KR101654789B1 - Composition for diagnosing organ injury comprising exosome or its protein, and method for diagnosing organ injury using the same - Google Patents

Abstract

The present invention relates to a biomarker composition for prolonged toxicity diagnosis comprising exosome or a protein thereof, a kit for a long-term toxicity diagnosis including the composition, a method for providing information for diagnosis of a long-term toxicity using the composition, To a screening method. The present invention can rapidly diagnose the long-term toxicity using the exosome according to the present invention and, in particular, can diagnose the toxicity of a specific organ using the organ specific exosome protein, And may be useful in diagnosing prolonged toxicity due to toxicity or predicting prognosis.

Description

TECHNICAL FIELD The present invention relates to a biomarker composition for diagnosing organ toxicity including exosome or a protein thereof, and a diagnostic method using the exosome or its protein,

The present invention relates to a biomarker composition for diagnosing organ toxicity comprising exosome or a protein thereof and a diagnostic method using the same.

Drug safety is an important factor in the public health that affects the pharmaceutical industry, regulators, physicians and patients. DILI is a hepatotoxicity caused by a drug, and many approved drugs are withdrawn and prohibited by DILI. In particular, among many drugs, acetaminophen (APAP) is potentially limited to concentrations that can cause liver damage. Acetaminophen is one of the top selling drugs in the world and causes problems due to side effects. Hepatic toxicity caused by acetaminophen is mainly caused by overdosage. At this time, excessive amounts of acetaminophen accumulate NAPQI due to insufficient supply of glutathione, which eventually kills hepatocytes and causes liver toxicity. NAC is one of the most effective drugs to protect hepatocytes from toxicity and helps metabolism by binding directly to glutathione recharge and NAPQI. If NAPQI accumulates, it should be treated within a short time (8-10 hours) after drug metabolism. However, since there are no symptoms at the beginning, it is difficult to handle them quickly. Therefore, there is a need for a biomarker capable of diagnosing hepatotoxicity in blood at an early stage.

On the other hand, exosomes are small vesicles (membrane vesicles) secreted from most cells. In exosomes, various types of proteins derived from the cells, genetic material (DNA, mRNA, miRNA) and lipids are reported to be contained. It has also been reported that tissue-derived exosomes can be used for the diagnosis of diseases because they reflect the state of the tissue that secretes exosome.

The present inventors have continued to develop a novel biomarker composition for long-term toxicity diagnosis and a diagnostic method using the same, and as a result, it has been found that when exosomes are used, the toxicity of organs can be diagnosed and predicted accurately and quickly, The present inventors completed the present invention by confirming that the toxicity of each organ can be diagnosed by analyzing the exosome protein.

It is an object of the present invention to provide a biomarker composition for diagnosing organ toxicity comprising exosome or a protein thereof.

It is also an object of the present invention to provide a long-term toxicity diagnostic kit comprising the composition.

It is also an object of the present invention to provide a method for detecting an exosome comprising: (a) isolating an exosome from a biological sample; And (b) confirming the amount of the exosome. The present invention also provides a method for providing information for a long-term toxicity diagnosis.

It is also an object of the present invention to provide a method for treating blood, comprising: (a) treating a candidate for blood; (b) measuring the amount of exosome in the blood treated with the candidate substance; And (c) comparing the amount of the exosome with a control. The present invention also provides a method for screening a long-term toxicity therapeutic substance.

In order to solve the above problems, the present invention provides a biomarker composition for diagnosing organ toxicity comprising exosome or a protein thereof.

The present invention also provides a kit for the long-term toxicity diagnosis comprising the composition.

(A) separating the exosome from the biological sample; And (b) confirming the amount of the exosome.

The present invention also relates to a method for the treatment of cancer, comprising the steps of: (a) treating a candidate substance to blood; (b) measuring the amount of exosome in the blood treated with the candidate substance; And (c) comparing the amount of the exosome with a control.

The present invention can rapidly diagnose the long-term toxicity using the exosome according to the present invention, and in particular, can diagnose the toxicity of a specific organ using the exosome protein, It can be used to diagnose toxicity or to predict prognosis.

FIG. 1 shows the result of measurement of hepatic toxicity of a hepatic toxic mouse model treated with acetaminophen.
Figure 2 shows the results of Fig.
FIG. 3 is a graph showing the results of measurement of the amount of exosome in a hepatotoxic mouse model treated with acetaminophen. FIG.
Figure 4 FIG. 5 shows the result of measurement of the amount of exosome in a hepatotoxic cell model treated with acetaminophen. FIG.
FIG. 5 is a graph showing the results of measuring the amount of exosomes in the liver toxic mouse model and hepatotoxic cell model treated with DGAL and TAA. FIG.
6 is a diagram showing the result of ELISA enzyme immunoassay of exosome protein in a long-term toxicity model.
FIG. 7 is a graph showing the results of analysis of exosome microRNA expression in a liver toxicity model. FIG.
8 is a graph showing the results of measurement of the amount of exosome after NAC treatment in the liver toxicity state.
FIG. 9 is a graph showing the results of measurement of expression of liver-specific exosome protein after NAC treatment in a hepatic toxic state. FIG.
FIG. 10 is a graph showing the results of measuring the amount of exosomes after quercetin treatment in a kidney toxic state. FIG.
11 is a graph showing the results of analysis of exosome microRNA expression after NAC or quercetin treatment in a long-term toxic state.

The present invention provides a biomarker composition for diagnosing organ toxicity comprising exosome or a protein thereof.

The term "diagnosing" as used herein means identifying the presence or characteristic of a pathological condition. For the purpose of the present invention, it is to confirm whether or not it is toxic for a long time. Since the amount of exosome is increased in a long-term toxic condition, the amount of exosome of the present invention can be confirmed from a sample of an individual, so that the long-term toxicity of the subject can be predicted.

In the present invention, "exosome" refers to a small form of an endoplasmic reticulum secreted from a cell. Preferably, it is a circulating exosome present in blood such as whole blood, serum and plasma.

The present invention also provides a biomarker composition for the diagnosis of specific toxicity of liver, kidney, heart, muscle or brain of a subject. The expression of exogenous long-term exosome protein in exosomes is increased in the long-term toxic state, and more specifically in the liver toxic state, the expression of albumin, haptoglobin or fibrinogen of exosomes increases (Kidney injury molecule 1) or neutrophil gelatinase-associated lipocalin (NGAL) in the kidney toxic state, and in the cardiac toxicity state, the expression of exosomal cardiotope The expression of cardiac troponin I (cTnI) is increased, the expression of skeletal muscle troponin (sTnI) is increased in the muscle toxic state, and in the brain toxic state, S100 calcium binding protein (S100 calcium binding protein B (S100B) expression is increased, the expression of the organ specific exosome protein is measured, This is possible.

Accordingly, the biomarker composition of the present invention can be used for diagnosing liver toxicity by measuring the expression of at least one protein selected from the group consisting of albumin, haptoglobin and fibrinogen in exosomes.

In addition, the biomarker composition of the present invention can be used to diagnose renal toxicity by measuring protein expression of Kim-1 (kidney injury molecule 1) or neutrophil gelatinase-associated lipocalin (NGAL) in exosomes have.

In addition, the biomarker composition of the present invention can be used for diagnosing cardiac toxicity by measuring protein expression of cardiac troponin I (cTnI) in exosomes.

In addition, the biomarker composition of the present invention can be used to diagnose muscle toxicity by measuring protein expression of skeletal muscle troponin (sTnI) in exosomes.

In addition, the biomarker composition of the present invention can be used to diagnose brain toxicity by measuring protein expression of S100 calcium binding protein (S100B) in exosomes.

The present invention also provides a kit for the diagnosis of long-term toxicity comprising the composition.

The kit includes, but is not limited to, RT-PCR kits, microarray chip kits, DNA kits, or protein chip kits.

The kit of the present invention can be used to predict the long-term toxicity of a drug by confirming the amount of exosomes.

In addition, the kit of the present invention can be used in the treatment of exosomes, such as albumin, haptoglobin, fibrinogen, kidney injury molecule 1, neutrophil gelatinase-associated lipocalin ; NGAL), cardiac troponin I (cTnI), skeletal muscle troponin (sTnI), and S100 calcium binding protein B (S100B). And can be used to predict the long-term specific toxicity of the drug.

The kit of the present invention may include one or more other component compositions, solutions, or devices suitable for the assay, as well as antibodies that selectively recognize the marker for long-term toxicity prediction.

In addition, the kit for measuring the level of protein expression in the present invention may include a substrate, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, and a chromogenic substrate for immunological detection of the antibody. The substrate may be a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from polystyrene resin, a slide glass made of glass, etc. The coloring enzyme may be peroxidase, Alkaline phosphatase and the like can be used. As the fluorescent material, FITC, RITC and the like can be used. The coloring substrate solution is ABTS (2,2'-azino-bis- (3-ethylbenzothiazoline- ), Or OPD (O-phenylenediamine), TMB (tetramethylbenzidine) can be used.

Further, according to the present invention,

(a) separating the exosome from the biological sample; And

and (b) confirming the amount of the exosome.

Further, according to the present invention,

(c) an albumin, a haptoglobin, a fibrinogen, a kidney injury molecule 1, a neutrophil gelatinase-associated lipocalin (NGAL) in the exosome, , Cardiac troponin I (cTnI), skeletal muscle troponin (sTnI), and S100 calcium binding protein B (S100B). The present invention provides a method for providing information for a long-term toxicity diagnosis.

The biological sample of step (a) may be selected from the group consisting of whole blood, serum, and plasma, but is not limited thereto, and is preferably plasma.

The method for confirming the expression level of the exosome protein in step (b) and the expression level of the exosome protein in step (c) may be Western blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, Immunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, Ouchterlony immunoelectrophoresis, tissue immuno staining, immunoprecipitation assay, complete fixation assay, FACS (Flow Cytometry ) And a protein chip, but the present invention is not limited thereto.

Further, according to the present invention,

(a) treating the candidate for blood;

(b) measuring the amount of exosome in the blood treated with the candidate substance; And

(c) comparing the amount of the exosome with a control.

In the present invention, the term "control" means a group not treated with a candidate substance.

Specifically, it can be usefully used for screening a long-term toxic agent by comparing the increase or decrease of the amount of exosomes in the presence or absence of a candidate for long-term toxicity treatment. A substance that indirectly or directly reduces the amount of exosome can be selected as a long-term toxicity therapeutic agent disclosed in the present invention. That is, the amount of exosome disclosed in the present invention is specified in the absence of a candidate for long-term toxicity treatment, the amount of exosome of the present invention is specified in the presence of a candidate organ for long-term toxicity treatment, A substance capable of reducing the amount of exosome in the presence of the substance to the amount of exosome in the absence of the prolonged toxic treatment candidate can be predicted by the long-term toxic agent disclosed in the present invention.

In addition,

(a) treating the candidate for blood;

(b) the exosome isolated from the candidate substance-treated blood is treated with albumin, haptoglobin, fibrinogen, Kim-1 (kidney injury molecule 1), neutrophil gelatinase-bound lipocalin Selected from the group consisting of Neutrophil gelatinase-associated lipocalin (NGAL), Cardiac troponin I (cTnI), Skeletal muscle troponin (sTnI) and S100 calcium binding protein B (S100B) Measuring the level of expression of the protein; And

(c) comparing the expression level of the protein with a control.

Specifically, it can be useful for screening long-term specific toxic therapeutic agents by comparing the increase or decrease of the amount of exosome protein in the presence and absence of long-term specific toxic treatment candidate substances. When the expression of albumin, haptoglobin or fibrinogen of exosomes is decreased in the presence of a candidate substance, the therapeutic agent for hepatotoxicity, Kim-1 (kidney injury molecule 1) of exosomes or neutrophil gella When the expression of Neutrophil gelatinase-associated lipocalin (NGAL) is decreased, the expression of cardiac troponin I (cTnI) in the renal toxic agent and exosomes is decreased. A decrease in the expression of skeletal muscle troponin (sTnI) is a therapeutic agent for muscle toxicity. When the expression of S100 calcium binding protein (S100 calcium binding protein B; S100B) in exosomes decreases, will be.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined by the appended claims. It will be obvious to you.

Example 1. Preparation of a long-term toxicity model

1-1. Preparation of long-term toxic mouse models

All mouse experiments were approved by Kyungpook National University and used in the experiment. The mouse was a 6-week-old Balb / C model. To produce a liver toxic mouse model with several drugs, each mouse received acetaminophen (APAP) (300 mg / kg, LD50 single dose), DGAL (1,000 mg / kg); (20 mg / kg), or PRX (peroxiredoxin) (20 mg / kg), or TAA (200 mg / kg) mg / kg) was intraperitoneally injected for 24 hours. In addition, each mouse was first treated with acetaminophen and then treated with N-acetyl cysteine (NAC) (100 mg / kg) every 12 hours for 72 days to generate a mouse model of hepatic toxicity. Cisplatin (20 mg / kg) to make a kidney toxicity model, 0.5% BPVC to make a muscle toxicity model, cyclophosphamide (200 mg / kg) to make a cardiac toxicity model, brain toxicity model (30 mg / kg) was treated with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) for 24 hours in order to establish the Parkinsonian disease model.

The hepatic toxicity of the acetaminophen-treated liver model mice prepared by the above method was measured, and the results are shown in FIG.

As shown in Fig. 1A, it was confirmed that the mice treated with acetaminophen caused liver damage.

1B, the ALT / AST enzyme activity was increased in the plasma of acetaminophen-treated mice, and as shown in FIG. 1C, expression of TNF-a mRNA in relation to liver injury was increased, It was confirmed that liver damage was caused by treatment with aminophen.

1-2. Production of long-term toxic cell models

HepG2, Hep3B and Hepa1-6 cells were cultured in DMEM (Eagle's minimal essential medium) / high glucose medium supplemented with 10% FBS and acetaminophen (APAP), DGAL, TAA, MTX, FLX or PRX RTI ID = 0.0 > IC50 < / RTI > for 24 hours.

Example 2 Isosomal Isolation

ExoQuick exosome precipitation solution (SBI System Biosciences cat. No. EXOQ50A-1) was used to isolate circulating exosomes from the blood isolated from the long-term toxic mouse model of Example 1-1 and the cells of the cells cultured in Example 1-2 above. 1) was used to obtain the circulating exosome according to the method proposed by the manufacturer. The resulting exosome was washed twice with PBS (phosphate buffered saline) to obtain a yellowish exosome pellet. MiRNA was isolated from the circulating exosomes using QIAzole (Qiagen). The circulating exosomes isolated from the long-term toxic mouse model of Example 1-1 were observed, and the results are shown in FIG.

As shown in FIG. 2, it was confirmed through TEM image and size analysis that circulating exosomes of about 100 nm were well isolated in both the control and acetaminophen-treated groups.

Example 3. Measurement of the amount of circulating exosomes

After administering each drug to the hepatotoxic mouse model of Example 1-1 and the cells of Example 1-2 by concentration and time, circulating exosomes were isolated in the same manner as in Example 2-2 Increased amount of exocose circulating was confirmed. The isolated circulating exomes were separated by radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris.HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X- (Sodium deoxycholate) supplemented with Complete Protease Inhibitor Mixture tablets) and stored at -80 ° C. In order to measure the amount of circulating exosomes, the amount of protein was analyzed by BCA (bicinchoninic acid) method. The results of measurement of the amount of circulating exosomes of the control group and acetaminophen-treated group measured by the above method are shown in FIG. 3 and FIG.

As shown in FIGS. 3A and 3B, when the acetaminophen was treated at various concentrations for 1 hour, the amount of circulating exosomes increased in the treated group of 150 and 300 mg / kg, especially in the treated group of 300 mg / kg ALT increased, histopathologic changes were observed, and liver toxicity was observed. In addition, as shown in FIGS. 3C and 3D, when the acetaminophen was treated at various concentrations for 3 hours, the amount of circulating exosomes increased in the 75 mg / kg treatment group at the low concentration, and 75 mg / kg treatment in the ALT treatment , But it was significantly increased in the treated group at 150 and 300 mg / kg. It was confirmed that early diagnosis of hepatotoxicity was possible by measuring the amount of circulating exosomes.

As shown in FIGS. 4A and 4B, it was confirmed that the amount of circulating exosome increased and the number of circulating exosomes also increased when hepatotoxic condition was treated with acetaminophen in HepG2, Hep3B, and Hepa1-6 hepatocytes , As shown in Figs. 4C and 4D, it was confirmed that the amount of circulating exosomes increased in the HepG2 hepatocyte treated with acetaminophen. In addition, it is possible to diagnose hepatotoxicity by increasing the amount of circulating exocytes in the cell model.

DGAL (1,000 mg / kg) or TAA (200 mg / kg) was administered to each mouse as in Example 1-1 to confirm whether or not the circulating exosome was increased in other liver toxic mouse models and liver toxic cell models , And each cell was treated with DGAL or TAA as in Example 1-2 above. As controls, MTX, FLX or PRX, a safe drug with no liver toxicity, was treated. The amounts of circulating exosomes, ALT, and AST in the mouse model and the cell model were measured, and liver tissues were observed, which is shown in FIG. 5,

As shown in FIG. 5, when DGAL or TAA was treated in the hepatotoxic mouse model, the amount of circulating exosome was increased, and ALT and AST in the blood were also increased. In addition, in the hepatotoxic cell model, it was confirmed that the amount of circulating exosome increased in hepatotoxic state by treating DGAL and TAA with HepG2, Hep3B and Hepa1-6 cells. On the other hand, when MTX, FLX and PRX, which are safe drugs that do not cause hepatotoxicity in the liver toxic mouse model, were treated, there was no change in the amount of circulating exosomes and ALT and AST in the blood, and HepG2, Hep3B And Hepa1-6 cells treated with MTX, FLX and PRX did not change the amount of circulating exosomes. Therefore, it was confirmed that the amount of circulating exosomes could be used as a marker for diagnosing hepatic toxicity.

Example 3. Protein analysis of exosomes related to organ specific toxicity using ELISA (Enzyme-Linked Immunosorbent Assay)

ELISA enzyme immunoassay was used to identify proteins associated with organ specific toxicity of the circulating exosomes. A 96-well polyclonal antibody was coated on the 96-well plate by O / N, and each well was blocked with blocking solution. The exosomes isolated from the long-term toxic mouse blood of Example 1 were reacted for 2 hours, washed thoroughly, and incubated with the detection antibody for albumin, haptoglobin, (1), fibrinogen, kidney injury molecule 1, neutrophil gelatinase-associated lipocalin (NGAL), cardiac troponin I (cTnI), skeletal muscle troponin ; sTnI) and S100 calcium binding protein (S100B) were measured. The results are shown in FIG.

As shown in FIG. 6, the amount of circulating exosomes increased in various long-term toxicity models and the expression of albumin, haptoglobin and fibrinogen increased in the circulating exosomes of the liver toxic mouse model The expression of Kidney injury molecule 1 (Kim-1) and neutrophil gelatinase-associated lipocalin (NGAL) was increased in the circulating exosome of the kidney-toxic mouse model, The expression of cardiac troponin I (cTnI) was increased in the moxa and the expression of skeletal muscle troponin (sTnI) was increased in the circulating exosomes of the muscle-toxic mouse model, and brain toxicity (Parkinson's disease) (S100 calcium binding protein B; S100B) expression in the exosome of the model that caused the S100 calcium binding protein.

Example 4. Exosomal microRNA analysis

Total RNA was isolated by adding QIAzole (Qiagen) to the exosomes circulating with the blood of the organ toxic mouse of Example 1-1. The experiments were performed according to the miRNeasy protocol (Qiagen), and the synthetic Caenorhabditis elegans (cel) -miR-39 was added thereto. Expression of miR-122, miR-192, miR-155, miR-7, miR-146a, miR-208 and miR-206 was confirmed using the miScript SYBR Green PCR Kit (Qiagen) ≪ / RTI > The results are shown in Fig.

As shown in FIG. 7, it was confirmed that miR-122, 192, and 155 were all increased in the circulating exosome of the liver toxicity model. In addition, miR-7 decreased in the exosome of the brain toxicity model (Parkinson's model), miR-146a increased in the exosome of the renal toxicity model, miR-208 decreased in the exosome of the cardiac toxicity model, It was confirmed that miR-206 increased in toxic exosomes. These results suggest that exosomes derived from various organs-specific toxic conditions may affect the expression of circulating exosome miRNA as well as organ specific exosome proteins.

Example 5. Measurement of liver toxicity by NAC treatment

Changes in the amount of circulating exosomes were measured when NAC was treated to treat toxicity by acetaminophen. The results are shown in FIG.

As shown in FIG. 8, it was confirmed that the amount of circulating exosomes decreased in the liver toxicity state during NAC treatment.

In addition, the expression of liver-specific exo-somatic proteins albumin, haptoglobin and fibrinogen was measured in the case of treating hepatic toxicity of acetaminophen with NAC to treat liver toxicity. The results are shown in Fig.

As shown in FIG. 9, it was confirmed that the expression of albumin, haptoglobin and fibrinogen decreased in the liver toxicity by NAC treatment. This confirms that not only the amount of circulating exosome, but also the liver-specific exosome protein is restored when hepatic toxicity is treated.

Example 7. Measurement of Renal Toxicity by Quercetin Treatment

The change in the amount of circulating exosomes when treating the toxicity of quercetin with respect to the renal toxicity by cisplatin was measured and the results are shown in FIG.

As shown in FIG. 10, it was confirmed that the amount of circulating exosomes decreased during the quercetin treatment in the kidney toxicity state.

In addition, changes in circulating exosomes miR-122, miR-192, miR-155 and miR-146a were measured when NAC and quercetin were treated with long-term toxicity by acetaminophen and cisplatin, The results are shown in Fig.

As shown in FIG. 11, miR-122 and miR-192 were significantly restored in circulating exosomes after NAC treatment, and miR-155 and miR-146a were significantly higher in circulating exosomes after quercetin treatment .

Claims (14)

delete delete delete delete delete delete delete Wherein the biomarker composition comprises a biomarker composition containing an exosome and is at least one selected from the group consisting of RT-PCR kits, microarray chip kits, DNA kits, and protein chip kits. Kit for simultaneous diagnosis of brain toxicity. (a) separating the exosome from the biological sample; And
(b) confirming the amount of the exosome. The method for simultaneous diagnosis of liver, kidney, heart, muscle and brain toxicity. 10. The method according to claim 9, wherein the information providing method comprises the steps of (c) selecting an albumin, haptoglobin, fibrinogen, Kim-1 (kidney injury molecule 1), neutrophil gelatinase A group consisting of Neutrophil gelatinase-associated lipocalin (NGAL), cardiac troponin I (cTnI), skeletal muscle troponin (sTnI) and S100 calcium binding protein B (S100B) And determining the level of expression of the protein selected from the group consisting of: (1) the expression level of the protein selected from the group consisting of the liver, the kidney, the heart, the muscle and the brain. 10. The method according to claim 9, wherein the biological sample in step (a) is at least one selected from the group consisting of whole blood, serum, and plasma. Information delivery method. The method according to claim 9, wherein the amount of exosome in step (b) is determined by Western blotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, radioimmunodiffusion Ouchterlony immuno-electrophoresis, tissue immuno staining, immunoprecipitation assay, complete fixation assay, FACS (flow cytometry), and protein chip protein chip). The present invention provides a method for simultaneous diagnosis of toxicity to liver, kidney, heart, muscle and brain. delete delete

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