JP2013044698A - Biomarker for cell stress condition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell stress condition determination method or cell stress condition detection kit which is more simplified and accurate than the prior arts, or a screening method of prevention or therapeutic agents for diseases caused by cell stress.SOLUTION: The present invention is completed by discovering that 1-methyladenosine, 5-methylcytidine, and pseudouridine can be utilized as an early-stage marker of a cell stress condition. As a solution, one or two or more modified nucleic acids selected from among 1-methyladenosine, 5-methylcytidine, and pseudouridine of a specimen are detected to determine the cell stress condition of a tissue. Furthermore, a cell stress condition detection kit is provided which contains anti-1-methyladenosine monoclonal antibody, anti-pseudouridine monoclonal antibody, or anti-5-methylcytidine monoclonal antibody.

Description

  The present invention relates to a method for determining a cell stress state by detecting one or more modified nucleic acids selected from 1-methyladenosine, 5-methylcytidine and pseudouridine from a specimen, an anti-1-methyladenosine antibody, anti-5 -It relates to a cell stress state detection kit comprising a methylcytidine antibody or an anti-pseudouridine antibody, a method for screening a preventive or therapeutic agent for diseases caused by cell stress, and the like.

  Ischemia refers to a state where the inflow of blood into tissues or organs is reduced or disrupted, such as cerebral infarction, myocardial infarction, renal infarction, renovascular hypertension, inflammatory bowel disease, mitochondrial disease, neuromuscular degenerative disease, Occurs in various diseases such as diabetes. Conventionally, this ischemic state has been diagnosed using ultrasound, electrocardiogram, cerebral ischemia scintigraphy, and the like, but these methods have a problem that a dedicated expensive and large-scale device is required. . Further, methods for measuring high molecular substances such as proteins, lipids, enzymes, and hormones have been proposed as site and disease non-specific ischemic markers, but they have not been put into practical use. Therefore, a method that can easily determine tissue ischemia has been desired.

  Endoplasmic reticulum (ER) stress refers to a state in which abnormal proteins accumulate inside the organelle (endoplasmic reticulum), which is the field of protein synthesis. It is known that apoptosis is induced when cells cannot respond to endoplasmic reticulum stress, and it is becoming clear that this apoptosis is involved in various diseases such as neurodegenerative diseases and diabetes (Patent Document 1). . In addition to ischemia and endoplasmic reticulum stress, cell stress such as mitochondrial damage, radiation damage, oxidative stress, and hypoxic stress leads to a decrease in cell function, and if cell function does not recover, apoptosis is induced. Causes death. If the effects of cell stress can be detected, it will be possible to quickly detect a malfunction of the body and start early treatment before subjective symptoms appear.

  Adenosine is a nucleotide composed of adenine and ribose, which are one of purine nucleosides, and 1-methyladenosine is a compound in which the 1-position of adenosine is methylated. In human tRNA, the 58th adenosine is a methylated 1-methyladenosine present in the L-shaped joint (also called elbow) of the 4th loop (also called T loop or TΨC loop). It is thought to be involved in binding to ribosome with a positive charge (FIG. 1). In addition, 1-methyladenosine is known to be excreted in blood and urine by apoptosis and autophagy. Apoptosis is observed in mitochondrial diseases, Alzheimer's disease, and other diseases with tissue loss and necrosis. Autophagy is infectious diseases, neurodegenerative diseases with abnormal protein accumulation (Alzheimer's disease, Parkinson's disease, Batten's disease, etc.), empty It is found in muscle diseases with cystization (such as Danone disease), Crohn's disease and other diseases with autophagy. Furthermore, blood 1-methyladenosine concentration tends to be highly correlated with renal function (GFR; glomerular filtration rate), and is disclosed as one of early diagnostic markers for renal failure (Patent Document 2). ), The relationship with ischemia is completely unknown.

  Uridine is a nucleoside composed of uracil and ribose, one of pyrimidine nucleosides. Pseudouridine cleaves the N-glycoside bond of uridine, rotates the uracil ring 180 degrees, and a new C1'-C5 CC bond. Pseudouridine is formed by an isomerization reaction to form Pseudouridine is responsible for stabilization of the higher-order structure of RNA molecules and molecular recognition functions. Pseudouridine synthase contributes to RNA maturation by converting uridine at a specific position in RNA into pseudouridine. It is believed that Cytidine is a nucleotide composed of cytosine and ribose, which is one of pyrimidine nucleosides, and 5-methylcytidine is a compound in which the 5-position of cytidine is methylated.

  So far, Patent Document 3, Patent Document 4, and Non-Patent Document 1 have anti-1-methyladenosine antibodies, Patent Document 5 has anti-pseudouridine monoclonal antibodies, and Patent Document 6 has anti-5-methylcytidine monoclonal antibodies. Although antibodies have been disclosed, it has never been clear that 1-methyladenosine, pseudouridine, and 5-methylcytidine are early markers of cellular stress. Further, in Patent Document 7, cells are cultured by gradually changing the concentration of the endoplasmic reticulum stress inducer, and the endoplasmic reticulum stress of the test substance is changed based on the difference in BiP detection start concentration depending on the presence or absence of the test substance. However, there is a problem in that a plurality of cell extract samples are prepared and a complicated operation requiring technical skill is required. Further, Patent Document 8 discloses that in a cell or tissue by detecting human heme oxygenase-1 in a cell or tissue using a monoclonal antibody that reacts with human heme oxygenase-1 or an antibody fragment containing an antibody binding site consisting of a variable region thereof. Although a method for detecting stress is disclosed, if protein is the target of detection, it is necessary to pay special attention to the time and effort required to perform special processing on the sample and quality control in order to keep the protein in the sample stable. There were problems such as. Therefore, there has been a demand for a simple and easy method for detecting a cell stress state by easily measuring a stable substance in a sample using urine or blood as a sample.

JP 2011-037722 A WO / 2011/027573 open brochure JP-A-62-299766 Japanese Patent Laid-Open No. 3-154867 Japanese Patent Publication No. 4-21479 Special table 2008-502332 gazette JP 2008-131899 A JP 2002-112791 A

Masuda M., et al., Cancer, 72, 3571-3578 (1993)

  The present inventors made it a subject to provide a method for easily determining a cell stress state, a cell stress state detection kit, and a screening method for a preventive or therapeutic agent for a cell stress state.

  We are selected from 1-methyladenosine, 5-methylcytidine and pseudouridine from tissue sections using anti- 1-methyladenosine monoclonal antibody, anti-pseudouridine monoclonal antibody or anti-5-methylcytidine monoclonal antibody. Since the cell stress state can be detected by detecting one or more modified nucleic acids, it was found that 1-methyladenosine, 5-methylcytidine and pseudouridine can be used as an early marker of the cell stress state, and this study is completed. It came to.

  That is, the present invention provides [1] (a) a step of detecting one or more modified nucleic acids selected from 1-methyladenosine, 5-methylcytidine and pseudouridine in a specimen; (b) a modified nucleic acid in step (a) A step of determining a cell stress state when there is an increase or localization change; a method of determining a cell stress state comprising the steps (a) and (b) of [2] The method for determining a cellular stress state according to the above [1], wherein the specimen is blood, urine, tissue, cultured cell, or cell culture, characterized by being stressed by ischemia, hypoxia, or radiation [1] to [3], wherein the method of determining a cellular stress state according to [1] or [2], or [4] detection is an immunoassay ] The cell in any one of A method for determining a tress state, [5] a method for determining a cell stress state according to [4], wherein the immunological measurement method is an ELISA method or an immunostaining method, and [6] an immunological measurement An anti-1-methyladenosine monoclonal antibody produced by a hybridoma having the accession number FERM P-22120, an anti-5-methylcytidine monoclonal antibody produced by a hybridoma having the accession number FERM P-22122, or a hybridoma having the accession number FERM P-22121 It comprises the method for determining a cell stress state according to the above [4] or [5], wherein an anti-pseudouridine monoclonal antibody to be produced is used.

  The present invention also provides a cell stress state detection kit comprising [7] an anti-1-methyladenosine monoclonal antibody, an anti-5-methylcytidine monoclonal antibody, or an anti-pseudouridine monoclonal antibody, and [8] accession number FERM. An anti-1-methyladenosine monoclonal antibody produced by a hybridoma of P-22120, an anti-5-methylcytidine monoclonal antibody produced by a hybridoma of accession number FERM P-22122, or an anti-pseudouridine produced by a hybridoma of accession number FERM P-22121 [8] The kit for detecting a cell stress state according to [7] above, which contains a monoclonal antibody, [9] The cell stress is ischemia, hypoxia, or radiation stress ] The cell strike described (10) (A) a step of administering a test substance to a non-human animal or a cultured cell before or after loading cell stress, or during cell stress loading; A step of collecting a specimen from the non-human animal or cultured cell of A) and detecting one or more modified nucleic acids selected from 1-methyladenosine, 5-methylcytidine and pseudouridine in the specimen; (C) Step ( (B) When the modified nucleic acid does not increase or the modified nucleic acid does not change in the intracellular localization, the test substance is evaluated as a candidate drug for the prevention or treatment of diseases caused by cellular stress. C), and a screening method for a preventive or therapeutic agent for diseases caused by cell stress.

  Furthermore, the present invention provides [11] a hybridoma with the accession number FERM P-22120 that produces an anti-1-methyladenosine monoclonal antibody, and [12] a hybridoma with the accession number FERM P-22122 that produces an anti-5-methylcytidine monoclonal antibody. [13] A hybridoma having the accession number FERM P-22121 that produces an anti-pseudouridine monoclonal antibody.

  According to the present invention, by detecting one or more modified nucleic acids selected from 1-methyladenosine, 5-methylcytidine, and pseudouridine in a specimen, it is possible to determine a cell stress state or to result from cell stress. Screening for preventive or therapeutic agents for diseases can be performed. In addition, a kit for detecting a cell stress state comprising an anti-1-methyladenosine monoclonal antibody, an anti-5-methylcytidine monoclonal antibody or an anti-pseudouridine monoclonal antibody, or an accession number FERM P-22120 that produces an anti-1-methyladenosine monoclonal antibody A hybridoma having the accession number FERM P-22122 producing an anti-5-methylcytidine monoclonal antibody or a hybridoma having the accession number FERM P-22121 producing an anti-pseudouridine monoclonal antibody can be provided.

It is a figure shown about tRNA and 1-methyl adenosine. It is a figure which shows the structure and antigen presentation site | part of 1-methyladenosine and pseudouridine, and the structure of 5-methylcytidine. Anti-1-methyladenosine antibody was used to stain kidney tissue sections before ischemia (upper stage), immediately after ischemia release (middle stage), and 1 hour after reperfusion (lower stage). The cortex (left side) and outer medulla layer (right side) are shown. Arrows indicate cTAL (cortical thick ascending limbs of Henle's Loop), and white arrows indicate glomeruli. A kidney tissue section of a rat that has not been subjected to ischemia treatment is treated with an anti-1-methyladenosine antibody (first and second rows from the top), an anti-5-methylcytidine antibody (third row from the top), or an anti-pseudouridine antibody ( Dyeing was performed using the 4th stage from the top). Inner medullary layer (left side), outer medullary layer (middle), and cortex (right side) are shown. A 1-hour post-ischemic reperfusion rat kidney tissue section was prepared from an anti-1-methyladenosine antibody (first and second rows from the top), anti-5-methylcytidine antibody (third row from the top), or anti-pseudouridine antibody. (4th stage from the top) was used for staining. Inner medullary layer (left side), outer medullary layer (middle), and cortex (right side) are shown. The arrow indicates the staining site. A rat kidney tissue section 8 hours after ischemia-reperfusion was prepared from an anti-1-methyladenosine antibody (first row from the top), anti-5-methylcytidine antibody (second row from the top), or anti-pseudouridine antibody (top). To the third stage). Inner medullary layer (left side), outer medullary layer (middle), and cortex (right side) are shown. The arrow indicates the staining site. The immunoelectron microscope observation result of the ischemic rat kidney TAL using an anti 1-methyl adenosine antibody is shown. The results of staining brain tissue sections of a rat cerebral infarction model with HE (hematoxin eosin) (upper), anti-1-methyladenosine antibody (middle), or anti-5-methylcytidine antibody (lower) are shown. The cerebral infarction region in the rat brain is shown in a schematic diagram in the upper right, and the boundary between the ischemic region and the normal region (first and second from the left), ischemic region (third from the left), normal region (fourth from the left) ). The result of having measured the 1-methyl adenosine density | concentration in the renal venous blood and arterial blood of a renal ischemia model pig is shown. The vertical axis of the graph represents 1-methyladenosine concentration (ng / ml), and the horizontal axis is the time axis. The results of triple staining of 1-methyladenosine, mitochondria, and nucleus of human proximal tubular epithelial cells RPTEC cells are shown. The result of having measured the 1-methyl adenosine density | concentration in the blood collected near the renal vein of the patient under arch replacement | replacement surgery is shown. The vertical axis of the graph represents 1-methyladenosine concentration (ng / ml), and the horizontal axis is the time axis. The result of having measured the 1-methyl adenosine density | concentration and creatine value in the peripheral blood of the patient after a renal artery dilatation is shown. The vertical axis of the graph represents 1-methyladenosine concentration (ng / ml), and the horizontal axis is the time axis. The result of having fractionated the serum sample extract | collected from the patient under arch replacement surgery by the ultrafiltration by molecular weight (50K, 30K, and 10K), and measuring the 1-methyl adenosine density | concentration value in a filtrate is shown. “Peripheral” represents a serum sample collected from the peripheral portion, and “1” represents a serum sample collected from the vicinity of the renal vein. Normal cultivation (control) or 1% after hypoxia culture 24 hours of oxygen 20% oxygen (O _2), shows the results of measurement of 1-methyl-adenosine concentration value in a cell lysate or culture medium. The result of having observed the intracellular localization of 1-methyladenosine in the peripheral blood lymphocyte before and behind X-ray irradiation (radiation) by the immunofluorescent dyeing | staining method is shown. It is a figure which shows that the presence or absence of the radiation disorder in a biological body can be detected by detecting 1-methyladenosine. rad12hr represents a mouse irradiated with X-ray, and control represents a mouse not irradiated with X-ray.

  In the present invention, “cell stress” refers to harmful stimuli (stresses) applied to cells from the environment, and such environments may be in vitro or in vivo. “Cell stress state” refers to a cell that is adversely affected by the load of cell stress. The state received. For example, when subjected to cellular stress such as genetic mutation, viral infection, inflammation, harmful chemicals, denatured protein is produced in the cell, and this denatured protein accumulates in the endoplasmic reticulum, which has an adverse effect on the cell. As a result, it is known to fall into a cell stress state called endoplasmic reticulum stress. In addition, it is known that when cells are exposed to cell stress such as heat, the expression of heat shock proteins that function as molecular chaperones is increased, causing a mechanism for protecting cells. As described above, when cells fall into a cell stress state due to cell stress, various cell stress response mechanisms are triggered, and adaptation to cell stress and recovery of cell functions can be achieved. In the absence, apoptosis is ultimately induced. The cell stress in the present invention is not particularly limited, and may include harmful stimuli given to cells by in vitro culture conditions and the like, in vitro harmful stimuli given to cells by living body modulation, etc. Cell stress can include stimuli that are objective (pressure, temperature, hypoxia, radiation, etc.), chemical (drugs, oxidation, etc.) and biological (bacterial or viral infection, endoplasmic reticulum stress, etc.). These cell stresses may be one type or a combination of two or more types. Specifically, all the cell stresses may not be specified and clarified. In addition, examples of cell stress to which tissues and cells in the living body are exposed include living body diseases, and cells in the living body affected by the disease may be in a state of cell stress caused by the disease. The disease causing cellular stress in the present invention is not particularly limited, and may be an endogenous disease or an extrinsic disease, a disease involving ischemia such as infarction, mitochondrial disease, Alzheimer's disease, or other diseases with tissue loss or necrosis Diseases with apoptosis, neurodegenerative diseases with abnormal protein accumulation, muscle diseases with vacuolation, Crohn's disease and other diseases with autophagy, anemia, malnutrition, allergies In addition to diseases such as autoimmune response diseases such as leukemia, cancer, kidney disease, lifestyle-related diseases such as hypertension and high cholesterol, diabetes, bacterial and viral infections, symptoms caused by trauma and surgical treatment, Examples include radiation damage due to radiation exposure and hypoxia due to hypoxia. Among them, ischemia, hypoxia, and / or radiation damage can be preferably exemplified.

  The ischemia in the present invention may be local anemia due to a decrease in arterial blood volume, and may be caused by an intrinsic disease or artificially caused during a surgical operation or the like. That is, by detecting the cell stress state, the presence or absence of a disease causing cell stress, for example, a disease causing ischemia, or the state of ischemia artificially caused by a surgical operation or the like is monitored. can do. In addition, the hypoxia in the present invention may be a symptom caused by exposure to hypoxia, and the hypoxia is preferably 1/2 or less of the oxygen concentration in a normal growth environment such as a living body or a cell. More preferred is an oxygen concentration of 1/5 or less, most preferably 1/10 or less. Specifically, for organisms living in the atmosphere, such as mammals and birds, and cells derived therefrom, hypoxia is an oxygen concentration lower than 20%, which is a general oxygen concentration in the atmosphere, and preferably 0. An oxygen concentration of -10%, more preferably 0-4%, most preferably 0-2% can be mentioned. Furthermore, the radiation damage in the present invention may be symptoms caused by exposure to radiation, and the type and amount of radiation are not particularly limited. Alpha rays (α rays) and beta rays ( β-rays), gamma rays (γ-rays), X-rays (X-rays), ultraviolet rays (UV), etc., and any one of them may be used alone or a mixture of two or more types. But you can. For example, if it is determined that a cell stress state occurs when ischemia, hypoxia, or radiation damage is expected, prevention and / or treatment at an early stage, prevention and / or improvement of environmental investigations and causes Measures can be taken.

  The specimen in the present invention is not particularly limited, and examples thereof include tissues such as tissues and body fluids collected from living bodies, artificial culture cells or culture media thereof, blood, urine, tissue sections, culture cells, and / or A cell culture solution can be preferably exemplified. When blood is used as a sample, blood coagulants, preservatives, fungicides, etc. can be added as appropriate, specific components such as red blood cells, white blood cells, and plasma can be removed, and serum can be used. It is also possible to extract only specific components. Moreover, the components in the sample can be fractionated based on the molecular weight by ultrafiltration or the like to obtain a specimen. When urine is used as a sample, a preservative, a fungicide, or the like may be added as appropriate. When a tissue is used as a specimen, it can be a tissue section that has been appropriately fixed. The cultured cells may be established cell lines, primary cultured cells of cells collected from blood or tissue collected from a living body, and the like. The origin of the specimen is not particularly limited, and examples thereof include humans, monkeys, pigs, sheep, goats, cows, horses, dogs, cats, rabbits, rats, mice, hamsters, birds, etc., preferably humans and mice. can do. Since the method for determining a cell stress state of the present invention can determine a cell stress state from a small amount of sample, there is an advantage that a burden on a living body for collecting a sample is small, and an experimental animal such as a small mouse can be used. Even when used, it is possible to collect a small amount of blood over time and effectively use it to monitor the state of cell stress.

  In the present invention, “detecting a modified nucleic acid” means confirming the presence or absence of the modified nucleic acid, quantifying the modified nucleic acid, or visualizing the modified nucleic acid, and a known method can be used. The detection method in the present invention is not particularly limited as long as it is a method capable of detecting one or two or more modified nucleic acids selected from 1-methyladenosine, 5-methylcytidine and pseudouridine in a specimen, and is immunological. Measurement methods, HPLC (high performance liquid chromatography) methods, CE (capillary electrophoresis) methods, HPLC / MS (mass spectrometry) methods, CE / MS methods, MS / MS methods, LC / MS / MS methods, etc. Preferably, an immunological assay can be mentioned. Further, as an immunological measurement method, a method capable of detecting one or more modified nucleic acids selected from 1-methyladenosine, 5-methylcytidine and pseudouridine using an antibody specific for these modified nucleic acids. The ELISA method, the immunostaining method, the Western blotting method, the flow cytometry method, the immunoprecipitation method, the immunoelectrophoresis method and the like can be exemplified, and the ELISA method and the immunostaining method can be preferably exemplified. . As the ELISA method, any of a direct adsorption method, a competitive method, and a sandwich method can be used, and among them, a competitive method suitable for detecting a small amount of sample can be preferably exemplified. The immunostaining method may be any method that stains a target molecule using an antibody. For example, the immunostaining method can be observed and detected using a stereoscopic microscope, a phase contrast microscope, a confocal fluorescence microscope, an electron microscope, or the like. In immunohistochemical staining, the modified nucleic acid of the tissue section can be detected using a labeled primary antibody against the modified nucleic acid, or using a primary antibody against the modified nucleic acid and a labeled secondary antibody. The labeling of the primary antibody or the secondary antibody may be any detectable labeling substance, such as FITC, Cy3, Cy5, Rhodamine, Alexa fluor (registered trademark, manufactured by Invitrogen), peroxidase, alkaline phosphatase, etc. Enzymes, proteins such as biotin, haptens such as DIG (digoxigenin), colloidal gold, and radioisotope elements. The peroxidase label can also be detected by the DAB (diaminobenzidi) method or nickel DAB method, and the alkaline phosphatase label can also be detected by the NBT / BCIP reaction. In addition, a TSA method (tyramide signal amplification), a sensitization method using a biotin / avidin reaction, and the like can be used in appropriate combination.

  The antibody used in the immunological assay in the present invention may be any antibody that can specifically recognize the modified nucleic acid 1-methyladenosine, 5-methylcytidine, or pseudouridine, such as a monoclonal antibody or a polyclonal antibody. In addition, antibodies such as single chain antibodies, humanized antibodies, chimeric antibodies, and antibodies derived from mice, rats, pigs, goats, sheep, etc. can be mentioned, and mouse monoclonal antibodies are preferred. Among them, the anti-1-methyladenosine monoclonal antibody produced by the hybridoma (AMA-2) having the deposit number FERM P-22120, the anti-methylcytidine monoclonal antibody produced by the hybridoma (FMC9) having the deposit number FERM P-22122, or the deposit number FERM A preferred example is an anti-pseudouridine monoclonal antibody produced by a hybridoma of P-22121 (APU-6). These antibodies may be labeled or unlabeled. Examples of the labeling substance include fluorescent substances such as FITC, Cy3, Cy5, Rhodamine, Alexa fluor (registered trademark, manufactured by Invitrogen), peroxidase and alkali. Examples thereof include enzymes such as phosphatase, proteins such as biotin, haptens such as DIG (digoxigenin), colloidal gold, and radioisotope elements.

As a method for determining the cell stress state of the present invention,
(A) detecting one or more modified nucleic acids selected from 1-methyladenosine, 5-methylcytidine and pseudouridine in a specimen;
(B) a step of determining a cell stress state when there is an increase or localization change of the modified nucleic acid in step (a);
As long as the steps (a) and (b) are provided, there is no particular limitation, and a diagnostic action by a doctor is usually excluded. Moreover, the method of collecting the data for the diagnosis of the cell stress state provided with process (a) and (b) as one Embodiment can also be illustrated. Although the modified nucleic acid in the measurement sample and the control sample can be detected and compared in the step (a), the modified nucleic acid in the measurement sample is detected in the step (a), and the control sample is separately modified from the standard sample. You may compare with the reference | standard value of the amount of modified nucleic acid set from the detection result of the nucleic acid, or the standard localization of a modified nucleic acid. In addition, a control sample and a living body may be separately prepared and a control specimen may be collected. However, two or more types of specimens, that is, a control specimen and a measurement specimen may be collected from the same sample or living body. In particular, examples of collecting samples from the same part of the living body, or from different parts of the living body at once or multiple times can be given. For example, the cell stress state of a diseased tissue can be examined using a sample collected from a normal tissue as a control sample and a sample collected from a diseased tissue as a measurement sample. By comparing a sample derived from one organ with a sample derived from another organ, the cell stress state of a specific organ can also be examined. In addition, a sample collected before or at the start of treatment or immediately after treatment is used as a control sample, and a sample collected at one or more time points during or after treatment is used as a measurement sample for evaluation of therapeutic effects. Data can be collected. In addition, a sample collected before surgery or before examination is used as a control specimen, and a cell stress state is examined using a specimen collected during surgery or examination or at one or more time points after surgery or examination as a measurement specimen. Thus, it can be used as an index for monitoring the influence of surgery or examination on a patient. The cell stress state can be used as an early marker of a disease or disorder, and by examining the cell stress state, treatment can be started before symptoms appear or before symptoms become serious.

In addition, as a method for determining ischemia using the method for determining a cell stress state of the present invention,
(A1) a step of detecting one or more modified nucleic acids selected from 1-methyladenosine, 5-methylcytidine and pseudouridine in a specimen;
(B1) a step of determining ischemia when the modified nucleic acid is increased in step (a1);
As long as the steps (a1) and (b1) are provided, there is no particular limitation, and a diagnostic action by a doctor is usually excluded. Moreover, the method of collecting the data for the diagnosis of ischemia provided with process (a1) and (b1) as one Embodiment can also be illustrated. Examples of specimens that can be preferably used include blood, urine, and tissue sections. Among them, an example of staining a modified nucleic acid in a tissue section is preferable, and an example of measuring a modified nucleic acid concentration in blood is more preferable. be able to. Data for diagnosis of ischemia include data for detecting ischemia, data for evaluating ischemic conditions, and for monitoring and evaluating the state of living bodies in surgical operations involving ischemia. Other data that can be used as data, for example, diseases with apoptosis such as mitochondrial disease, Alzheimer's disease, other diseases with tissue loss or necrosis, infectious diseases, neurodegenerative diseases with abnormal protein accumulation, muscles with vacuolation It can also be used as data for diagnosis of diseases in which autophagy is observed, such as diseases, Crohn's disease and other diseases involving autophagy.

Furthermore, as a method for determining hypoxia using the method for determining a cellular stress state of the present invention,
(A2) a step of detecting one or more modified nucleic acids selected from 1-methyladenosine, 5-methylcytidine and pseudouridine in a specimen;
(B2) a step of determining hypoxia when the number of modified nucleic acids increases in step (a2);
As long as the steps (a2) and (b2) are provided, there is no particular limitation, and a diagnostic action by a doctor is usually excluded. Moreover, the method of collecting the data for the diagnosis of hypoxia provided with process (a2) and (b2) as one Embodiment can also be illustrated. An example in which the modified nucleic acid released to the outside of the cell is detected can be preferably exemplified, and examples of the sample include a cell culture solution, blood and urine. Data for diagnosis of such hypoxia include respiratory disturbance due to alveolar ventilation failure, reduction of blood oxygen transport due to carbon monoxide poisoning, cyanide poisoning hypoxic encephalopathy, respiratory center disorder, neuromuscular disorder It can also be used as data for diagnosis involving hypoxia such as chronic obstructive pulmonary disease.

As a method for determining radiation damage using the method for determining a cellular stress state of the present invention,
(A3) a step of detecting one or more modified nucleic acids selected from 1-methyladenosine, 5-methylcytidine and pseudouridine in a specimen;
(B3) a step of determining a radiation injury when the number of modified nucleic acids is increased or the localization of the modified nucleic acids is changed in step (a3);
As long as the steps (a3) and (b3) are provided, there is no particular limitation, and a diagnostic action by a doctor is usually excluded. Preferred examples of the sample include blood and urine, cells contained in blood and urine, and tissue sections. Among them, the intracellular localization of the modified nucleic acid in lymphocytes collected from peripheral blood is immunized. Examples of observing that the modified nucleic acid aggregates and migrates to the cell membrane by staining with a staining method, and an example of measuring the modified nucleic acid concentration in blood can be given. In addition, as an embodiment, a method of collecting data for diagnosis of radiation damage including steps (a3) and (b3) can be exemplified.

  As the cell stress state detection kit of the present invention, an antibody capable of recognizing 1-methyladenosine, 5-methylcytidine, or pseudouridine as a modified nucleic acid, for example, a monoclonal antibody, a polyclonal antibody, or a chimeric antibody is particularly suitable. Without limitation, a monoclonal antibody is preferable, and among them, an anti-1-methyladenosine mouse monoclonal antibody produced by a hybridoma having accession number FERM P-22120, an anti-5-methylcytidine mouse monoclonal antibody produced by a hybridoma having accession number FERM P-22122, or A preferred example is an anti-pseudouridine mouse monoclonal antibody produced by a hybridoma having the deposit number FERM P-22121. The cell stress state detection kit may contain a fixing solution, a washing solution, a reaction container, a detection reagent and the like in addition to these antibodies. In addition, these antibodies in the cell stress state detection kit of the present invention may be labeled or unlabeled. Examples of labeling substances include FITC, Cy3, Cy5, Rhodamine, Alexa fluor (registered trademark, Invitrogen). And fluorescent substances such as peroxidase and alkaline phosphatase, proteins such as biotin, haptens such as DIG (digoxigenin), colloidal gold, and radioisotope elements. Since 1-methyladenosine is excreted in blood and urine by apoptosis and autophagy, the anti-1-methyladenosine monoclonal antibody produced by the cell stress state detection kit of the present invention or the hybridoma of the accession number FERM P-22120 In addition to ischemia, radiation damage, hypoxia, mitochondrial disease with apoptosis, Alzheimer's disease, other diseases with tissue loss or necrosis, infection with autophagy, abnormal protein accumulation It is possible to detect the presence or absence of cellular stress including diseases such as neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, Batten's disease, etc.), muscle diseases with vacuolation (Danon's disease, etc.), Crohn's disease and the like.

As a screening method for a preventive or therapeutic agent for diseases caused by cellular stress of the present invention,
(A) A step of administering a test substance to a non-human animal or cultured cell before or after loading cell stress or during cell stress loading;
(B) A step of collecting a specimen from the non-human animal or cultured cell in step (A) and detecting one or more modified nucleic acids selected from 1-methyladenosine, 5-methylcytidine and pseudouridine in the specimen;
(C) When the modified nucleic acid does not increase in step (B) or the modified nucleic acid does not change in the intracellular localization, the test substance is evaluated as a candidate drug for prevention or treatment of a disease caused by cellular stress;
The steps (A) to (C) are not particularly limited, and may include other steps. The type of cell stress, the method of loading the cell stress, the presence or absence of the recovery time after loading the cell stress The length and the like can be appropriately selected. Although the modified nucleic acid in the measurement sample and the control sample can be detected and compared in the step (B), the modified nucleic acid in the measurement sample is detected in the step (B), and the control sample is separately modified from the standard sample. You may compare with the reference | standard value of the amount of modified nucleic acid set from the detection result of the nucleic acid, or the standard localization of a modified nucleic acid. In addition, a control sample and a living body may be separately prepared and a control specimen may be collected. However, two or more kinds of specimens such as a control specimen and a measurement specimen may be collected from the same sample or living body. Examples of collecting samples from the same part of the living body at different times or from different parts of the living body at once or multiple times can also be given. Moreover, it is preferable that the control sample and the measurement sample have the same origin in the living body. When a non-human animal is used, the control sample and the measurement sample are preferably derived from the same tissue in the living body. Examples of the non-human animal in the present invention include monkeys, pigs, sheep, goats, cows, horses, dogs, cats, rabbits, rats, mice, hamsters, birds, etc., and rats and mice are preferably exemplified. It is also possible to use disease model animals and the like. The administration method of the test substance to the non-human animal can be appropriately selected, and examples thereof include oral, injection, infusion, and application. In the case of using cultured cells, the type of cultured cells such as animals and organs from which the cultured cells are derived is not particularly limited, and HeLa cells, HEK293 cells, MCF-7 cells, HepG2 cells, PC12 cells, Jurkat cells, COS7 cells, Cell lines such as CHO cells, NIH3T3 cells, L cells, MDCK cells, S2 cells, ES cells, iPS cells, primary culture cells, etc. may be used, and the test substance is added to the culture medium, or the test substance is a peptide. In the case of proteins and proteins, these expression vectors can be introduced into cells. The test substance can be selected as appropriate, and can include chemical substances, proteins, peptides, steroids, etc. These can be prepared by synthesis or extraction, etc., and can also be purchased commercially. Can also be used.

Screening for an agent for preventing or treating ischemia comprising the following steps (A1) to (C1) can also be performed using the method for screening a agent for preventing or treating a disease caused by cell stress of the present invention.
(A1) A step of administering a test substance to a non-human animal during or after ischemia-reperfusion;
(B1) A step of collecting a specimen from the non-human animal in step (A1) and detecting one or more modified nucleic acids selected from 1-methyladenosine, 5-methylcytidine and pseudouridine in the specimen;
(C1) a step of evaluating a test substance as a candidate drug for prevention or treatment of ischemia when the modified nucleic acid does not increase in step (B1);
The steps (A1) to (C1) are not particularly limited, and may include other steps. The method of performing ischemia, tissue, ischemic time, time after ischemia reperfusion, and the like can be selected as appropriate. As the sample, blood, urine, and tissue section can be preferably exemplified, and examples of staining the tissue section and examples of measuring the modified nucleic acid concentration in blood can be given. Since 1-methyladenosine is excreted in the blood and urine by apoptosis and autophagy, candidate drugs for prevention or treatment of ischemia obtained by screening include, for example, mitochondria, as well as diseases involving ischemia. Diseases such as Alzheimer's disease, Alzheimer's disease, other diseases with tissue loss or necrosis, infections, neurodegenerative diseases with abnormal protein accumulation, muscle diseases with vacuolation, Crohn's disease and other autophagy It can also be evaluated as a candidate drug for the prevention or treatment of diseases in which autophagy is observed, such as associated diseases.

Screening for a prophylactic or therapeutic agent for hypoxia comprising the following steps (A2) to (C2) can also be performed using the method for screening a prophylactic or therapeutic agent for diseases caused by cellular stress of the present invention.
(A2) A step of administering a test substance to cultured cells during or after hypoxic treatment;
(B2) A step of collecting the cultured cell culture solution of step (A2) as a sample and quantifying one or more modified nucleic acids selected from 1-methyladenosine, 5-methylcytidine and pseudouridine in the sample;
(C2) a step of evaluating the test substance as a candidate drug for prevention or treatment of hypoxia when the modified nucleic acid does not increase in step (B2);
The steps (A2) to (C2) are not particularly limited, and may include other steps, including the type of cultured cells, the oxygen concentration, the time for performing the hypoxic treatment, and the recovery time after the hypoxic treatment. Presence / absence, length thereof, and the like can be selected as appropriate.

Using the screening method for a preventive or therapeutic agent for a disease caused by cell stress of the present invention, a screening agent for a prophylactic or therapeutic agent for radiation damage comprising the following steps (A3) to (C3) can also be performed.
(A3) a step of administering a test substance to cells or non-human animals during or before irradiation;
(B3) A step of collecting a sample from the cell or non-human animal in step (A3) and detecting one or more modified nucleic acids selected from 1-methyladenosine, 5-methylcytidine and pseudouridine in the sample;
(C3) When the modified nucleic acid does not change in localization or increase in step (B3), the test substance is evaluated as a candidate drug for the prevention or treatment of radiation damage;
The steps (A3) to (C3) are not particularly limited, and may include other steps, and may be performed by collecting a sample a plurality of times. The number of times, the tissue to be irradiated, the presence or absence of a recovery time after radiation irradiation, the length thereof, and the like can be selected as appropriate. Examples of using non-human animals include measuring the concentration of modified nucleic acids in peripheral blood collected from non-human animals, and examining the subcellular localization of modified nucleic acids in lymphocytes collected from such peripheral blood. Can do. As cells, screening can also be performed using lymphocytes collected from peripheral blood or cultured cells.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

[Staining of kidney tissue of renal ischemia model rats]
Rats (wister 8 weeks old (Charles)) bilateral renal arteries were clamped for 1 hour for ischemia, then unclamped and reperfused. The rat was sacrificed without ischemic treatment, immediately after reversal of ischemia and 1 hour after reperfusion, and the right kidney was removed. The kidney was fixed by immersion in 10% formalin, a paraffin specimen was prepared, and immunohistochemical staining was performed using an anti-1-methyladenosine antibody (1: 100) produced from a hybridoma with the accession number FERM P-22120. Color was developed with a stain kit (manufactured by Nichirei) (FIG. 3). Immediately after the release of ischemia and 1 hour after reperfusion, cTAL (cortical Thick Ascending Limb of Henle's Loop) was strongly stained, and the glomeruli were not stained.

  Similarly, bilateral renal arteries of rats were clamped for 1 hour for ischemia, and then unclamped and reperfused. Rats without ischemia treatment (sham), 1 hour after reperfusion, 3 hours after reperfusion, and 8 hours after reperfusion were sacrified and the right kidney was removed. The kidney was fixed by immersion in 10% formalin to prepare a paraffin specimen, and anti-1-methyladenosine antibody (Accession No. FERM P-22120) (1: 100), anti-pseudouridine antibody (Accession No. FERM AP-22121) (1 : 100) or anti-5-methylcytidine antibody (Accession No. FERM P-22122) (1: 100) was used for immunohistochemical staining and developed with a simple stain kit (manufactured by Nichirei). Weak non-specific staining was observed in the cortical proximal tubules even in rats without ischemia treatment (FIG. 4). After 1 hour of reperfusion, staining was mainly observed in the outer medulla layer, and the tubules were stained in the inner layer even when any of the anti-1-methyladenosine antibody, anti-pseudouridine antibody, and anti-5-methylcytidine antibody was used. The cortical staining positive part was cTAL (FIG. 5). After 8 hours of reperfusion, the overall staining decreased. The portion of the tubules (arrows) observed in the cortex 1 hour after reperfusion may have a possibility that the cells are thinned and the staining property is lost after 8 hours of reperfusion, and the cells are dead (FIG. 6). It may be that the outer medullary layer, which is considered to be vulnerable to ischemia, causes cell damage due to ischemia, and RNA is degraded and degenerated RNA is produced 1 hour after reperfusion. However, after 8 hours after reperfusion, the cells have already died and there is no denatured RNA, so it is possible that staining was reduced. In addition, an anti-1-methyladenosine antibody (Accession No. FERM P-22120) (1: 100) of a rat kidney TAL (Thick Ascending Limb of Henle's Loop) 1 hour after reperfusion was used. The result of the immunoelectron microscope is shown in FIG. Observation by immunoelectron microscopy also showed that 1-methyladenosine was present in abundance in the kidney TAL after reperfusion. From the above, it can be seen that strong staining with anti-1-methyladenosine antibody, anti-pseudouridine antibody, and anti-5-methylcytidine antibody is obtained in tissue sections by ischemia treatment. These 1-methyladenosine, pseudouridine, 5-methyl It was shown that cytidine can be used as an ischemic marker.

[1-methyladenosine staining of brain tissue of cerebral infarction model rats]
The cerebral infarction model rat in which the middle cerebral artery was ischemic for 1 hour to induce cerebral infarction was sacrified after reperfusion for 1 hour, and the brain was removed. The brain was fixed by immersion in 10% formalin to prepare a paraffin specimen. Tissue sections were stained with HE, or using anti 1-methyladenosine antibody (Accession No. FERM P-22120) (1: 100) or anti-5-methylcytidine antibody (Accession No. FERM P-22122) (1: 100), Color was developed with a simple stain kit (manufactured by Nichirei Co., Ltd.) and immunohistochemical staining was performed (FIG. 8). When comparing staining in the ischemic region and normal region, it was shown that staining of 1-methyladenosine and 5-methylcytidine was stronger in the ischemic region, and ischemia in the brain could also be detected. The same observation results were obtained when cerebral infarction model rats that were ischemic for 2 hours and were not reperfused were used.

[1-methyladenosine concentration in peripheral arterial blood of renal ischemia model pig]
In a porcine renal ischemia model in which bilateral renal arteries were ischemic for 1 hour, blood from peripheral arteries and renal veins before ischemia, immediately after reperfusion, 15 minutes after reperfusion, 1 hour after reperfusion, and 2 hours after reperfusion Were collected. The concentration of 1-methyladenosine in the collected blood was measured by ELISA (competitive method) as follows. 1-methyladenosine-BSA conjugate (0.125 ng / ml in 50 μl per well) dissolved in PBS was solidified overnight at 4 ° C., and then 100 μl of 1% BSA-PBS was added to perform blocking at 37 ° C. . After 1 hour, 1-methyladenosine standard solution and anti-1-methyladenosine antibody (Accession No. FERM AP-22120) prepared in a dilution series (1, 5, 10, 50, 100, 500 ng / ml) 0.02 μg / ml 25 μl was added and reacted at 4 ° C. for 1 hour. Each well was washed 5 times with 0.05% Tween-PBS, 50 μl of ALP rabbit anti-mouse IgG Fc diluted 1: 500 with 0.05% Tween-PBS was added and reacted at 4 ° C. for 1 hour. . After washing in the same manner as described above, a substrate (paranitrophenyl phosphate dissolved in 1M diethanolamine buffer pH 9.8 at a concentration of 1 mg / ml) was added and allowed to react for 15 minutes, and the absorbance at 405 nm was measured to obtain a calibration curve. It was created. Similarly, the serum of the collected blood (centrifugal ultrafiltration using Amicon Ultra-4 (10,000 NMWL) and the filtrate obtained by further diluting 2-fold, 4-fold, 8-fold) was measured, The amount of 1-methyladenosine was calculated from the calibration curve. The results are shown in FIG. The 1-methyladenosine concentration in arterial blood rose to a value about 5 times that before ischemia 2 hours after reperfusion. Also, at any time point, the concentration of 1-methyladenosine in renal venous blood is always higher than the concentration of 1-methyladenosine in arterial blood, which contradicts the interpretation that increased 1-methyladenosine in the blood is derived from the kidney. The result is not. Therefore, it was shown that ischemia in organs such as kidneys can be detected by measuring the concentration of 1-methyladenosine in blood collected from peripheral arteries.

[1-Methyladenosine staining of human proximal tubular epithelial cells RPTEC cells]
It is known that mitochondria are damaged by ischemia reperfusion. Therefore, the intracellular localization of 1-methyladenosine in cultured cells of proximal tubules rich in mitochondria was examined. Human proximal tubule epithelial cells RPTEC cells, which are primary cultured cells, were obtained from Takara, Inc. using OPTI-MEM medium (manufactured by Invitrogen) supplemented with 10% FBS (manufactured by Invitrogen) at 37 ° C., 5 ° C. % CO ₂ and maintained under. RPTEC cells were seeded on a 12-well chamber at 300,000 cells / well, fixed with 4% paraformaldehyde / PBS after 24 hours, washed and blocked with 1% BSA-0.05% Tween-PBS. Anti-1-methyladenosine antibody is used to stain 1-methyladenosine, Mitotracker (registered trademark) deep red (manufactured by Invitrogen), mitochondrion, and DAPI (manufactured by Invitrogen) to stain nuclei, and washing and mounting Then, it was observed with a confocal microscope (LSM5, manufactured by Zeiss) (FIG. 10). It was confirmed that 1-methyladenosine and mitochondria showed colocalization.

[1-methyladenosine concentration in blood near the renal vein during arch replacement]
During a human arch replacement operation for a heart patient, 200 μl of a serum sample in an ischemic state was collected from the periphery and the vicinity of the renal vein of the patient before the ischemic open distal operation (and before the start of the cardiopulmonary pump). During the arch replacement operation for a heart patient, blood near the renal vein was collected and the 1-methyladenosine concentration was measured. Blood from the renal vein was collected at a total of 7 points from the start of the cardiopulmonary pump to ischemia due to arch aortic blockade, distal open vessel anastomosis, and 2 hours after reperfusion. FIG. 11 shows the results of measuring blood 1-methyladenosine concentration by ELISA (competitive method) in the same manner as described above. At 2 hours after surgery and reperfusion, a 1-methyladenosine concentration higher than 3 times before the start of surgery was measured. Measurement of 1-methyladenosine showed that the ischemic state during surgery can also be monitored in real time.

[Concentration of 1-methyladenosine in terminal blood during treatment of human renal artery stenosis]
Terminal blood from patients with renal artery stenosis was collected at a total of 5 points before and after renal artery dilation, and the 1-methyladenosine concentration in the blood was measured. The blood 1-methyladenosine concentration was measured by ELISA (competitive method) as described above. The value of creatine, one of the kidney function markers, was also measured from the same sample using LC-MS. The results are shown in FIG. While the concentration of 1-methyladenosine in the blood continued to increase after renal infarction, the value of creatine did not change before and after renal infarction. Therefore, it was shown that the cell stress determination method of the present invention that detects 1-methyladenosine can accurately reflect the state of the kidney, rather than creatine that has been used as a marker for kidney function.

[Detection of 1-methyladenosine in peripheral blood during arch replacement]
During a human arch replacement operation for a heart patient, a serum sample of 200 μl was prepared from the peripheral blood and catheter blood of the patient before ischemic open distal surgery (and before the start of the heart-lung pump). This serum sample was diluted 2-fold with 1 × PBS, centrifugal ultrafiltration device (Amicon Ultra-4 centrifugal filter unit (Millipore)): molecular weight cut-off 10,000 (UFC801008), fractionation Molecular weight 30,000 (UFC 803008) and molecular weight cut off 50,000 (UFC 805008)), respectively, using a micro refrigerated centrifuge (KUBOTA3700, manufactured by Kubota Corporation), 4 ° C., 7,500 × g (6, Centrifuge at 830 rpm) for 90 minutes. The result of measuring the amount of 1-methyladenosine in the obtained filtrate (about 320 μl) by ELISA is shown in FIG.

  In FIG. 13, “peripheral” indicates a peripheral blood sample collected from the arm, and “1” indicates a serum sample collected by inserting a catheter into the renal vein. In FIG. 13, serum, 10K, 30K, and 50K are serum that is not ultrafiltered, ultrafiltrate with a molecular weight cut off of 10,000, ultrafiltrate with a molecular weight cut off of 30,000, and a molecular weight cut off of 50,000, respectively. Represents the ultrafiltrate. 1-methyladenosine exists as a free nucleoside in a cell or is contained in tRNA. Since the molecular weight of tRNA is about 25,000, tRNA is recovered in an ultrafiltrate of 50K (50,000) in the complete form, but 30K (30,000) and 10K (10,000). Is not filtered. Moreover, if tRNA is in the middle of decomposition, it is recovered in 50K and 30K ultrafiltrate and is not filtered at 10K. When the decomposition further proceeds and the oligonucleoside is decomposed, the oligonucleoside is recovered in the 10K ultrafiltrate. Therefore, 1-methyladenosine in various states can be recovered by changing the molecular weight cutoff of the ultrafiltration device to obtain ultrafiltrate with different molecular weights. In FIG. 13, since 1-methyladenosine was measured in all samples of serum and 10K, 30K and 50K ultrafiltrate, free 1-methyladenosine was in the process of degradation using the antibody of the present invention. It was shown that tRNA (including 1-methyladenosine) can also be detected together. Furthermore, as shown in FIG. 13, 1-methyladenosine (detected in 50K ultrafiltrate) contained in tRNA is more than free 1-methyladenosine (detected in 10K ultrafiltrate). Therefore, the method of the present invention using an antibody can detect 1-methyladenosine in serum more sensitively than a system using a conventional mass spectrometer that can detect only 1-methyladenosine. It was shown to be useful.

  In the process of cell death due to ischemia or the like, cells are broken and tRNA also leaks out of the cell. Therefore, it is considered that apoptosis or cell stress state can be detected by detecting the amount of blood tRNA. In the method of the present invention using an antibody, not only free 1-methyladenosine but also tRNA (including 1-methyladenosine) before and during degradation can be detected together. Compared with a measurement method using a mass spectrometer capable of detecting only adenosine, cell death can be detected with high sensitivity and accuracy, and is highly useful. That is, it is considered that a large amount of tRNA is detected in the serum as the state of ischemia or cell stress is severe, and compared with the amount of free 1-methyladenosine (in 10K ultrafiltrate), free and in tRNA When the amount of 1-methyladenosine (in the 50K ultrafiltrate) is significantly higher than that of a healthy person (normal), it can be determined that the subject is in an apoptotic or cellular stress state.

[Detection of MDCK cell 1% O ₂ hypoxia load]
MDCK cells (cultured cells derived from canine tubules) were seeded in 6-well dishes at 250,000 cells / well. After 12 hours, the cells became 50% confluent, washed once with PBS, and then washed with OPTI-MEM in 2 ml medium. % _{O 2} in normally carried out culture (control) or 1% 24 hour hypoxia culture _{O 2,} both control and 1% _{O 2} and 95% confluent. Thereafter, the medium (supernatant) and cells were collected by the following method, and the amount of 1-methyladenosine in the cells and the medium was measured.

(Production of Cell lysate)
After washing 3 wells twice with PBS, the cells were removed by trypsin / EDTA treatment and collected, and the number of cells was counted. Both the control and 1% O ₂ low oxygen load samples were 1.05 million to 1.1 million / well. Next, after centrifugation at 1,000 g × 5 minutes, the supernatant was removed, the cells were solubilized with 500 μl of lysis buffer, and ultrasonically disrupted (H × 5 minutes) using Bioruptor (manufactured by Toago Denki Co., Ltd.). After centrifugation at 1,000 g × 5 minutes, the supernatant was recovered as cell lysate and stored at −80 ° C.
Lysis buffer composition 1% TritonX-100
50 mM Tris-HCl pH 7.6
150 mM NaCl
0.02% Sodium azide
Protease inhibitor (Roche: Complete mini EDTA free)
VRC (Vanadyl-ribonucleotide complex) 10 mM

(Total RNA extraction)
Three wells were washed twice with PBS, 2 ml / well of TriPure Isolation Reagent (Roche) was added, and 1 ml was dispensed into 1.5 ml Eppendorf tubes. 200 μl of chloroform was added to the Eppendorf tube, stirred by vortex, and centrifuged at 15,000 rpm for 10 minutes. Transfer 500 μl of the water layer after centrifugation to another tube, precipitate with 500 μl of 2-propanol, rinse twice with 70% ethanol, dissolve with 200 μl of RNase free distilled water, spectrophotometer Nanodrop (trademark registered, stock) RNA concentration was measured with a company LMS Co., Ltd. and stored at -80 ° C. The RNA concentration of the control sample is 80.8 ng / μl, 87.4 ng / μl, 85.8 ng / μl, and the RNA concentration of the 1% O ₂ hypoxia sample is 75.0 ng / μl, 72.4 ng / μl, 71 4 ng / μl. As described above, the concentration of 1-methyladenosine in lysate and supernatant was measured by ELISA (competitive method). The result of correcting the measured value by the RNA concentration is shown in FIG. As a result, it was shown that the 1-methyladenosine concentration in the cell lysate was not changed by a low oxygen load of 1% O ₂ , but the 1-methyladenosine concentration in the medium was significantly increased. Therefore, it was shown that cell stress due to low oxygen load can be detected by detecting 1-methyladenosine.

[Detection of radiation damage in peripheral blood lymphocytes]
Human peripheral blood lymphocytes were collected by the following method, and 1-methyladenosine was stained and observed.
(Collecting lymphocytes from peripheral blood)
On an empty stomach, 10 ml of blood was collected from the subject using a heparin blood collection tube. Blood is slowly transferred from the syringe to a 50 cc tube containing 10 ml (equal volume) of Ficoll (Becton) so as not to disturb the liquid level of Ficoll, and centrifuged at 1,500 rpm for 30 minutes at room temperature so as not to disturb the liquid level. The operation was performed. Next, the serum and the PBL layer were collected from above with a pipette, and an equal amount of PBS was added, followed by centrifugation at 1,200 rpm × 10 minutes. The supernatant was aspirated, the precipitate was suspended in 5 ml of PBS, and a series of operations involving centrifugation at 1,500 rpm × 5 minutes was performed twice, and the precipitate was suspended in 2 ml of RPMI containing 10% FCS (fetal calf serum). . The lymphocytes having a PBL of 1 × 10 ⁷ cells / 2 ml were dispensed into 24-well plates (no coat) and cultured at 37 ° C. and 5% CO ₂ .

(Preparation of 1-methyladenosine stained specimens of lymphocytes)
The peripheral blood lymphocytes were irradiated with 287 rads for about 3,000 rads for 10 minutes using an X-ray apparatus Softex (Softex Corporation). Thereafter, the cells were cultured at 37 ° C. and 5% CO _2. Over time, 80 μl (4 × 10 ⁵ lymphocytes) of lymphocytes were transferred to a 1.5 ml tube and collected as a specimen sample. The specimen sample is centrifuged at 2,000 rpm × 5 minutes, the supernatant is discarded, and the washing operation is repeated twice with 200 μl of PBS. The cells are suspended in 20 μl of PBS and placed on a coated slide to prepare a smear specimen. Fixing treatment was performed with PFA (paraformaldehyde). The 4% PFA-fixed smear sample was solubilized with PBS containing 0.2% TritonX-100 for 2 minutes at room temperature, and then treated with TBS-T containing 3% NGS (usually goat serum) for 10 minutes at room temperature. Blocking treatment was performed. Primary for 1 hour at room temperature using a primary antibody prepared by diluting an anti-1-methyladenosine antibody (1 mg / ml) produced by the hybridoma with the accession number FERM P-22120 at 1: 100 with TBS-T containing 3% NGS An antibody reaction is performed, and then a secondary antibody reaction is performed at room temperature for 1 hour using a secondary antibody in which Alexa Fluor ™ 488 anti-mouse IgG (manufactured by Invitrogen) is diluted 1: 500 with 3% NGS TBS-T. After performing, the specimen was encapsulated and observed with a fluorescence microscope. The result is shown in FIG.

  As a result, in peripheral blood lymphocytes before X-ray irradiation, 1-methyladenosine is distributed in the cytoplasm, but after X-ray irradiation, the stained portion of 1-methyladenosine changes to the cell membrane margin, and the cells It was confirmed that the inside also changed to granular aggregated localization. In addition, changes in cell morphology were observed in which cells were broken after X-ray irradiation, suggesting that mitochondrial damage was caused by X-ray irradiation. Therefore, it was shown that radiation damage can also be detected by detecting 1-methyladenosine.

[Detection of radiation damage in irradiated mice]
A mouse (C57BL / 6) (n = 3) was irradiated with 10 Gy of X-rays using an X-ray apparatus Softex (Softex Corporation). After irradiation, blood was collected and the 1-methyladenosine concentration in the blood was measured by the ELISA method in the same manner as described above. The results of the average value of 1-methyladenosine concentration in blood are shown in FIG. In FIG. 16, control indicates the concentration of 1-methyladenosine in the blood of mice not irradiated with X-rays. rad12hr represents the concentration of 1-methyladenosine in the blood of a mouse 12 hours after X-ray irradiation. Mice irradiated with X-rays (rad12hr) were shown to have a 1.4-fold higher concentration of 1-methyladenosine in blood compared to mice not irradiated with X-rays (control). Therefore, it was shown that the influence of radiation and the detection of radiation damage can be detected by detecting 1-methyladenosine even in a living body such as an experimental animal.

  The present invention can be suitably used in the fields of blood tests, urine tests, and tissue tests. Further, it can be suitably used in medical / research fields relating to diseases related to ischemia and medical / research fields relating to detection of apoptosis, autophagy, exposure to hypoxia and radiation.

Claims (13)

A cell stress state determination method comprising the following steps (a) and (b):
(A) detecting one or more modified nucleic acids selected from 1-methyladenosine, 5-methylcytidine and pseudouridine in a specimen;
(B) a step of determining a cell stress state when there is an increase or localization change of the modified nucleic acid in step (a);   The cell stress state determination method according to claim 1, wherein the cell stress is ischemia, hypoxia, or radiation stress.   The method for determining a cell stress state according to claim 1 or 2, wherein the specimen is blood, urine, tissue, cultured cells, or cell culture supernatant.   The method for determining a cell stress state according to any one of claims 1 to 3, wherein the detection is an immunological assay.   The method for determining a cell stress state according to claim 4, wherein the immunological measurement method is an ELISA method or an immunostaining method.   Anti-1-methyladenosine monoclonal antibody produced by a hybridoma having an accession number of FERM P-22120, anti-5-methylcytidine monoclonal antibody produced by a hybridoma of the accession number FERM P-22122, or accession number FERM P- The method for determining a cell stress state according to claim 4 or 5, wherein an anti-pseudouridine monoclonal antibody produced by 22121 hybridoma is used.   A kit for detecting a cell stress state, comprising an anti-1-methyladenosine monoclonal antibody, an anti-5-methylcytidine monoclonal antibody, or an anti-pseudouridine monoclonal antibody.   Produced by an anti-1-methyladenosine monoclonal antibody produced by a hybridoma with accession number FERM P-22120, an anti-5-methylcytidine monoclonal antibody produced by a hybridoma with accession number FERM P-22122, or a hybridoma with accession number FERM P-22121 The kit for detecting a cellular tres state according to claim 7, comprising an anti-pseudouridine monoclonal antibody.   The kit for detecting a cellular stress state according to claim 8, wherein the cellular stress is ischemia, hypoxia, or radiation stress. A screening method for a preventive or therapeutic agent for diseases caused by cell stress, comprising the following steps (A) to (C).
(A) A step of administering a test substance to a non-human animal or cultured cell before or after loading cell stress or during cell stress loading;
(B) A step of collecting a specimen from the non-human animal or cultured cell in step (A) and detecting one or more modified nucleic acids selected from 1-methyladenosine, 5-methylcytidine and pseudouridine in the specimen;
(C) When the modified nucleic acid does not increase in step (B) or the modified nucleic acid does not change in the intracellular localization, the test substance is evaluated as a candidate drug for prevention or treatment of a disease caused by cellular stress;   A hybridoma with accession number FERM P-22120 producing an anti-1-methyladenosine monoclonal antibody.   A hybridoma with accession number FERM P-22122 producing an anti-5-methylcytidine monoclonal antibody.   A hybridoma with accession number FERM P-22121 that produces an anti-pseudouridine monoclonal antibody.