JP7007665B2 - Method for determining the tissue regeneration state of a living body - Google Patents

Description

The present invention relates to a method for determining a tissue regeneration state. The present invention also relates to a method comprising a step of detecting Opioid growth factor receptor-like 1 (OGFRL1) in a body fluid collected from a subject.

When tissue is injured or defective, the remaining cells of the injured or defective tissue proliferate, resulting in regeneration and repair. If it is insufficient, tissue cells derived from embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells) can be transplanted to regenerate or repair damaged or defective tissue due to their proliferation. Attempted. The degree of cell proliferation in the injured or defective tissue reflects the regeneration state of the tissue and can be an important judgment material for determining the therapeutic effect and predicting the prognosis of the patient.

As an example of a marker for evaluating tissue regeneration, Non-Patent Document 1 shows that the production of alpha-fetoprotein (AFP) is increased during the recovery period from acute liver injury in rats by administration of D-galactosamine. Clinically, it is also known that the serum AFP level increases in the recovery period from acute severe hepatic disorder (Non-Patent Document 2) and in liver cirrhosis (Non-Patent Document 3), which is the final image of chronic hepatic disorder.
It has been reported that the expression of OGFRL1 is enhanced in the cancer stem cell fraction (Patent Documents 1 and 2).

International Publication No. 2013/122140 Japanese Unexamined Patent Publication No. 2015-107918

Takehide Noguchi "Study of α-Fetoprotein-producing cells in the recovery phase of D-Galactosamine rat acute liver injury-combination of enzyme antibody method and Autoradiography method-" Journal of Japanese Society of Gastroenterology 75: 1972-1982, 1978 Kakisaka K, et al. “Alpha-fetoprotein: A biomarker for the recruitment of progenitor cells in the liver in patients with acute liver injury or failure.” Hepatology Research 45: E12-E20, 2015 Liu YR, et al. “Alpha-fetoprotein level as a biomarker of liver fibrosis status: a cross-sectional study of 619 consecutive patients with chronic hepatitis B.” BMC Gastroenterol 14: 145, 2014

Since the expression of AFP is frequently and remarkably increased in hepatocellular carcinoma cells, it cannot be determined whether the increase in serum AFP level is due to recovery from severe liver injury or production by hepatocellular carcinoma cells. Therefore, AFP is not suitable as a marker for determining the growth of non-tumor cells, and is currently used exclusively as a tumor marker. As described above, there is no specific marker for evaluating the regeneration or repair state of the liver suffering from acute or chronic injury, and it is difficult to determine the therapeutic effect and predict the prognosis for patients with severe liver injury. Therefore, it has been desired to develop a tissue regeneration marker that can be distinguished from the growth of tumor cells.

In addition, a method (tissue biopsy) has been used to determine the cell proliferation state in tissue regeneration by collecting solid tissue from the patient's body and inspecting it. However, tissue biopsy is highly invasive and painful, and sometimes has serious complications such as massive bleeding. Therefore, a method for evaluating cell proliferation with low invasiveness is eagerly desired.

In view of such problems, it is an object of the present invention to provide a tissue regeneration marker that reflects the cell proliferation state. Another object of the present invention is to provide a tissue regeneration marker that is not affected by the growth of tumor cells and can be evaluated with minimal invasiveness.

The first embodiment of the present invention relates to a method for determining a tissue regeneration state. In the method, a step of measuring Opioid growth factor receptor-like 1 (OGFRL1) in a body fluid collected from a subject, a step of comparing an OGFRL1 measured value with a reference value, and a step of comparing the OGFRL1 measured value with the reference value, and the OGFRL1 measured value being the reference value. In the above case, the step including a step suggesting that the regeneration of the tissue is promoted in the subject is included.
According to this aspect, by measuring OGFRL1 in body fluid, the regeneration state of tissue can be determined using cell proliferation as an index.

A second aspect of the present invention is a method comprising a step of detecting Opioid growth factor receptor-like 1 (OGFRL1) in a body fluid collected from a subject, wherein the detection of the OGFRL1 causes the tissue of the subject to be detected. It relates to a method characterized by suggestion of enhanced regeneration.
According to this aspect, it is possible to suggest the enhancement of tissue regeneration by detecting OGFRL1 in the body fluid.

According to the present invention, a tissue regeneration marker can be provided.

A shows Western blotting images of OGFRL1 present in the sera of carbon tetrachloride-administered mice and diethylnitrosamine (DEN) -administered mice. B shows a graph of emission intensity showing the relative expression ratio of OGFRL1 detected in A. In the figure, "-" indicates a mouse to which carbon tetrachloride was not administered, or a mouse to which DEN was administered but liver cancer did not develop. “+” Indicates a mouse to which carbon tetrachloride was administered or a mouse in which liver cancer was developed by administration of DEN. A shows a Western blotting image of OGFRL1 present in serum exosomes or liver tissues of carbon tetrachloride-administered mice. B shows a graph of emission intensity showing the relative expression ratio of OGFRL1 detected in A. In the figure, "-" indicates a mouse not administered carbon tetrachloride. "+" Indicates a mouse to which carbon tetrachloride was administered. A shows a Western blotting image of OGFRL1 present in serum exosomes of mice treated with different concentrations of carbon tetrachloride. B shows a graph of emission intensity showing the relative expression ratio of OGFRL1 detected in A. The numbers in the figure indicate individual numbers of each mouse. The luminescence intensity when the serum OGFRL1 using the anti-OGFRL1 (344-362) antibody and the anti-CD9 antibody was measured by the sandwich Enzyme-linked immunosorbent assay (ELISA) method is shown. In the figure, "-" indicates a mouse not administered carbon tetrachloride. "+" Indicates a mouse to which carbon tetrachloride was administered. The numbers in the figure indicate individual numbers of each mouse.

1. 1. First Embodiment The first embodiment of the present invention comprises a step of measuring Opioid growth factor receptor-like 1 (OGFRL1) in a body fluid collected from a subject.

In the case of humans, Opioid growth factor receptor-like 1 (OGFRL1) can be mentioned as a protein registered in GenBank provided by The National Center for Biotechnology Information under accession number Q5TC84.1, and its isoform. In the case of mice, proteins registered in GenBank with accession number Q8VE52.2 and their isoforms can be mentioned. Further, in the present invention, OGFRL1 includes a variant of the protein registered by the accession number and an ortholog.

The subject is an individual whose state of tissue regeneration is evaluated. The individual is not particularly limited, and examples thereof include mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, cows, horses, goats, sheep and pigs, and birds such as chickens. Mammals such as humans, monkeys, mice, rats, dogs, cats, cows, horses and pigs are preferable, humans, monkeys, mice, rats, dogs or cats are more preferable, and humans are more preferable. It is a monkey, mouse, or rat, most preferably a human. In addition, the age and gender (male or female) of the individual are not limited.

The subject may include both individuals with and without disease. The subject is preferably an individual having no tumor, more preferably no malignant tumor, and even more preferably no malignant tumor in the liver. Moreover, it is preferable that the subject has a non-neoplastic disease. The non-neoplastic disease is not limited as long as it is a disease other than a tumor. For example, the non-neoplastic disease is a disease presenting a liver disorder, and examples of the type of liver disorder include acute hepatitis, chronic hepatitis, cirrhosis, and steatohepatitis. In addition, the causes of hepatic disorder include viral, alcoholic, drug-induced, autoimmune, glucose metabolism disorder, lipid metabolism disorder, amino acid metabolism disorder, metal metabolism disorder, cholestasis, hepatic blood flow disorder, etc. Can be done.

Body fluids can be collected from the subject and are not restricted as long as OGFRL1 can be detected. For example, body fluids include blood samples, ascites, pleural effusion, pericardial fluid, cerebrospinal fluid, interstitial fluid, lymph, urine and the like. Blood samples include whole blood, serum, and plasma. The body fluid is more preferably serum or plasma.

In addition, the body fluid preferably contains extracellular vesicles. The extracellular vesicles are also called extracellular vesicles and include exosomes, microvesicles, extracellular vesicles and the like. Recovery of extracellular vesicles can be performed using, for example, a commercially available exosome precipitation reagent such as ExoQuick (System Biosciences).

Tissue is not restricted as long as it is present in the subject's body. Preferably, it is the tissue of the organ in which the non-neoplastic disease is present. More preferably, the organ is the liver.

Tissue regeneration refers to the process of quantitatively and functionally returning to the original state of tissue that has been injured or defective, or the state of returning to the original state. In order to return to the original state quantitatively and functionally, the residual cells or transplanted cells constituting the tissue proliferate to recover the size as an organ and to perform a function specific to the tissue. It is desirable to restore the function that has been expressed and impaired.

Tissue regeneration is enhanced, which means that in the injured or defective tissue, the residual cells or transplanted cells constituting the tissue proliferate and are quantitatively and functionally original. It indicates that the biological reaction to return to the state of is active, which means that the tissue repair is proceeding smoothly, that is, the subject is in the recovery period from the tissue damage.

The method of measuring OGFRL1 is not limited as long as the measured value of OGFRL1 can be obtained. Here, the measured value of OGFRL1 is a value that reflects the amount or concentration of OGFRL1. For example, in order to obtain a measured value of OGFRL1, an antibody that can specifically bind to the OGFRL1 protein, that is, an anti-OGFRL1 antibody can be used. Examples of the method for obtaining the measured value of the OGFRL1 protein by the anti-OGFRL1 antibody include the ELISA method and the Western blotting method.

For example, when the measured value of OGFRL1 is obtained by the ELISA method, the order of mixing the extracellular vesicles collected from the body fluid or the body fluid and the anti-OGFRL1 antibody is not particularly limited, and these are mixed substantially at the same time. It may be mixed sequentially. Hereinafter, this embodiment will be described by taking as an example a method for detecting the OGFRL1 protein in a body fluid.

In this embodiment, the complex of the anti-OGFRL1 antibody and the OGFRL1 protein in the body fluid is first formed and then immobilized on the solid phase, or the anti-OGFRL1 antibody is immobilized on the solid phase in advance. , It is possible to form a complex of immobilized anti-OGFRL1 antibody and OGFRL1 protein in body fluid. More preferably, it is an embodiment in which the complex is first formed and then the complex is immobilized on a solid phase. Then, the measured value of the OGFRL1 protein contained in the body fluid is obtained by detecting the complex immobilized on the solid phase or the complex formed on the solid phase by a method known in the above technique. Can be done.

When a complex of anti-OGFRL1 antibody and OGFRL1 protein in body fluid is first formed and then the complex is immobilized on a solid phase, the anti-OGFRL1 antibody modified with biotin or the like is brought into contact with the OGFRL1 protein in body fluid. To form a complex. By separately binding avidin or streptavidin (hereinafter, also referred to as "avidins") to the solid phase in advance, the complex can be immobilized on the solid phase via the binding between biotin and avidins. can.

Further, when the anti-OGFRL1 antibody is fixed to the solid phase in advance, the mode of fixing the anti-OGFRL1 antibody to the solid phase is not particularly limited. For example, the anti-OGFRL1 antibody and the solid phase may be directly bound, or the anti-OGFRL1 antibody and the solid phase may be indirectly bound via another substance. Examples of the direct bond include physical adsorption. Examples of the indirect binding include binding via a combination with avidins. In this case, the anti-OGFRL1 antibody is modified with biotin in advance, and the avidins are bound to the solid phase in advance, whereby the anti-OGFRL1 antibody and the solid phase are indirectly bound via the binding between biotin and the avidins. Can be made to. In this embodiment, the binding between the anti-OGFRL1 antibody and the solid phase is preferably an indirect binding via biotin and avidins.

The material of the solid phase is not particularly limited, and can be selected from, for example, an organic polymer compound, an inorganic compound, a biopolymer and the like. Examples of the organic polymer compound include latex, polystyrene, polypropylene and the like. Examples of the inorganic compound include magnetic substances (iron oxide, chromium oxide, ferrite, etc.), silica, alumina, glass, and the like. Examples of the biopolymer include insoluble agarose, insoluble dextran, gelatin, cellulose and the like. Two or more of these may be used in combination. The shape of the solid phase is not particularly limited, and examples thereof include particles, membranes, microplates, microtubes, and test tubes. Among them, particles are preferable, and magnetic particles are particularly preferable.

In this embodiment, Bound / Free (B / F) is used to remove unreacted free components that have not formed a complex, after the formation of the complex, preferably after the formation of the complex and before the detection of the labeling substance. ) Separation may be performed. The unreacted free component means a component that does not form a complex. For example, an anti-OGFRL1 antibody that did not bind to the OGFRL1 protein can be mentioned. The means for B / F separation is not particularly limited, but if the solid phase is particles, B / F separation can be performed by recovering only the solid phase in which the complex is captured by centrifugation. If the solid phase is a container such as a microplate or microtube, B / F separation can be performed by removing the liquid containing unreacted free components. When the solid phase is magnetic particles, B / F separation can be performed by suctioning and removing the liquid containing unreacted free components with a nozzle while the magnetic particles are magnetically constrained by a magnet. The method is preferable from the viewpoint of automation. After removing the unreacted free components, the solid phase containing the complex may be washed with a suitable aqueous medium such as phosphate buffered saline (PBS).

In this embodiment, the detection of the complex can be carried out using an anti-OGFRL1 antibody labeled with a labeling substance, or a labeling substance capable of binding to an unlabeled anti-OGFRL1 antibody and the unlabeled anti-OGFRL1 antibody. Although it can be carried out using an anti-immunoglobulin antibody labeled with, it is preferable to use a labeled anti-OGFRL1 antibody. Further, it is preferable that the epitope in the OGFRL1 protein of the labeled anti-OGFRL1 antibody and the epitope in the OGFRL1 protein of the anti-OGFRL1 antibody that binds to the solid phase are different.

The labeling substance used for the labeled anti-OGFRL1 antibody or the labeled anti-immunoglobulin antibody is not particularly limited as long as a detectable signal is generated. For example, the substance itself may be a substance that generates a signal (hereinafter, also referred to as a “signal generating substance”), or may be a substance that catalyzes the reaction of another substance to generate a signal. Examples of the signal generating substance include a fluorescent substance and a radioisotope. Examples of substances that catalyze the reaction of other substances to generate a detectable signal include enzymes. Examples of the enzyme include alkaline phosphatase (ALP), peroxidase, β-galactosidase, luciferase and the like. Examples of the fluorescent substance include fluorescent dyes such as fluorescein isothiocyanate (FITC), Rhodamine and Alexa Fluor (registered trademark), and fluorescent proteins such as Enhanced green fluorescent protein (EGFP). Examples of the radioisotope include ¹²⁵ I, ¹⁴ C, ³² P and the like. Among them, an enzyme is preferable as a labeling substance, and ALP is particularly preferable.

The labeled anti-OGFRL1 antibody is obtained by labeling the anti-OGFRL1 antibody with the above-mentioned labeling substance by a labeling method known in the field of immunoassay technology. Alternatively, labeling may be performed using a commercially available labeling kit or the like. Further, as the labeled immunoglobulin antibody, the same method as that for labeling the anti-OGFRL1 antibody may be used, or a commercially available product may be used.

In this embodiment, the measured value of OGFRL1 contained in the body fluid can be obtained by detecting the signal generated by the labeling substance of the labeled anti-OGFRL1 antibody contained in the complex. Here, "detecting a signal" includes qualitatively detecting the presence or absence of a signal, quantifying the signal intensity, and semi-quantitatively detecting the signal intensity. Semi-quantitative detection means that the intensity of a signal is shown stepwise, such as "no signal generated", "weak", "medium", "strong", and the like.

The method of detecting a signal itself is known in the field of immunoassay technology. In this embodiment, the measuring method according to the type of the signal derived from the above-mentioned labeling substance may be appropriately selected. For example, when the labeling substance is an enzyme, signals such as light and color generated by reacting a substrate with the enzyme can be measured by using a known device such as a luminometer or a spectrophotometer. can.

The substrate of the enzyme can be appropriately selected from known substrates according to the type of the enzyme. For example, when ALP is used as the enzyme, CDP-Star® (4-chloro-3- (methoxyspiro [1,2-dioxetane-3,2'-(5'-chloro) trixilo] [3] is used as the substrate. 3.1.13,7] decane] -4-yl) disodium phenylphosphate), CSPD® (3- (4-methoxyspiro [1,2-dioxetane-3,2- (5') -Chemiluminescent substrate such as tricyclo [3.3.1.13,7] decane] -4-yl) disodium phenylphosphate), 5-bromo-4-chloro-3-indrill phosphate (BCIP) ), 5-bromo-6-chloro-indrill phosphate disodium, p-nitrophenyl phosphate and other chromogenic substrates can be mentioned. Particularly preferred is CDP-Star®. It is preferable to detect the luminescence of the substrate with a luminometer.

When the labeling substance is a radioisotope, the radiation as a signal can be measured using a known device such as a scintillation counter. When the labeling substance is a fluorescent substance, the fluorescence as a signal can be measured using a known device such as a fluorescent microplate reader. The excitation wavelength and the fluorescence wavelength can be appropriately determined according to the type of the fluorescent substance used.

The signal detection result can be used as a measured value of OGFRL1 protein. For example, when the signal intensity is quantitatively detected, the measured value of the signal intensity itself or the value calculated from the measured value of the signal intensity can be used as the measured value of the OGFRL1 protein. The value calculated from the measured value of the signal intensity is, for example, a value obtained by subtracting the measured value of the signal intensity of the negative control sample from the measured value of the signal intensity, and the measured value of the signal intensity is the signal intensity of the positive control sample. A value divided by a measured value, a combination thereof, and the like can be mentioned. Examples of the negative control sample include body fluids of healthy individuals. Examples of the positive control sample include body fluids containing the OGFRL1 protein at a predetermined concentration.

For the measured value of OGFRL1 protein in body fluid, a calibration curve is prepared from the measured value of the signal intensity of the positive control containing the known concentration of OGFRL1 protein, and the measured value of the signal intensity of OGFRL1 protein in body fluid is measured on the calibration curve. Can be calculated by applying. In addition, without creating a calibration curve, a regression equation was obtained from the measured value of the signal intensity of the positive control containing the OGFRL1 protein at a known concentration, and the measured value of the signal intensity of the OGFRL1 protein in the body fluid was applied to the regression equation. , The measured value of OGFRL1 protein in body fluid can be calculated.
When the measured value is expressed as a concentration, it may be a molar concentration or a mass ratio (mass / volume) per fixed volume of the biological sample.

The anti-OGFRL1 antibody is not limited as long as it specifically binds to the OGFRL1 protein, and a polyclonal antibody, a monoclonal antibody, and a fragment thereof obtained by immunizing an animal other than human with the OGFRL1 protein or a part thereof as an antigen. Any of (for example, Fab, F (ab) ₂ , etc.) can be used. Further, the class and subclass of immunoglobulin are not particularly limited. It may also be a chimeric antibody. Further, it may be scFv or the like.

The antigens used to produce the anti-OGFRL1 antibody include the OGFRL1 protein registered in GenBank as accession number Q5TC84.1 for humans and accession number Q8VE52.2 for mice. can. For example, a peptide consisting of 19 amino acids from the 344th to the 362nd amino acids on the amino acid sequence of mouse OGFRL1 represented by the accession number Q8VE52.2 can be used as an antigen. The peptide can be synthesized by a known method.

Further, in the present embodiment, either the antibody that binds to OGFRL1 in the body fluid or the labeled anti-OGFRL1 antibody may be replaced with an antibody that binds to a protein or the like present in extracellular vesicles. Examples of the protein present in the extracellular vesicle include CD9 and the like.
This aspect further includes a step of comparing the measured value with the reference value.

The reference value can be set in advance. For example, a body fluid group collected from an individual whose regeneration is promoted in a tissue other than a tumor is defined as a positive control group, and a body fluid group collected from a healthy individual is defined as a negative control group. Then, a value that can classify the positive control group and the negative control group with the highest accuracy can be adopted as a reference value. Here, the "value that can be classified most accurately" can be appropriately set based on an index such as sensitivity, specificity, positive predictive value, and negative predictive value depending on the purpose of the test. Furthermore, the reference value can be determined by the ROC curve (Receiver Operatorating Characteristic curve), discriminant analysis method, mode method, Kittler method, 3σ method, p-tile method, or the like. The reference value is preferably set for each body fluid.

In this embodiment, when the measured value is equal to or higher than the reference value, it is suggested that the tissue regeneration is promoted in the subject. Here, suggesting that the tissue regeneration is enhanced in the subject includes determining that the tissue regeneration is enhanced in the subject.

Furthermore, this aspect may suggest that the subject is in the recovery phase from the damage to the tissue when it is suggested that the tissue regeneration is enhanced in the subject. Here, suggesting that the subject is in the recovery phase from the tissue disorder includes determining that the subject is in the recovery phase from the tissue disorder.
2. 2. Second embodiment

A second embodiment of the present invention relates to a method comprising a step of detecting Opioid growth factor receptor-like 1 (OGFRL1) in a body fluid collected from a subject. It is possible to detect OGFRL1 in body fluids by the method described in the first embodiment. Here, for detection, in addition to the method for obtaining the measured value of OGFRL1 in the body fluid described in the first embodiment, it is qualitative whether or not OGFRL1 is present in the body fluid by the method described in the first embodiment. Includes judgment.

In this embodiment, when OGFRL1 is detected in the body fluid of the subject, it is suggested that the tissue of the subject is promoted to be regenerated. Here, suggesting that the tissue regeneration is enhanced in the subject includes determining that the tissue regeneration is enhanced in the subject.

Furthermore, this aspect may suggest that the subject is in the recovery phase from the damage to the tissue when it is suggested that the tissue regeneration is enhanced in the subject. Here, suggesting that the subject is in the recovery phase from the tissue disorder includes determining that the subject is in the recovery phase from the tissue disorder.
For the terms used in the second embodiment, the description in the first embodiment is incorporated.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not construed as being limited to Examples.

1. 1. Example 1
To verify that OGFRL1 can be used as a marker for determining cell proliferation status other than tumors, detection of OGFRL1 in serum using mice treated with carbon tetrachloride-administered liver injury and mice treated with DEN, a liver carcinogen. Was done.

1-1. Preparation of anti-OGFRL1 antibody Of the mouse OGFRL1 protein consisting of 464 amino acids in total length, a peptide consisting of 19 amino acids from 344th to 362nd was synthesized, and this synthetic peptide was immunized in rabbits. The antibody titer was confirmed by ELISA. The above rabbit serum was column-purified using the synthetic peptide used as an immunogen.

1-2. Preparation of carbon tetrachloride-administered liver-damaged mice Carbon tetrachloride was mixed with a 3-fold volume of olive oil and diluted to prepare a 25% carbon tetrachloride solution. Using the prepared carbon tetrachloride solution, 1 mL of carbon tetrachloride per kg of body weight was subcutaneously injected into C57BL / 6J male mice (Claire Japan) weighing 20 to 25 g under isoflurane inhalation anesthesia. Liver tissues were collected from carbon tetrachloride non-administered mice and mice 24 hours after carbon tetrachloride administration (3 each) under isoflurane inhalation anesthesia.

1-3. Induction of Liver Cancer by DEN Administration Three-week-old C57BL / 6J male mice received a single intraperitoneal dose of 10 μg DEN per kg body weight. Ten months after DEN administration, the abdomen was opened under isoflurane anesthesia, and the presence of a tumor on the liver surface was visually observed. The presence or absence of liver cancer was histologically confirmed in the removed liver, and the DEN-administered mice were divided into two groups, tumor-free and tumor-developed.

1-4. Serum collection To collect serum, mice were laparotomized under isoflurane anesthesia and blood was collected from the inferior vena cava. After allowing the collected blood to stand at room temperature for 30 minutes or more, the serum and blood clot were separated by centrifugation at 1,200 × g for 20 minutes, and the obtained serum was stored at -20 ° C until use.

1-5. Western blotting serum homogenization buffer (final concentration: 20 mM Tris-HCl pH 7.5, 60 mM beta-glycerophosphate, 10 mM wicking glycol tetraacetic acid, 10 mM magnesium chloride, 10 mM sodium fluoride, 2 mM dithiothreitol, 1.0% NP-40 , 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1 × proteinase inhibitor buffer [Aprotinin, Leupeptin, Pepstatin A]) was added and mixed, and the mixture was centrifuged at 4 ° C, 13,500 rpm for 10 minutes. An equal amount of 2 × Laemmli sample buffer (final concentration: 32.9 mM Tris-HCl pH 6.8, 1.05% SDS, 2.5% 2-mercaptoethanol, 13.15% glycerol, 0.005% bromophenol blue) was added to the obtained supernatant and mixed. Above, it was heated at 98 ° C. for 5 minutes. A sample equivalent to 50 μg of protein was injected into a 10% polyacrylamide / SDS gel, electrophoresed and then transferred to a PVDF membrane. Shake the membrane in blocking buffer (3% bovine serum albumin [BSA] / PBS) for 1 hour at room temperature, add 3,000-fold diluted anti-OGFRL1 (344-362) antibody, and add anti-OGFRL1 (344-362) antibody at 4 ° C for 16 hours. It was reacted. After washing the membrane, a peroxidase-labeled secondary antibody diluted 10,000-fold was added, and the mixture was reacted at room temperature for 45 minutes. After washing the membrane, it was reacted with Immobilon Western chemiluminescent HRP reagent (Merck-Millipore) to detect the band showing OGFRL1 protein.

1-6. Results Western blotting of sera obtained from carbon tetrachloride non-administered mice and mice 24 hours after carbon tetrachloride administration (3 animals each), and liver cancer non-developed mice and liver cancer-developed mice 10 months after DEN administration (3 animals each). The results are shown in FIG. As is clear from FIG. 1, OGFRL1 was not detected in the sera of carbon tetrachloride non-administered mice, but OGFRL1 in the serum was increased by the administration of carbon tetrachloride. In contrast, OGFRL1 was not detected in the serum of DEN-induced liver cancer mice. This suggests that OGFRL1 detected in serum can be used as a marker for determining cell proliferation status other than tumors.

2. 2. Example 2
OGFRL1 was detected in liver tissue and serum exosomes in carbon tetrachloride-administered mice.

2-1. Extraction of protein from liver tissue The collected liver tissue was homogenized in the homogenization buffer described in Example 1, and heat-treated by adding 2 × Laemmli sample buffer.

2-2. Collection of serum exosomes and detection of OGFRL1
A carbon tetrachloride-administered mouse was prepared according to the method described in Example 1 above. Exosomes were recovered from the mouse serum using an exosome precipitation reagent (ExoQuick [System Biosciences]) according to the attached protocol. Specifically, serum from which living cells and cell debris had been removed by centrifugation was mixed with ExoQuick reagent and reacted at 4 ° C. for 30 minutes. Centrifugation at 1,500 xg for 30 minutes gave a precipitate containing exosomes. To the precipitate, the homogenization buffer described in Example 1 was added to dissolve the precipitate, an equal amount of 2 × Laemmli sample buffer was added, and then the mixture was heated at 98 ° C. for 5 minutes.

2-3. Western blotting Western blotting was performed according to the method described in Example 1.

2-4. Results The results are shown in FIG. As is clear from FIG. 2, OGFRL1 was significantly increased in serum exosomes 24 hours after carbon tetrachloride administration. When the amount of OGFRL1 protein in the liver tissue at this time was examined per unit liver weight, it was rather decreased in the liver tissue 24 hours after carbon tetrachloride administration as compared with the liver tissue not administered with carbon tetrachloride. This suggests that an increase in OGFRL1 concentration in serum or serum exosomes more closely reflects the proliferative state of cells than the OGFRL1 content in liver tissue.

3. 3. Example 3
Different amounts of carbon tetrachloride were administered to mice and the amount of OGFRL1 in serum exosomes in each mouse was compared.

3-1. Administration of carbon tetrachloride to mice and detection of OGFRL1 in serum exosomes Using a carbon tetrachloride solution prepared in the same manner as in Example 1, 1 mL of carbon tetrachloride per kg of body weight was used to induce moderate liver damage. In addition to the group to which was administered, a group to which carbon tetrachloride was administered at a dose of 0.5 mL / kg body weight, which was half the dose, or 2 mL / kg body weight, which was double the dose, was set. The amount of OGFRL1 in serum exosomes of each group of mice at 24 hours after administration was detected by Western blotting according to the methods described in Examples 1 and 2.

Here, according to a study using other mice, all the mice survived and recovered by administration of 0.5 or 1 mL / kg of carbon tetrachloride, whereas 2 mL / kg of carbon tetrachloride was used. When administered, many mice suffer from severe liver damage and die. That is, in the case of administration of 2 mL / kg body weight of carbon tetrachloride, sufficient tissue regeneration does not occur and recovery from liver damage cannot be achieved.

3-2. Results The results are shown in FIG. From FIG. 3, in mice with a carbon tetrachloride dose of 0.5 mL to 1.0 ml / kg body weight, the OGFRL1 concentration in serum exosomes was significantly increased, whereas carbon tetrachloride at 2.0 mL / kg body weight was significantly increased. It was found that there was no increase in OGFRL1 concentration in serum exosomes of mice treated with. That is, it was shown that the serum OGFRL1 concentration increased in mice that can recover from liver damage by cell proliferation, and conversely, the serum OGFRL1 concentration does not increase in mice that cannot recover from liver damage. .. This suggests that measuring the OGFRL1 concentration in serum exosomes makes it possible to determine the therapeutic effect and predict the prognosis in subjects with hepatic disorder.

4. Example 4
Serum OGFRL1 in carbon tetrachloride non-administered mice (2) and carbon tetrachloride-administered mice (3) was detected by sandwich ELISA. An anti-OGFRL1 (344-362) antibody was used as the capture antibody, and an anti-CD9 antibody (BioLegend # 312102) that detects CD9 known to be present in exosomes was used as the detection antibody.

4-1. ALP Labeling of Detected Antibodies Alkaline Phosphatase Labeling Kit-NH2 (Dojindo kit # LK12) was used to label the detected antibody with ALP. The labeling method followed the protocol attached to the kit. The labeled antibody was stored at 4 ° C. by adding the storage buffer attached to the kit.

4-2. Purification of capture antibody
500 μL of PBS was added to Amicon ultra 50 kDa and centrifuged at 14000 × g for 5 minutes. The above centrifugation was repeated by adding 500 μL of PBS again, and the column was washed 5 times in total. The column was turned upside down and centrifuged at 1000 xg for 10 minutes. As a capture antibody, 22.4 μg of anti-OGFRL1 (344-362) antibody was permeated into the column, and 492 μL of carbonate buffer was added. After centrifugation at 14000 × g for 5 minutes, 400 μL of carbonic acid buffer was added and the mixture was centrifuged again. This operation was repeated twice. The column was turned upside down and centrifuged at 1000 xg for 10 minutes to recover the purified antibody. The protein concentration of the purified antibody was confirmed by measuring the absorbance with Nanodrop (Thermo Fisher Scientific). The results were as follows.
Concentration: 0.5154 μg / μL (average of 5 measurements)
Yield: 33 μL
Total protein content: 17.0082 μg

4-3. Fixation of capture antibody to solid phase
Anti-OGFRL1 (344-362) antibody as a capture antibody was adjusted to 5 μg / mL with a carbonate buffer on an ELISA plate and added to each well to 90 μL / well. After allowing to stand at 4 ° C. for 15 hours, the cells were washed 3 times with 300 μL / well of PBS (PBS-T) containing 0.05% (w / v) Tween 20.

4-4. ELISA measurement 4-3. PBS-T containing 1% BSA was added to the capture antibody solid phase plate prepared in 1. at 200 μL / well, blocked at room temperature for 1.5 hours, and then washed 3 times with PBS-T 300 μL / well.

To 50 μL of serum, 5 μL of PBS was added, the mixture was lightly stirred with vortex, and then allowed to stand for 5 minutes. A 3-fold dose of 1% BSA / PBS was added to this sample to dilute the serum. 50 μL of the diluted sample was added to the capture antibody solid phase plate and shaken at 37 ° C. for 2 hours at 400 rpm. After the reaction, the plates were washed 3 times with PBS-T 300 μL / well.

ALP-labeled detection antibody was diluted 2400-fold with 1% BSA / PBS in each well, 50 μL was added, and the mixture was shaken at 37 ° C. for 1.5 hours at 400 rpm. After the reaction, the plates were washed 3 times with PBS-T at 300 μL / well.
CDP-star 100 μL / well was added to the plate and reacted at room temperature for 10 minutes, and the emission intensity was measured.

4-5. Results The measurement results are shown in FIG. As is clear from FIG. 4, it was revealed that the OGFRL1 concentration in serum showed a remarkable increase in the mice treated with carbon tetrachloride.

Claims (7)

It is a method for determining the tissue regeneration state,
Including the step of measuring Opioid growth factor receptor-like 1 (OGFRL1) in the body fluid collected from the subject.
When the measured value of O GFRL1 is equal to or higher than the reference value, it is suggested that the tissue regeneration is promoted in the subject.
A method for determining the tissue regeneration status. When it is suggested that the tissue regeneration is enhanced, it further suggests that the subject is in the recovery phase from the tissue damage.
The method according to claim 1. The method of claim 1 or 2, wherein the tissue is tissue in an organ in which a non-neoplastic disease is present. The method of claim 3, wherein the organ is the liver. The method according to claim 4, wherein the non-neoplastic disease present in the liver presents liver damage. The method according to any one of claims 1 to 5, wherein the subject is an individual having no malignant tumor in the liver. The method according to any one of claims 1 to 6, wherein in the measurement step, OGFRL1 in serum, OGFRL1 in plasma, or OGFRL1 in extracellular vesicles present in body fluid is detected.

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