome.
  • RNF213 another biomarker taken up as an index of vascular aging in the present invention, has a specific gene polymorphism of a gene encoding this, which forms an abnormal vascular network of blood vessels in the brain, and is susceptible to "moyamoya disease".
  • a gene "Moyamoya disease” is a rare disease and is a designated intractable disease, but it is estimated that about 3 million people, which is 2 to 3% of the domestic population, carry this polymorphism gene.
  • Recent findings (National Cerebral and Cardiovascular Research Center (abbreviation: National Circulation), Department of Neurology, Director Masafumi Inohara, Dr.
  • RNF213 the rationale for the direct link between vascular aging and RNF213 has not yet been clarified, but since a clear correlation is observed between the two as shown in Examples described later, RNF213 It has been shown by the present invention that it can also be used as a biomarker for vascular aging.
  • the present inventors do not use inflammatory cytokines and various other known methods that have been attracting attention as a method for detecting vascular aging, but as a completely different age-related factor. Focusing on CMPK2 and RNF213 and the gene mRNA encoding each of them, they have found that they can be used as biomarkers in biological samples, leading to the present invention.
  • CMPK2 and RNF213 and the gene mRNA encoding each of them they have found that they can be used as biomarkers in biological samples, leading to the present invention.
  • the index of vascular aging correlates with the progress of arteriosclerosis and is thought to lead to use as an index of arteriosclerosis. Be done.
  • the present invention is also considered to be an index for the expression of CMPK2 associated with chronic inflammation, it can be widely used as an index for chronic inflammation associated with aging. Furthermore, it can be used as an index of various age-related diseases such as diabetes, metabolic syndrome, chronic kidney disease, and Alzheimer's disease.
  • the present inventors performed database analysis on mRNA expressed by vascular aging, and used databases such as BioGPS, Array Express, NCBI GEO, ReFex, Human proteome map, The human protein atlas, and Wikimedia commons as databases.
  • databases such as BioGPS, Array Express, NCBI GEO, ReFex, Human proteome map, The human protein atlas, and Wikimedia commons as databases.
  • a set of genes included in E-GEOD-13712 Transcription profiling by array of human young and senescent HUVECs under static and laminar shear stress conditions
  • GSE98081 Neoroarray analysis of TNF ⁇
  • IFI44L IFIT3, IFI44, MX1, IFITM1, OAS2, EPSTI1, HERC6, XAF1, RSAD2, OASL, CCNA1, HSD17B2, ADRB1, SFTA1P (SFTPF), CMPK2, RNF213, OAS3, It was 20 mRNAs of.
  • CMPK2 was selected and narrowed down to a total of 5 genes.
  • the setting was performed for 45 cycles with 50 ° C.-2 minutes, 95 ° C.-20 seconds, 95 ° C.-1 second, and 60 ° C.-20 seconds as one cycle.
  • a comparative CT method was used with ⁇ -actin as an internal standard.
  • FIG. 1 shows the expression levels of CMPK2 gene mRNA contained in saliva collected from seniors and young adults for each group. Expression of CCNA1 and HSD17B2 was not confirmed in saliva. In addition, it was found that ADRB1 has a large variation in measured values and is difficult to use as a biomarker. Furthermore, we also tried to detect the adhesion factor VCAM1, which is known to be upregulated on vascular endothelial cells in arteriosclerosis and the like, but could not be detected in this example. From the results shown in FIG. 1, the relationship between the amount of the CMPK2 gene expressed in saliva and aging was clearly shown, and it was clarified that the expression of CMPK2 was enhanced with aging. Similarly, it was revealed that the expression of the RNF213 gene contained in saliva of RNF213 is enhanced by aging.
  • CMPK2 Based on the above results for CMPK2, the expression levels of IF144L, HERC6, IFIT1 and OASL genes were also confirmed in saliva as other genes that could be correlated with the expression of the CMPK2 gene. The results shown are obtained. 2 (1) to (5) show a comparison of the expression levels of CMPK2, IF144L, HERC6, IFIT1 and OASL genes in saliva, respectively, for healthy adults (A: ID number: St008) and elderly people (B: ID). The result of comparison for three persons (number: Kr138) and the elderly (C: ID number: Kr144) is shown. From the results of FIGS.
  • CMPK2 the expression of CMPK2 and any of the genes correlated with the CMPK2 tend to be consistent with each other, and can be used as a biomarker for vascular aging, which is the object of the present invention.
  • the upregulation of CMPK2 expression was the most prominent among them, so that it can be understood that CMPK2 is the most excellent biomarker among them.
  • CMPK2 reflects the state of the vascular endothelium at that time and sensitively expresses the progress of vascular aging
  • PWV is not only the vascular endothelium but also the entire vascular matrix. Since it reflects the mechanical structural change, it is considered to represent the secular change up to the time of measurement. Therefore, since there is a possibility that vascular aging is progressing due to the high value of CMPK2, in this example, the subject whose PWV was low despite the high value of CMPK2 will soon be distant. Since there is a possibility that the value of PWV will rise, it will be an opportunity to take preventive measures to delay vascular aging such as reviewing lifestyle habits.
  • CMPK2 can be extremely useful as a biomarker as an index of vascular aging because it can be tested by collecting saliva of a subject, unlike the conventional test method by blood sampling.
  • Example 1 saliva was collected from 5 healthy adults and 4 elderly people, and the correlation between the concentration of the RNF213 gene contained in each saliva and the PWV value was confirmed. The result will be explained.
  • FIG. 3 shows the expression level of the RNF213 gene mRNA contained in saliva collected from the elderly (Senior) and the healthy adult (Young) for each group.
  • 2 out of 3 patients with high RNF213 values had high PWV values, and conversely, 2 patients with low RNF213 values had low PWV values. ..
  • RNF213 was observing the progress of vascular aging at this time, whereas PWV was aging. I guess this is to reflect it.
  • RNF213 is correlated with vascular aging for healthy adults. However, for the elderly, it is only used as an index for estimating the progress of vascular aging at the time of measuring the numerical value of RNF213.
  • the present invention can numerically indicate the progress of vascular aging of a subject using saliva or blood sample from the subject, it can be used as a biomarker for age-related diseases associated with vascular aging. It is useful as a biomarker for the diagnosis and prevention of age-related diseases. It is also useful as a biomarker for the diagnosis and prevention of chronic inflammation such as metabolic syndrome, diabetes, chronic kidney disease, and Alzheimer's disease.

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Abstract

Provided are: a biomarker which can be used in the diagnosis of vascular aging in a subject and is included in a biological sample taken from the subject; and a method and a biomarker for not only evaluating the degree of progression of vascular aging in the subject by measuring an expression level of the biomarker but also determining the presence or absence of arteriosclerotic disease, cerebrovascular disease, cardiovascular disease, or other aging-related diseases of the subject. The gene expression level of one or both of CMPK2 and RNF213, which are contained in the biological sample taken from the subject as a biomarker for predicting vascular aging, are measured. In addition, the gene expression level of one or both of CMPK2 and RNF213, which are contained in the biological sample taken from the subject as a biomarker for arteriosclerotic disease, cerebrovascular disease, or cardiovascular disease, are measured. Saliva can be suitably used as a biological sample taken from a subject.

Description

血管老化予測方法、疾患リスク予測方法、血管老化予測用バイオマーカー、疾患用バイオマーカー、測定キット、及び、診断装置Blood vessel aging prediction method, disease risk prediction method, vascular aging prediction biomarker, disease biomarker, measurement kit, and diagnostic device
　本発明は、被験者から採取した生体試料中に含まれるバイオマーカーに関し、該バイオマーカーの発現レベルを測定することで、被験者の血管老化の進行の程度を評価するとともに被験者の動脈硬化性疾患、脳血管疾患、心血管疾患、その他の加齢関連疾患の有無を判定する方法とバイオマーカーに関する。 The present invention relates to a biomarker contained in a biological sample collected from a subject, and by measuring the expression level of the biomarker, the degree of progression of vascular aging of the subject is evaluated, and the subject's arteriosclerosis and brain Related to methods and biomarkers for determining the presence or absence of vascular disease, cardiovascular disease, and other age-related diseases.
　ヒトの加齢に伴う老化の程度は必ずしも暦年齢に比例せず個人差が大きいことから、老化の程度を正確に数値で表すことのできる尺度が求められている。ヒトの細胞や組織は加齢による変化を受けるが、特に、血管は加齢に伴い構造的・機能的変化が明確に表れやすいことが知られている。血管老化として現れる構造的変化には、動脈血管内膜の肥厚や石灰化およびコラーゲン／エラスチン比の増大などが現れ、機能的変化として血管の伸展性が低下し、動脈硬化や動脈の閉塞等を引き起こす要因となる。
　また、血管老化は加齢だけでなくメタボリックシンドロームの起因となる糖尿病や高血圧、高脂血症、肥満などによっても老化が加速されることから、暦上の年齢だけでなく「血管年齢」と表現される血管老化の程度を評価することにより、脳梗塞や心筋梗塞、閉塞性動脈硬化症などの重篤で不可逆的な組織障害の発生を予知し、これを予防するための対策を講じることが期待される。 Since the degree of aging associated with human aging is not necessarily proportional to the calendar age and varies greatly among individuals, a scale capable of accurately expressing the degree of aging is required. Human cells and tissues are subject to changes with aging, but it is known that structural and functional changes are particularly likely to appear in blood vessels with aging. Structural changes that appear as vascular aging include thickening and calcification of the intima of arteries and an increase in the collagen / elastin ratio, and functional changes such as decreased vascular extensibility, arteriosclerosis, and occlusion of arteries. It becomes a factor that causes it.
In addition, vascular aging is expressed not only as aging but also as "vascular age" because aging is accelerated not only by aging but also by diabetes, hypertension, hyperlipidemia, obesity, etc. that cause metabolic syndrome. By evaluating the degree of vascular aging, it is possible to predict the occurrence of serious and irreversible tissue disorders such as cerebral infarction, myocardial infarction, and obese arteriosclerosis, and take measures to prevent them. Be expected.
　近年、血管老化の測定方法として、ＦＭＤ（Flow-Mediated Dilatation）測定が利用されているが、この測定方法は安静時における上腕動脈の血管径を測定後、５分間駆血し駆血解除後の最大拡張期における血管径の比率から血管の柔らかさを数値化して表すものである。ＦＭＤ測定のための装置は比較的高価であり、普及はあまり進んでおらず、特別な病院のみで診断可能であるため、より簡便で費用の掛からない検査方法の確立が望まれている。 In recent years, FMD (Flow-Mediated Dilatation) measurement has been used as a method for measuring blood vessel aging. In this measurement method, after measuring the blood vessel diameter of the brachial artery at rest, the blood vessel is aerated for 5 minutes and the avascularization is released. The softness of blood vessels is quantified from the ratio of blood vessel diameter in the maximum diastolic period. Since the device for FMD measurement is relatively expensive, has not been widely used, and can be diagnosed only in a special hospital, it is desired to establish a simpler and less costly examination method.
　かかる状況下、血管老化の検出方法として、眼底動脈と眼底静脈の容積脈波を測定することで末梢血管の老化を検出するシステムが開示されている（特許文献１を参照）。特許文献１に開示されたシステムの場合、容積脈波を検出する方法として、血管に超音波エコーを当て、その反射波に認められるドップラー効果から血流の伝達速度を求める方法や、血液中に浮遊する赤血球などの血液細胞にレーザー照射して、それから跳ね返ってくる光を検出してその間の時間差を測定する方法などによって容積脈波を測定しており、簡便に血管老化を測定する方法とは言い難い。 Under such circumstances, as a method for detecting vascular aging, a system for detecting aging of peripheral blood vessels by measuring volume pulse waves of the fundus artery and the fundus vein is disclosed (see Patent Document 1). In the case of the system disclosed in Patent Document 1, as a method of detecting a voluminous pulse wave, a method of applying an ultrasonic echo to a blood vessel and obtaining the transmission speed of blood flow from the Doppler effect observed in the reflected wave, or in the blood The volume pulse wave is measured by irradiating blood cells such as floating red blood cells with a laser, detecting the light that bounces off the blood cells, and measuring the time difference between them. What is a simple method for measuring blood vessel aging? It's hard to say.
　上記のような方法以外にも、例えばプレスチモグラフィー（plethysmography）と称される容積脈波を用い、上腕の静脈灌流を停止させた状態で前腕容積の変化を測定する方法が挙げられる。この測定方法を利用するに際し、アセチルコリンやブラジキニンなどの血管作動性物質を血管内に直接注入して、それらの影響を評価するなど主に研究用途として用いられてきたが、反復測定ができず、また、操作に熟練を要するなどの理由から、実臨床検査には用いられていない。
　また、ＲＨ－ＰＡＴ（末梢動脈容積脈波を用いた反応性充血指数）の測定方法が挙げられるが、これは両方の人差し指（示指）に専用のカフを装着して指尖脈波を測定する方法である。上述したＦＭＤが、上腕動脈の反応性血管拡張を見るのに対し、この測定方法では、示指の指尖容積を見ることから、被験者の自律神経活動の影響を敏感に受ける可能性が指摘されており、未だ十分なエビデンスの蓄積もないことから、現状では一般的に用いる方法とは言い難い。 In addition to the above methods, for example, a method of measuring a change in forearm volume while venous perfusion of the upper arm is stopped by using a volume pulse wave called plethysmography can be mentioned. When using this measurement method, it has been mainly used for research purposes such as injecting vasoactive substances such as acetylcholine and bradykinin directly into blood vessels and evaluating their effects, but repeated measurement is not possible. In addition, it is not used in actual clinical examinations because it requires skill in operation.
In addition, there is a method of measuring RH-PAT (reactive hyperemic index using peripheral arterial volume pulse wave), which measures fingertip pulse wave by attaching a dedicated cuff to both index fingers (index fingers). The method. While the above-mentioned FMD looks at the reactive vasodilation of the brachial artery, it is pointed out that this measurement method may be sensitive to the influence of the autonomic nerve activity of the subject because it looks at the fingertip volume of the index finger. At present, it is hard to say that it is a commonly used method because there is not enough evidence accumulated yet.
　上記のような様々な装置を利用して血管老化または血管内皮機能検査を行う方法とは別に、血液中に存在し、被験者の血管内皮の状態を反映するバイオマーカーとして利用可能な様々な生化学物質の測定方法も挙げることができる。例えば、血管の拡張作用は血管内皮細胞に存在するｅＮＯＳの作用で産生されるＮＯ（一酸化窒素）の働きによるが、様々な原因で血管内皮機能が障害されるとｅＮＯＳの機能が低下し、血管の拡張が阻害されることが知られている。血中のＮＯ濃度の測定は、ＮＯが化学的に不安定で短寿命であるため測定は困難であるため、ＮＯが代謝して生成するＮＯｘを測定する方法が可能である。この指標は血管老化に特異的ではなく、それ以外の原因で数値が変化するため、補助的な指標としての利用に限られる。 Apart from the method of performing vascular aging or vascular endothelial function test using various devices as described above, various biochemistrys that are present in blood and can be used as biomarkers that reflect the condition of the vascular endothelium of the subject. A method for measuring a substance can also be mentioned. For example, the vasodilatory action depends on the action of NO (nitric oxide) produced by the action of eNOS existing in vascular endothelial cells, but when the vascular endothelial function is impaired due to various causes, the function of eNOS decreases. It is known that vasodilation is inhibited. Since it is difficult to measure the NO concentration in blood because NO is chemically unstable and has a short life span, a method of measuring NOx produced by metabolism of NO is possible. This index is not specific to vascular aging, and its numerical value changes due to other causes, so it is limited to use as an auxiliary index.
　また、ＰＴＸ３（Pentraxin 3）として知られる急性期反応タンパクは、古くから炎症マーカーとして利用されており、先のＦＭＤと強い逆相関関係があることから、血管内皮機能検査の一つとして利用可能である。しかしながら、ＰＴＸ３は、必ずしも血管老化を反映するものではなく、広く循環器系全般のバイオマーカーとして利用されるといった位置づけである。これら以外にも多数の血管内皮機能関連のバイオマーカーが知られており、例えば、接着分子であるＩＣＡＭやＶＣＡＭは、ＦＭＤと相関することが知られており、ｖｏｎ　Ｗｉｌｌｅｂｒａｎｄ因子なども挙げられるが、これらは血管内皮の障害による血栓凝固などに係る因子であり血管老化と直接結びつくものではない。 In addition, the acute phase reaction protein known as PTX3 (Pentraxin 3) has been used as an inflammation marker for a long time and has a strong inverse correlation with the previous FMD, so it can be used as one of the vascular endothelial function tests. is there. However, PTX3 does not necessarily reflect vascular aging, and is widely used as a biomarker for the entire circulatory system. In addition to these, many biomarkers related to vascular endothelial function are known. For example, adhesion molecules ICAM and VCAM are known to correlate with FMD, and von Willebrand factor and the like are also mentioned. These are factors related to thrombus coagulation due to damage to the vascular endothelium and are not directly linked to vascular aging.
　これらに対して、血管老化の予知因子および血管老化に起因する早期病変の検査方法が知られており（特許文献２を参照）、かかる検査方法では、血液試料中のヒトアポリポタンパクＢ１００の特定のチオール基のグルタチオン化を指標として、この濃度を測定することにより、血管老化に対するバイオマーカーとして利用する。特許文献２の検査方法は、アポリポタンパクＢ１００におけるグルタチオン化したチオール基が存在するドメインに特異的に結合する特異抗体を反応させることにより、被験者の血清中に含まれるＢ１００の検出を行う方法であるが、モノクロナール抗体あるいはポリクロナール抗体の作製や測定方法が煩雑であり、長時間を要することに加えて被験者から採血して血清を分離するため、より簡便で被験者に対する侵襲性の低い検査方法が求められている。 On the other hand, a predictor of vascular aging and a method for examining early lesions caused by vascular aging are known (see Patent Document 2), and in such an examination method, a specific human apolypoprotein B100 in a blood sample is specified. Glutathione formation of thiol groups is used as an index, and by measuring this concentration, it is used as a biomarker for vascular aging. The test method of Patent Document 2 is a method of detecting B100 contained in the serum of a subject by reacting a specific antibody that specifically binds to a domain in which a glutathioneized thiol group is present in apolypoprotein B100. However, the preparation and measurement method of monoclonal antibody or polyclonal antibody is complicated, and in addition to requiring a long time, blood is collected from the subject and serum is separated. Therefore, a simpler and less invasive test method for the subject is required. Has been done.
国際公開パンフレットＷＯ２００７／１３２８６５号公報International Pamphlet WO2007 / 132865 国際公開パンフレットＷＯ２００７／０６３６６４号公報International Pamphlet WO2007 / 063664
　かかる状況に鑑みて、本発明は、被験者における血管老化の診断に利用可能であり、該被験者から採取した生体試料中に含まれるバイオマーカーと、該バイオマーカーの発現レベルを測定することにより、被験者の血管老化の進行の程度を評価するとともに、被験者の動脈硬化性疾患、脳血管疾患、心血管疾患、その他の加齢関連疾患の有無を判定するための該バイオマーカーの利用方法を提供することを目的とする。 In view of this situation, the present invention can be used for diagnosing vascular aging in a subject, and by measuring the biomarker contained in the biological sample collected from the subject and the expression level of the biomarker, the subject. To provide a method of using the biomarker to evaluate the degree of progression of vascular aging in a subject and to determine the presence or absence of arteriosclerosis, cerebrovascular disease, cardiovascular disease, and other age-related diseases of a subject. With the goal.
　本発明者らは、被験者における血管老化や、動脈硬化性疾患、脳血管疾患、心血管疾患などの判別に利用可能なバイオマーカーについて鋭意検討した結果、被験者から採取した生体試料に含まれるＣＭＰＫ２とＲＮＦ２１３の何れかの遺伝子、または両方の遺伝子の発現レベルを測定することにより、判別することができるという知見を得た。 As a result of diligent studies on biomarkers that can be used to discriminate vascular aging, arteriosclerosis, cerebrovascular disease, cardiovascular disease, etc. in subjects, the present inventors have found that CMPK2 contained in biological samples collected from subjects. It was found that it can be discriminated by measuring the expression level of any gene of RNF213 or both genes.
　すなわち、本発明は、血管老化予測用バイオマーカーとして、被験者から採取した生体試料に含まれるＣＭＰＫ２とＲＮＦ２１３の何れかの遺伝子、または両方の遺伝子の発現レベルを測定することを特徴とし、かかる測定により、血管老化を予測する。 That is, the present invention is characterized in that, as a biomarker for predicting vascular aging, the expression level of either or both genes of CMPK2 and RNF213 contained in a biological sample collected from a subject is measured by such measurement. , Predict vascular aging.
　また本発明の別の観点によれば、動脈硬化性疾患、脳梗塞などの脳血管疾患または心血管疾患の何れかの疾患用バイオマーカーとして、被験者から採取した生体試料に含まれるＣＭＰＫ２とＲＮＦ２１３の何れかの遺伝子、または両方の遺伝子の発現レベルを測定することを特徴とし、かかる測定により、それらの疾病の有無を予測する。
　上記の本発明の方法において、生体試料として唾液、血液、尿などを利用することができる。 Further, according to another aspect of the present invention, CMPK2 and RNF213 contained in a biological sample collected from a subject as biomarkers for any of arteriosclerosis, cerebrovascular disease such as cerebral infarction, and cardiovascular disease. It is characterized by measuring the expression level of either or both genes, and such measurement predicts the presence or absence of those diseases.
In the above method of the present invention, saliva, blood, urine and the like can be used as a biological sample.
　本発明の血管老化予測用バイオマーカーは、生体試料に含まれるＣＭＰＫ２とＲＮＦ２１３の何れかの遺伝子、または両方の遺伝子からなる。また、本発明の疾患用バイオマーカーは、生体試料に含まれるＣＭＰＫ２とＲＮＦ２１３の何れかの遺伝子、または両方の遺伝子からなる。上記の本発明のバイオマーカーにおいて、生体試料として唾液、血液、尿などを利用することができる。
　本発明の測定キットは、被験者から採取した生体試料について、ＣＭＰＫ２とＲＮＦ２１３の何れかの遺伝子、または両方の遺伝子の発現レベルを測定するための試薬を含む。
　本発明の診断装置は、被験者から得られた生体試料について、ＣＭＰＫ２とＲＮＦ２１３の何れかの遺伝子、または両方の遺伝子の発現レベルを測定して、生体試料を採取した被験者の血管に関する疾病を診断する。 The biomarker for predicting vascular aging of the present invention comprises one or both genes of CMPK2 and RNF213 contained in a biological sample. In addition, the biomarker for diseases of the present invention comprises one or both genes of CMPK2 and RNF213 contained in a biological sample. In the above-mentioned biomarker of the present invention, saliva, blood, urine and the like can be used as a biological sample.
The measurement kit of the present invention contains a reagent for measuring the expression level of either or both genes of CMPK2 and RNF213 in a biological sample collected from a subject.
The diagnostic apparatus of the present invention measures the expression level of either or both genes of CMPK2 and RNF213 in a biological sample obtained from a subject, and diagnoses a disease related to a blood vessel of the subject from whom the biological sample was collected. ..
　本発明によれば、被験者における血管老化の診断に利用可能であり、該被験者から採取した生体試料中に含まれるバイオマーカーと、該バイオマーカー発現レベルを測定することで、被験者の血管老化の進行の程度を評価するとともに、被験者の動脈硬化性疾患、脳血管疾患、心血管疾患、その他の加齢関連疾患の有無を判定できるといった効果がある。 According to the present invention, it can be used for diagnosing vascular aging in a subject, and the progress of vascular aging in the subject is measured by measuring the biomarker contained in the biological sample collected from the subject and the expression level of the biomarker. It has the effect of being able to evaluate the degree of the disease and determine the presence or absence of arteriosclerosis, cerebrovascular disease, cardiovascular disease, and other age-related diseases of the subject.
高齢者（Senior）と健康成人（Young）から採取した唾液中に含まれるＣＭＰＫ２遺伝子のｍＲＮＡのそれぞれのグループに対する発現量を示す図The figure which shows the expression level of the mRNA of CMPK2 gene contained in saliva collected from the elderly (Senior) and the healthy adult (Young) for each group. ＣＭＰＫ２、ＩＦ１４４Ｌ、ＨＥＲＣ６、ＩＦＩＴ１およびＯＡＳＬ遺伝子の唾液中における発現量の比較を、健康成人（Ａ:ＩＤ番号：Ｓｔ００８）、高齢者（Ｂ：ＩＤ番号：Ｋｒ１３８）および高齢者（Ｃ：ＩＤ番号：Ｋｒ１４４）の３名について比較した結果を示す図Comparison of the expression levels of CMPK2, IF144L, HERC6, IFIT1 and OASL genes in saliva was compared with healthy adults (A: ID number: St008), elderly people (B: ID number: Kr138) and elderly people (C: ID number: The figure which shows the result of having compared about 3 persons of Kr144) 高齢者（Senior）と健康成人（Young）から採取した唾液中に含まれるＲＮＦ２１３遺伝子ｍＲＮＡのそれぞれのグループに対する発現量を示す図The figure which shows the expression level for each group of the RNF213 gene mRNA contained in saliva collected from the elderly (Senior) and the healthy adult (Young).
　ヒトの血管は加齢に伴い次第にしなやかさを失い内膜が肥厚して血管コンプライアンスが低下する。このことは血管平滑筋細胞から分泌されるコラーゲンとエラスチンの比率が次第に増大し、伸展性に寄与するエラスチンの割合が低下することに加えて、血管壁への石灰質の沈着なども影響することが知られている。このことで、血管内を流れる血液の脈波速度は加齢に伴い上昇する。こうした血管老化による血管の構造的変化とともに生じる生化学的な変化としては、血管の拡張に寄与する内皮型ＮＯ合成酵素ｅＮＯＳの発現低下と体内で生成する血管収縮因子であるアンジオテンシンIIやエンドセリン、トロンボキサンチンＡ２などの産生は逆に増大することが知られている。さらに血管を構成する細胞レベルで血管老化を見ると、細胞老化に陥った細胞は***を停止し、それとともに各種炎症性サイトカインの発現が亢進することで、マクロファージなどの炎症細胞の浸潤が生じ、血管壁の組織リモデリングが進行することが知られている。 Human blood vessels gradually lose their suppleness with aging, the endometrium thickens, and vascular compliance declines. This means that the ratio of collagen and elastin secreted from vascular smooth muscle cells gradually increases, and the ratio of elastin that contributes to extensibility decreases. In addition, the deposition of calcareous substance on the blood vessel wall may also have an effect. Are known. As a result, the pulse wave velocity of blood flowing in the blood vessel increases with aging. Biochemical changes that occur along with structural changes in blood vessels due to aging of blood vessels include decreased expression of the endothelial NO synthase eNOS, which contributes to vasodilation, and angiotensin II, endothelin, and thrombo, which are vasoconstrictor factors produced in the body. It is known that the production of xanthin A2 and the like is conversely increased. Furthermore, looking at vascular senescence at the level of cells that make up blood vessels, cells that have undergone cellular senescence stop dividing, and at the same time, the expression of various inflammatory cytokines is enhanced, resulting in infiltration of inflammatory cells such as macrophages. It is known that tissue remodeling of the blood vessel wall progresses.
　血管老化は炎症性サイトカインの産生と分泌の亢進およびマクロファージの浸潤による構造リモデリングの進行によりさらに老化のプロセスに拍車がかかることになる。最近の報告では、炎症性サイトカインが細胞***を制御するテロメアに異常を来し、細胞の老化を促進させることが報告されている（Dayeon Shin, et al., J. Clin. Med. 2019, 8, 711; doi:10.3390/jcm8050711)。 Vascular aging further accelerates the aging process due to the increased production and secretion of inflammatory cytokines and the progress of structural remodeling due to infiltration of macrophages. Recent reports have reported that inflammatory cytokines cause abnormalities in telomeres that control cell division and accelerate cell aging (Dayeon Shin, et al., J. Clin. Med. 2019, 8). , 711; doi: 10.3390 / jcm8050711).
　マクロファージは、表面に存在するＴＬＲ（トル様受容体）やＲＬＲ、ＮＬＲｓなどのＰＲＲ（パターン認識受容体）が微生物由来のＰＡＭＰｓ（病原因子由来分子パターン）以外にも自己細胞の細胞質や核内物質が損傷された場合に、自己組織由来の分子パターンとしてＤＡＭＰｓ（ダメージ関連分子パターン）によっても活性化され、それによる炎症誘導性細胞死の結果、細胞内からＩＬ－１βが放出されることでその周辺において、炎症反応を惹起することが知られている。 Macrophages have TLRs (Toll-like receptors), RLRs, NLRs, and other PRRs (pattern recognition receptors) on the surface that are not only PAMPs (pathogen-associated molecular patterns) derived from microorganisms, but also cytoplasm and nuclear substances of autologous cells. When is damaged, it is also activated by DAMPs (damage-associated molecular patterns) as a self-tissue-derived molecular pattern, and as a result of inflammation-induced cell death, IL-1β is released from the cells. It is known to provoke an inflammatory response in the surrounding area.
　血管老化の進行により血管細胞のテロメアが短縮され、もはや細胞***が出来なくなると細胞死に至りアポトーシスにより細胞構成要素が分解を始める。この際生成するＤＡＭＰｓを血液中のマクロファージ表面のＰＲＲが認識することで、転写因子ＮＦ－κＢ経路を活性化することから炎症反応が開始される。炎症性サイトカインとしてインターロイキンＩＬ-１βが炎症反応において主要な働きを行うが、これの産生はＮＬＲＰ３インフラマソームがカスパーゼ１を活性化させることで亢進することが従来から知られている。最近明らかにされた知見としてミトコンドリアＤＮＡがＮＬＲＰ３インフラマソームを活性化させることが判明した（R.C.Coll, et al., Cell Research 28, 1046－1047 (2018)）。ＰＲＲの活性化により核内における転写因子が活性化され、ＣＭＰＫ２として知られるミトコンドリアに局在してミトコンドリアＤＮＡの合成を促進させる機能を有するタンパク質の合成にかかわる遺伝子ｍＲＮＡの発現が促進される。さらにミトコンドリアで生成したＲＯＳ（活性酸素種）がミトコンドリアＤＮＡを酸化し、これがＮＬＲＰ３インフラマソームを活性化させることが、現在までに明らかにされている知見である。 As vascular aging progresses, telomeres of vascular cells are shortened, and when cell division is no longer possible, cell death occurs and cell components begin to decompose due to apoptosis. The PRR on the surface of macrophages in the blood recognizes the DAMPs produced at this time, and activates the transcription factor NF-κB pathway, thereby initiating an inflammatory reaction. It has been conventionally known that interleukin IL-1β plays a major role in the inflammatory response as an inflammatory cytokine, and its production is enhanced by the activation of caspase 1 by the NLRP3 inflammasome. Recently revealed that mitochondrial DNA activates the NLRP3 inflammasome (R.C.Coll, et al., Cell Research 28, 1046-1047 (2018)). Activation of PRR activates transcription factors in the nucleus and promotes the expression of gene mRNA involved in the synthesis of proteins that localize to mitochondria known as CMPK2 and promote the synthesis of mitochondrial DNA. Furthermore, it has been clarified to date that ROS (reactive oxygen species) produced in mitochondria oxidize mitochondrial DNA, which activates the NLRP3 inflammasome.
　最近の老化関連研究において、加齢とともに全身性の慢性炎症の亢進との関係に注目が集まり、様々な炎症性サイトカインの産生が加齢とともに亢進することが報告されている（H.Y.Chung, et al, Aging and Disease, 10(2) 367-382 (2019)）。 In recent aging-related studies, attention has been focused on the relationship with the increase in systemic chronic inflammation with aging, and it has been reported that the production of various inflammatory cytokines increases with aging (HYChung, et al). , Aging and Disease, 10 (2) 367-382 (2019)).
　本発明における血管老化の指標として取り上げるもう一つのバイオマーカーであるＲＮＦ２１３は、これをコードする遺伝子の特異的な遺伝子多型が脳内血管の異常な血管網を形成し「もやもや病」の疾患感受性遺伝子として知られている。「もやもや病」は希少疾患で指定難病であるが、本多型遺伝子保有者は国内人口の２～３％に相当する約３００万人前後が保有していると推定される。最近の知見（国立循環器病研究センター（略称：国循）脳神経内科の猪原匡史部長、岡崎周平医長（現・大阪大学神経内科）を中心とする国循の研究グループ）ではアテローム血栓性脳梗塞の強力な感受性遺伝子であることが明らかにされている。遺伝子多型とは別に、本遺伝子の元々の機能は明らかでない点が多いが、血管形成と走行に関与しており、血管内の血液のハイドロダイナミックな特性に大きな影響を及ぼしていると考えられる（Sungjae An, et al., Scientific Reports volume 9, Article number: 8614 (2019)）。
　本発明において血管老化とＲＮＦ２１３の直接的な結びつきに対する論理的な根拠については未だ明確にされていないが、後述する実施例において示すように両者の間に明確な相関が認められることから、ＲＮＦ２１３についても血管老化に対するバイオマーカーとして利用することが可能であることが本発明により示された。 RNF213, another biomarker taken up as an index of vascular aging in the present invention, has a specific gene polymorphism of a gene encoding this, which forms an abnormal vascular network of blood vessels in the brain, and is susceptible to "moyamoya disease". Known as a gene. "Moyamoya disease" is a rare disease and is a designated intractable disease, but it is estimated that about 3 million people, which is 2 to 3% of the domestic population, carry this polymorphism gene. Recent findings (National Cerebral and Cardiovascular Research Center (abbreviation: National Circulation), Department of Neurology, Director Masafumi Inohara, Dr. Shuhei Okazaki (currently Department of Neurology, Osaka University), a research group of national circulation) show atherothrombotic cerebral infarction. Has been shown to be a potent susceptibility gene. Apart from the gene polymorphism, the original function of this gene is often unclear, but it is thought to be involved in angioplasty and running and have a great influence on the hydrodynamic properties of blood in blood vessels. (Sungjae An, et al., Scientific Reports volume 9, Article number: 8614 (2019)).
In the present invention, the rationale for the direct link between vascular aging and RNF213 has not yet been clarified, but since a clear correlation is observed between the two as shown in Examples described later, RNF213 It has been shown by the present invention that it can also be used as a biomarker for vascular aging.
　本発明者らは血管老化に関してこれを検出するための方法として従来から着目されてきた炎症性サイトカインやその他の様々な既知の手法を用いるのではなく、これとは全く異なる加齢関連因子としてのＣＭＰＫ２およびＲＮＦ２１３と、それぞれをコードする遺伝子ｍＲＮＡに着目し、これらを生体試料中におけるバイオマーカーとして利用することが可能であることを見出し、本発明に至ったものである。加齢とともに血管老化が進行すると次第に血管が固くなり動脈硬化へと進展する可能性が考えられるため、血管老化の指標は動脈硬化の進展と相関し、動脈硬化の指標としての利用に繋がると考えられる。更には、本発明は慢性炎症に伴うＣＭＰＫ２の発現についての指標であるとも考えられることから、老化に伴う慢性炎症の指標として広く利用することが可能である。さらには、糖尿病、メタボリックシンドローム、慢性腎臓病、アルツハイマー病などの様々な加齢関連疾患の指標として利用することが可能である。 The present inventors do not use inflammatory cytokines and various other known methods that have been attracting attention as a method for detecting vascular aging, but as a completely different age-related factor. Focusing on CMPK2 and RNF213 and the gene mRNA encoding each of them, they have found that they can be used as biomarkers in biological samples, leading to the present invention. As vascular aging progresses with aging, it is possible that the blood vessels gradually harden and progress to arteriosclerosis, so the index of vascular aging correlates with the progress of arteriosclerosis and is thought to lead to use as an index of arteriosclerosis. Be done. Furthermore, since the present invention is also considered to be an index for the expression of CMPK2 associated with chronic inflammation, it can be widely used as an index for chronic inflammation associated with aging. Furthermore, it can be used as an index of various age-related diseases such as diabetes, metabolic syndrome, chronic kidney disease, and Alzheimer's disease.
　以下、実施例に基づいて本発明をさらに詳細に説明する。本発明の範囲は、以下の実施例や図示例に限定されるものではなく、幾多の変更及び変形が可能である。 Hereinafter, the present invention will be described in more detail based on Examples. The scope of the present invention is not limited to the following examples and illustrated examples, and many modifications and modifications can be made.
（データベース解析による血管老化関連遺伝子候補の抽出）
　本発明者らは、血管老化で発現するｍＲＮＡに関するデータベース解析を実施し、データベースとしてBioGPS、Array Express、NCBI GEO、ReFex、Human proteome map、The human protein atlas、Wikimedia commonsなどのデータベースを利用した。それらの中から、Ｅ－ＧＥＯＤ－１３７１２（Transcription profiling by array of human young and senescent HUVECs under static and laminar shear stress conditions）に含まれる遺伝子のセットと、もう一方の遺伝子セットとして、ＧＳＥ９８０８１（Microarray analysis of TNFα‐induced senescence of HUVECs）の両方に共通する２０個のｍＲＮＡを候補遺伝子としてピックアップした。それらは、具体的には、ＩＦＩ４４Ｌ、ＩＦＩＴ３、ＩＦＩ４４、ＭＸ１、ＩＦＩＴＭ１、ＯＡＳ２、ＥＰＳＴＩ１、ＨＥＲＣ６、ＸＡＦ１、ＲＳＡＤ２、ＯＡＳＬ、ＣＣＮＡ１、ＨＳＤ１７Ｂ２、ＡＤＲＢ１、ＳＦＴＡ１Ｐ（ＳＦＴＰＦ）、ＣＭＰＫ２、ＲＮＦ２１３、ＯＡＳ３、ＩＦＩＴ１、ＩＳＧ１５の２０個のｍＲＮＡであった。 (Extraction of vascular aging-related gene candidates by database analysis)
The present inventors performed database analysis on mRNA expressed by vascular aging, and used databases such as BioGPS, Array Express, NCBI GEO, ReFex, Human proteome map, The human protein atlas, and Wikimedia commons as databases. Among them, a set of genes included in E-GEOD-13712 (Transcription profiling by array of human young and senescent HUVECs under static and laminar shear stress conditions) and another gene set, GSE98081 (Microarray analysis of TNFα). -Twenty mRNAs common to both induced senescence of HUVECs) were picked up as candidate genes. Specifically, they are IFI44L, IFIT3, IFI44, MX1, IFITM1, OAS2, EPSTI1, HERC6, XAF1, RSAD2, OASL, CCNA1, HSD17B2, ADRB1, SFTA1P (SFTPF), CMPK2, RNF213, OAS3, It was 20 mRNAs of.
　次に、これらのｍＲＮＡに対し、GeneMANIA prediction serverを利用して各遺伝子の共発現動向によるグループ分けを行ったところ、ＣＣＮＡ１、ＨＳＤ１７Ｂ２、ＡＤＲＢ１、ＲＮＦ２１３の４個の遺伝子とそれら以外の遺伝子の中から、特に、ＣＭＰＫ２を選択し、計５個の遺伝子に絞り込みを行った。 Next, when these mRNAs were grouped according to the co-expression trend of each gene using the GeneMANIA prediction server, the four genes CCNA1, HSD17B2, ADRB1, and RNF213 and other genes were selected. In particular, CMPK2 was selected and narrowed down to a total of 5 genes.
（高齢者と健康成人からなる被験者からの唾液採取とこれに含まれる候補遺伝子の発現についての検査と血管の硬さとの関係）
　本発明者らは、６５歳以上の高齢者１７名と年齢が２０～３０歳の健康成人１６名に対して、それぞれの唾液を採取し、その中に含まれるｍＲＮＡを抽出し、これからｃＤＮＡを作製した。ＲＮＡは、RNeasy Protect saliva mini kitを利用して採取し、ｍＲＮＡ発現解析には、Taqman gene expression assayを用いてｑＰＣＲ法により遺伝子増幅を行うことで、高齢者と健康成人のそれぞれのグループで上記５個の遺伝子の発現状態を比較したところ、ＣＭＰＫ２について高齢者に特異的に発現が認められた。 (Relationship between saliva collection from subjects consisting of elderly people and healthy adults, examination of expression of candidate genes contained therein, and blood vessel hardness)
The present inventors collected saliva from 17 elderly people aged 65 years or older and 16 healthy adults aged 20 to 30 years, extracted mRNA contained therein, and obtained cDNA from the saliva. Made. RNA was collected using the RNeasy Protect saliva mini kit, and for mRNA expression analysis, gene amplification was performed by the qPCR method using the Taqman gene expression assay. When the expression states of the individual genes were compared, the expression of CMPK2 was specifically observed in the elderly.
　唾液からｍＲＮＡ発現解析までの作業フローについて説明する。健康成人および高齢者より唾液２００μＬを採取後、RNeasy（登録商標） Protect Saliva Mini Kit(50)の定法に乗っ取り、Ｔｏｔａｌ　ｍＲＮＡを抽出した。その後、Invitrogen（登録商標） SuperScript（登録商標） IV ViLO（登録商標） Master Mix－Thermo Fisher（登録商標）を用いてｃＤＮＡの合成を行い、更に、TaqMan（登録商標） gene expression arrayの各プラーマー・プローブと、StepOnePlusとTaqMan（登録商標） Fast Advanced Master Mixを用いてリアルタイムＰＣＲを行った。設定は５０℃－２分、９５℃－２０秒、９５℃－１秒、６０℃－２０秒を１サイクルとして４５サイクル行った。発現定量には、β-actinを内部標準として比較ＣＴ法を用いた。 The work flow from saliva to mRNA expression analysis will be explained. After collecting 200 μL of saliva from healthy adults and the elderly, the total mRNA was extracted by taking over the standard method of RNeasy (registered trademark) Protect Saliva Mini Kit (50). After that, cDNA was synthesized using Invitrogen (registered trademark) SuperScript (registered trademark) IV ViLO (registered trademark) Master Mix-Thermo Fisher (registered trademark), and further, each plumer of TaqMan (registered trademark) gene expression array. Real-time PCR was performed using the probe, StepOnePlus and TaqMan® Fast Advanced Master Mix. The setting was performed for 45 cycles with 50 ° C.-2 minutes, 95 ° C.-20 seconds, 95 ° C.-1 second, and 60 ° C.-20 seconds as one cycle. For expression quantification, a comparative CT method was used with β-actin as an internal standard.
　図１は、高齢者（Senior）と健康成人（Young）から採取した唾液中に含まれるＣＭＰＫ２遺伝子ｍＲＮＡのそれぞれのグループに対する発現量を表す。ＣＣＮＡ１およびＨＳＤ１７Ｂ２に関しては、唾液中には発現は確認できなかった。また、ＡＤＲＢ１は測定値のばらつきが大きく、バイオマーカーとしての利用は困難であることが判明した。さらに、動脈硬化などにおいて血管内皮細胞上で発現が亢進することで知られる接着因子ＶＣＡＭ１についても検出を試みたが、本実施例においては検出できなかった。
　図１に示す結果から、唾液内に発現しているＣＭＰＫ２遺伝子の量と加齢との関係が明確に表れており、加齢とともにＣＭＰＫ２の発現が亢進することが明確となった。同様にＲＮＦ２１３についても唾液内に含まれるＲＮＦ２１３遺伝子の発現が加齢により亢進することが明らかとなった。 FIG. 1 shows the expression levels of CMPK2 gene mRNA contained in saliva collected from seniors and young adults for each group. Expression of CCNA1 and HSD17B2 was not confirmed in saliva. In addition, it was found that ADRB1 has a large variation in measured values and is difficult to use as a biomarker. Furthermore, we also tried to detect the adhesion factor VCAM1, which is known to be upregulated on vascular endothelial cells in arteriosclerosis and the like, but could not be detected in this example.
From the results shown in FIG. 1, the relationship between the amount of the CMPK2 gene expressed in saliva and aging was clearly shown, and it was clarified that the expression of CMPK2 was enhanced with aging. Similarly, it was revealed that the expression of the RNF213 gene contained in saliva of RNF213 is enhanced by aging.
　ＣＭＰＫ２についての上記の結果を受けて、さらにＣＭＰＫ２遺伝子の発現と相関が考えられる他の遺伝子として、ＩＦ１４４Ｌ、ＨＥＲＣ６、ＩＦＩＴ１およびＯＡＳＬ遺伝子についても同様に唾液中における発現量を確認した結果、図２に示す結果を得た。
　図２（１）～（５）は、それぞれ、ＣＭＰＫ２、ＩＦ１４４Ｌ、ＨＥＲＣ６、ＩＦＩＴ１およびＯＡＳＬ遺伝子の唾液中における発現量の比較を、健康成人（Ａ：ＩＤ番号：Ｓｔ００８）、高齢者（Ｂ：ＩＤ番号：Ｋｒ１３８）および高齢者（Ｃ：ＩＤ番号：Ｋｒ１４４）の３名について比較した結果を示す。図２（１）～（５）の結果から、ＣＭＰＫ２およびこれに相関が認められるいずれの遺伝子の発現も傾向が一致しており、本発明の目的とする血管老化に対するバイオマーカーとしての利用の可能性が示されたが、これらの内でＣＭＰＫ２の発現亢進が最も顕著であることから、これらの中ではＣＭＰＫ２が最も優れたバイオマーカーであると理解できる。 Based on the above results for CMPK2, the expression levels of IF144L, HERC6, IFIT1 and OASL genes were also confirmed in saliva as other genes that could be correlated with the expression of the CMPK2 gene. The results shown are obtained.
2 (1) to (5) show a comparison of the expression levels of CMPK2, IF144L, HERC6, IFIT1 and OASL genes in saliva, respectively, for healthy adults (A: ID number: St008) and elderly people (B: ID). The result of comparison for three persons (number: Kr138) and the elderly (C: ID number: Kr144) is shown. From the results of FIGS. 2 (1) to 2 (5), the expression of CMPK2 and any of the genes correlated with the CMPK2 tend to be consistent with each other, and can be used as a biomarker for vascular aging, which is the object of the present invention. Although the sex was shown, the upregulation of CMPK2 expression was the most prominent among them, so that it can be understood that CMPK2 is the most excellent biomarker among them.
　図１に示すように高齢者グループに対して唾液中に含まれるＣＭＰＫ２遺伝子の発現が多いことから、次にＣＭＰＫ２の発現量と血管老化との関係を調べるため、健康成人と高齢者のそれぞれについてＣＭＰＫ２の高い群とＣＭＰＫ２が低い群を選び、血圧脈波検査装置（オムロンコーリン株式会社製、ＯＦＦＩＲＩＯ　ＬＰ－Ｓ２１０）を使用して、それぞれについて上腕動脈－足首動脈間脈波伝達速度ＰＷＶ（ｂａＰＷＶ）を測定した。それぞれの年齢における標準値からのズレの程度を評価した結果、健康成人ついてはＣＭＰＫ２が高い群については、３名全ての被験者について標準値より１ＳＤ（標準偏差）より高く外れており、一方のＣＭＰＫ２の低い群では３名全ての被験者についての標準値からのズレは１ＳＤより低い値であった。 As shown in FIG. 1, since the expression of the CMPK2 gene contained in saliva is higher in the elderly group, next, in order to investigate the relationship between the expression level of CMPK2 and vascular aging, for each of healthy adults and elderly people. Select a group with high CMPK2 and a group with low CMPK2, and use a blood pressure pulse wave tester (OFFIRIO LP-S210 manufactured by Omron Korin Co., Ltd.) for each of them with the brachial artery-ankle artery pulse wave velocity PWV (baPWV). Was measured. As a result of evaluating the degree of deviation from the standard value at each age, in the group with high CMPK2 for healthy adults, all three subjects deviated from the standard value by more than 1 SD (standard deviation), and one of the CMPK2. In the low group, the deviation from the standard value for all three subjects was lower than 1SD.
　同様に、高齢者に対して試験を行った結果、ＣＭＰＫ２が高い群については４名の内２名の被験者については、標準値より１ＳＤより高く外れており、残りの２名については、ＣＭＰＫ２は高値であったが、ＰＷＶの数値は低い結果であった。一方のＣＭＰＫ２の低い群では、２名の被験者についての標準値からのズレは１ＳＤより逆に高い値であった。
　上記の結果より、健康成人についてはＣＭＰＫ２とＰＷＶの数値の間に良好な相関関係が認められ、健康成人の血管老化の指標としての利用が可能であることが明確となったが、一方で、高齢者に対する評価では両者の相関は必ずしも良くない結果であった。
　しかしながら、その理由として、ＣＭＰＫ２はその時々の血管内皮の状態を反映しており、血管老化の進行を敏感に表現するものであるのに対し、ＰＷＶは、血管内皮だけでなく血管の基質全体の力学的構造変化を反映するため測定時点に至るまでの経年的変化を表していると考えられる。したがって、ＣＭＰＫ２が高値であることで血管老化が進行している可能性があることから、本実施例で、ＣＭＰＫ２が高値であるにもかかわらずＰＷＶが低値であった被験者については、やがて遠からずＰＷＶの数値が上昇する可能性が挙げられるため、生活習慣などを見直すなどの血管老化を遅らせるための予防処置を行う契機となる。 Similarly, as a result of conducting a test on elderly people, 2 out of 4 subjects in the group with high CMPK2 deviated from the standard value by more than 1SD, and the remaining 2 subjects had CMPK2. Although it was a high value, the PWV value was a low value. On the other hand, in the group with low CMPK2, the deviation from the standard value for the two subjects was conversely higher than 1SD.
From the above results, it was clarified that a good correlation was observed between the values of CMPK2 and PWV for healthy adults, and that it could be used as an index of vascular aging in healthy adults. In the evaluation of the elderly, the correlation between the two was not always good.
However, the reason is that CMPK2 reflects the state of the vascular endothelium at that time and sensitively expresses the progress of vascular aging, whereas PWV is not only the vascular endothelium but also the entire vascular matrix. Since it reflects the mechanical structural change, it is considered to represent the secular change up to the time of measurement. Therefore, since there is a possibility that vascular aging is progressing due to the high value of CMPK2, in this example, the subject whose PWV was low despite the high value of CMPK2 will soon be distant. Since there is a possibility that the value of PWV will rise, it will be an opportunity to take preventive measures to delay vascular aging such as reviewing lifestyle habits.
　さらに、ＣＭＰＫ２の数値が低値であるが、ＰＷＶの数値が高値であった被験者に対しては、血管老化以外の原因によって、ＰＷＶの数値が上昇している可能性が高いことから、動脈硬化が様々な原因で進行している可能性があるため、別の観点からの再検査を促すことが勧められる。このようにしてＣＭＰＫ２は、血管老化の指標となるバイオマーカーとして、従来の採血による検査方法とは異なり、被験者の唾液を採取することで検査できることから極めて有用に利用できることが明らかとなった。 Furthermore, for subjects with low CMPK2 values but high PWV values, there is a high possibility that the PWV values are increasing due to causes other than vascular aging, and thus arteriosclerosis. May be progressing for a variety of reasons, so it is advisable to encourage re-examination from a different perspective. In this way, it has been clarified that CMPK2 can be extremely useful as a biomarker as an index of vascular aging because it can be tested by collecting saliva of a subject, unlike the conventional test method by blood sampling.
　次に、それぞれの被験者に対して実際に頸動脈の超音波診断装置（アロカ株式会社製、ＰＲＯＳＯＵＮＤα１０）を利用して、頸動脈のエコー診断を行ったところ、ＣＭＰＫ２の数値が高値であった高齢者４名中３名について、頸部プラークの存在が認められ、動脈硬化が実際に進行していることが確認された。さらにＣＭＰＫ２の数値が低い２名の高齢者について同様に検査した結果、両方とも頸部プラークの存在は確認されず、動脈硬化の進行は認められなかった。健康成人については、頸部プラークの存在は認められないことから、健康成人についてはＣＭＰＫ２を動脈硬化の判定に用いることは困難である。これらの結果より、ＣＭＰＫ２は高齢者に対して動脈硬化の指標としての利用が可能であることが示唆された。 Next, when an echo diagnosis of the carotid artery was actually performed for each subject using an ultrasonic diagnostic device for the carotid artery (PROSOUND α10 manufactured by Aloka Medical, Ltd.), the elderly with a high CMPK2 value. The presence of cervical plaque was observed in 3 of 4 patients, confirming that arteriosclerosis was actually progressing. Furthermore, as a result of similar examination of two elderly people with low CMPK2 values, the presence of cervical plaque was not confirmed in both cases, and no progression of arteriosclerosis was observed. Since the presence of cervical plaque is not observed in healthy adults, it is difficult to use CMPK2 for determining arteriosclerosis in healthy adults. These results suggest that CMPK2 can be used as an index of arteriosclerosis in the elderly.
（ＲＮＦ２１３をバイオマーカーとして利用）
　本実施例では、実施例１と同様に、健康成人５名、高齢者４名から唾液を採取し、それぞれの唾液中に含まれるＲＮＦ２１３遺伝子について、それらの濃度とＰＷＶの数値との相関を確認した結果について説明する。なお、唾液からｍＲＮＡ発現解析までの作業フローについては実施例１と同様である。
　図３は、同様に高齢者（Senior）と健康成人（Young）から採取した唾液中に含まれるＲＮＦ２１３遺伝子ｍＲＮＡのそれぞれのグループに対する発現量を表す。
　本実施例の場合、健康成人において、ＲＮＦ２１３の数値が高い３名中２名については、ＰＷＶの数値が高く、逆にＲＮＦ２１３が低かった２名は、両方ともＰＷＶの数値も低い結果であった。ＲＮＦ２１３が高値であるがＰＷＶは低値であった被験者１名については、上述の実施例１と同様に、ＲＮＦ２１３が現時点における血管老化の進行性を見ているのに対し、ＰＷＶは経年変化を反映するためであると推察する。 (Use RNF213 as a biomarker)
In this example, as in Example 1, saliva was collected from 5 healthy adults and 4 elderly people, and the correlation between the concentration of the RNF213 gene contained in each saliva and the PWV value was confirmed. The result will be explained. The work flow from saliva to mRNA expression analysis is the same as in Example 1.
FIG. 3 shows the expression level of the RNF213 gene mRNA contained in saliva collected from the elderly (Senior) and the healthy adult (Young) for each group.
In the case of this example, in healthy adults, 2 out of 3 patients with high RNF213 values had high PWV values, and conversely, 2 patients with low RNF213 values had low PWV values. .. For one subject whose RNF213 was high but PWV was low, as in Example 1 above, RNF213 was observing the progress of vascular aging at this time, whereas PWV was aging. I guess this is to reflect it.
　一方、高齢者４名中、ＲＮＦ２１３の数値が高値であった３名については、ＰＷＶの数値は１名のみが高値で、残り２名は低値であった。また、ＲＮＦ２１３の数値が低値であった高齢者１名は、ＰＷＶの数値が高値であった。
　上記の結果より、健康成人に対してＲＮＦ２１３の数値は血管老化との相関が認められる。しかしながら、高齢者に対しては、ＲＮＦ２１３の数値の測定時点での血管老化の進行を推測するための指標としての利用に留まる。 On the other hand, among the four elderly people, of the three who had a high RNF213 value, only one had a high PWV value and the remaining two had a low value. In addition, one elderly person with a low RNF213 value had a high PWV value.
From the above results, the value of RNF213 is correlated with vascular aging for healthy adults. However, for the elderly, it is only used as an index for estimating the progress of vascular aging at the time of measuring the numerical value of RNF213.
　さらに、頚部プラークの有無とＲＮＦ２１３の数値の相関についても検討を行った結果、ＲＮＦ２１３の数値が高い高齢者３名中２名にプラークが認められ、一方、ＲＮＦ２１３の数値が低値であった高齢者１名についてもプラークの存在が認められた。高齢者に対するＲＮＦ２１３の数値と動脈硬化の相関は、今回の実施例だけでは判断し難いものの、動脈硬化の進行を表すバイオマーカーとしての有効性を示唆する結果であった。 Furthermore, as a result of examining the correlation between the presence or absence of cervical plaque and the value of RNF213, plaque was observed in 2 out of 3 elderly people with a high value of RNF213, while the elderly with a low value of RNF213. The presence of plaque was also confirmed in one person. Although it is difficult to determine the correlation between the RNF213 value and arteriosclerosis for the elderly from this example alone, the results suggest its effectiveness as a biomarker indicating the progression of arteriosclerosis.
　本発明は、被験者からの唾液や血液試料を用いて、被験者の血管老化の進行を数値的に示すことができるため、血管老化に伴う加齢関連疾患のバイオマーカーとしての利用が可能であり、加齢関連疾患の診断や予防のバイオマーカーとして有用である。また、メタボリックシンドロームや糖尿病、慢性腎臓病、アルツハイマー病などの慢性炎症の診断や予防のバイオマーカーとして有用である。
  Since the present invention can numerically indicate the progress of vascular aging of a subject using saliva or blood sample from the subject, it can be used as a biomarker for age-related diseases associated with vascular aging. It is useful as a biomarker for the diagnosis and prevention of age-related diseases. It is also useful as a biomarker for the diagnosis and prevention of chronic inflammation such as metabolic syndrome, diabetes, chronic kidney disease, and Alzheimer's disease.

Claims (12)

  1. 　血管老化予測用バイオマーカーとして、生体試料に含まれるＣＭＰＫ２とＲＮＦ２１３の何れかの遺伝子、または両方の遺伝子の発現レベルを測定することを特徴とする血管老化予測方法。 A method for predicting vascular aging, which comprises measuring the expression level of either or both genes of CMPK2 and RNF213 contained in a biological sample as a biomarker for predicting vascular aging.
  2. 　動脈硬化性疾患、脳血管疾患または心血管疾患の疾患用バイオマーカーとして、生体試料に含まれるＣＭＰＫ２とＲＮＦ２１３の何れかの遺伝子、または両方の遺伝子の発現レベルを測定することを特徴とする疾患リスク予測方法。 Disease risk characterized by measuring the expression level of either or both of the CMPK2 and RNF213 genes contained in a biological sample as a biomarker for a disease of arteriosclerosis, cerebrovascular disease or cardiovascular disease. Prediction method.
  3. 　前記生体試料が、唾液、血液、または、尿である、請求項１の血管老化予測方法。 The method for predicting vascular aging according to claim 1, wherein the biological sample is saliva, blood, or urine.
  4. 　前記生体試料が、唾液、血液、または、尿である、請求項２の疾患リスク予測方法。 The disease risk prediction method according to claim 2, wherein the biological sample is saliva, blood, or urine.
  5. 　生体試料に含まれるＣＭＰＫ２とＲＮＦ２１３の何れかの遺伝子、または両方の遺伝子からなる、血管老化予測用バイオマーカー。 A biomarker for predicting vascular aging, which comprises either or both genes of CMPK2 and RNF213 contained in a biological sample.
  6. 　生体試料に含まれるＣＭＰＫ２とＲＮＦ２１３の何れかの遺伝子、または両方の遺伝子からなる、動脈硬化性疾患、脳血管疾患または心血管疾患の疾患用バイオマーカー。 A biomarker for diseases of arteriosclerosis, cerebrovascular disease or cardiovascular disease, which comprises one or both genes of CMPK2 and RNF213 contained in a biological sample.
  7. 　前記生体試料が、唾液、血液、または、尿である、請求項５の血管老化予測用バイオマーカー。 The biomarker for predicting vascular aging according to claim 5, wherein the biological sample is saliva, blood, or urine.
  8. 　前記生体試料が、唾液、血液、または、尿である、請求項６の疾患用バイオマーカー。 The disease biomarker according to claim 6, wherein the biological sample is saliva, blood, or urine.
  9. 　請求項５の血管老化予測用バイオマーカーの発現レベルを測定するための試薬を含む測定キット。 A measurement kit containing a reagent for measuring the expression level of the biomarker for predicting vascular aging according to claim 5.
  10. 　請求項６の疾患用バイオマーカーの発現レベルを測定するための試薬を含む測定キット。 A measurement kit containing a reagent for measuring the expression level of the biomarker for a disease according to claim 6.
  11. 　請求項５の血管老化予測用バイオマーカーの発現レベルに基づき、前記生体試料を採取した被験者の血管老化を予測診断する診断装置。 A diagnostic device that predicts and diagnoses vascular aging of a subject who has collected the biological sample based on the expression level of the biomarker for predicting vascular aging according to claim 5.
  12. 　請求項６の疾患用バイオマーカーの発現レベルに基づき、前記生体試料を採取した被験者の動脈硬化性疾患、脳血管疾患または心血管疾患を診断する診断装置。
          A diagnostic device for diagnosing arteriosclerosis, cerebrovascular disease or cardiovascular disease of a subject from whom the biological sample was collected, based on the expression level of the biomarker for disease according to claim 6.
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