KR102242999B1 - Early liver cirrhosis diagnostic composition and early liver cirrhosis diagnostic method using same - Google Patents

Abstract

The present invention relates to a diagnostic composition for early liver cirrhosis, and a diagnostic method for early liver cirrhosis using the same. Specifically, the present invention relates to a composition for diagnosing early cirrhosis comprising a microtubule acetylation measuring agent, and a method for diagnosing early cirrhosis using the same.

Description

Early liver cirrhosis diagnostic composition and early liver cirrhosis diagnostic method using same}

The present invention relates to a composition for diagnosing early cirrhosis and a method for diagnosing early cirrhosis using the same, and more particularly, to a composition for diagnosing early cirrhosis including an agent for measuring microtubule acetylation, and a method for diagnosing early cirrhosis using the same.

Liver cirrhosis refers to extensive fibrosis that forms nodules in the liver, and is a disease in which persistent and repetitive diffuse hepatic damage and consequently fibrosis and regenerated nodules of hepatocytes are formed as end-stage lesions of chronic interstitial rings. The main causes of hepatitis include viruses, drugs, alcohol, metabolic diseases, chronic cholestasis, hepato-venous blood flow obstruction, and the like. Among them, hepatitis B increases the risk of chronic hepatitis or cirrhosis. In adults, about 10% of cases of acute hepatitis and 90% of cases of vertical infection from parents become chronic hepatitis B carriers. After that, in their 40s and 50s, many transition to chronic hepatitis or cirrhosis. Meanwhile, hepatitis C virus is also believed to be a major factor in chronic hepatitis and cirrhosis, and 16.6% of all liver cancer patients are positive for hepatitis C virus antibody. In addition, liver cirrhosis is observed in 60-90% of liver cancer patients, and the occurrence of liver cancer in 5-20% of liver cirrhosis patients suggests that cirrhosis is a major risk factor for liver cancer. Therefore, early detection of cirrhosis and removal of factors causing cirrhosis are required. In addition to viral factors, factors that can cause cirrhosis include alcohol consumption due to continuous binge drinking, drugs, and hemoglobinosis. Therefore, early discrimination and elimination of these diseases and risk factors, and prevention of vaccination and transmission are important.

The most reliable test for cirrhosis is laparoscopy or liver biopsy. Laparoscopy is a test that directly observes the liver by making a small hole in the stomach and inserting an endoscope. If you show, you can make a diagnosis with that. In addition, if findings such as liver fibrosis are observed in the biopsy, a diagnosis of cirrhosis can be made. However, these tests are not only very burdensome to the patient, but can usually be diagnosed by combining examination findings, blood tests, ultrasound or CT findings. In the case of cirrhosis, serum alanine aminotransferase (ALT) (conventional GOT, GPT) level, that is, hepatitis level is not very high, and is usually normal or within 2 times of normal, so it is not effectively applied for diagnosis. I can't. In the case of targeted cirrhosis, the levels of albumin and bilirubin do not deviate significantly from normal because there are enough liver cells to function properly, but in the case of decompensated cirrhosis, albumin decreases or bilirubin increases. Can be seen. In patients with liver cirrhosis or advanced chronic liver disease, it is important to know how many functional hepatocytes exist, and albumin or bilirubin is an index that gives a rough estimate of this. In addition, if there are not enough hepatocytes that function to make blood clotting factors in hepatocytes, they are not made enough and blood clotting may be delayed. A test called prothrombin time (PT) is a test that directly measures blood clotting time and is also one of the indicators for evaluating residual liver function. When cirrhosis occurs, the spleen becomes enlarged, and platelets are trapped in the enlarged spleen, resulting in a low platelet count on a general blood test. If platelets are degraded without knowing the cause, the possibility of cirrhosis should be suspected. As in chronic hepatitis, serological marker testing for hepatitis virus is also important. About 60% of liver cirrhosis in Korea is caused by hepatitis B virus, and about 20% is caused by hepatitis C virus. Therefore, if the hepatitis B or C virus marker is positive, it is highly likely to have chronic liver disease, and if there are other findings suggesting cirrhosis, it is not unreasonable to attach a diagnosis of cirrhosis clinically.

In addition, in general, liver fibrosis, unlike cirrhosis, is reversible, consists of thin fibril, and is known to have no nodule formation, and if the cause of liver damage is lost, normal recovery may be possible. If the liver fibrosis process continues repeatedly, crosslinking between ECMs (extra cellular matrix) increases, leading to irreversible cirrhosis with nodules.

Liver fibrosis progresses further and cirrhosis occurs, and the pathogenesis is liver cell regeneration and fibrous tissue proliferation when hepatocyte necrosis occurs due to some cause. Cirrhosis occurs when this process is repeated over a long period of time. Liver cirrhosis formed by hepatic nodules or nodules regenerated by persistent or repetitive diffuse hepatic parenchymal damage, fibrous tissue proliferation and hepatocyte regeneration is a chronic disease accompanied by necrosis, inflammation, and fibrosis pathologically. Ultimately, it leads to liver cancer and death. In particular, since there is no subjective symptom in the early stage, it is discovered only after a considerable progression, so studies on effective diagnosis and treatment are needed.

Republic of Korea Patent Publication No. 10-2004-0101633

Accordingly, the present inventors have completed the present invention by confirming that the initial cirrhosis can be effectively diagnosed using acetylation of microtubules as a result of intensive research efforts to develop a test method for effective early diagnosis of cirrhosis.

Accordingly, an object of the present invention is to provide a method for providing information for early cirrhosis diagnosis, including the following steps.

(a) obtaining a biological sample of the subject; And

(b) measuring the level of microtubule acetylation in the biological sample.

Another object of the present invention is to provide a composition for diagnosing early cirrhosis, including an agent for measuring microtubule acetylation.

Another object of the present invention is to provide a kit for diagnosing early cirrhosis, comprising the composition.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention, the present invention provides a method for providing information for initial cirrhosis diagnosis, including the following steps.

(a) obtaining a biological sample of the subject; And

(b) measuring the level of microtubule acetylation in the biological sample.

In an embodiment of the present invention, the microtubule acetylation may be measured by detecting acetylated α-tubulin, but is not limited thereto.

In another embodiment of the present invention, (c) when the microtubule acetylation level is higher than that of the control group, it may include predicting that it is an initial cirrhosis, but is not limited thereto.

In another embodiment of the present invention, the measurement is Western blot, ELISA, radioimmunoassay, radiation immunodiffusion, Ouchterlony immunodiffusion, Rocket immunoelectrophoresis, immunostaining, immunoprecipitation assay. , Complement fixation analysis, mass spectrometry, FACS, and may be measured by one or more methods selected from the group consisting of a protein chip, but is not limited thereto.

In addition, the present invention provides a composition for diagnosing early cirrhosis, including an agent for measuring microtubule acetylation.

In one embodiment of the present invention, the agent may be an antibody or an aptamer, but is not limited thereto.

In addition, the present invention provides a kit for early cirrhosis diagnosis comprising the composition.

In addition, the present invention provides a method for diagnosing early cirrhosis using a composition comprising an agent measuring microtubule acetylation.

In addition, the present invention provides a use of a composition comprising an agent for measuring microtubule acetylation in early cirrhosis diagnosis.

The present invention relates to a composition for diagnosing early cirrhosis and a method for diagnosing early cirrhosis using the same. By measuring microtubule acetylation, the present invention is expected to provide information on early cirrhosis, thereby enabling diagnosis in an early stage of liver cirrhosis progression.

1A shows α-SMA and acetylated α-tubulin expression in liver sections obtained from mice treated for 2 and 4 weeks with corn oil or CCl _{4 (0.5 ml kg/body weight, intraperitoneal injection, twice weekly).} As a result of performing immunohistochemical (IHC) analysis of, the graph shows the percentage of acetylated α-tubulin and α-SMA positive regions in each liver section. Scale bar: 200 μm.
1B shows α-SMA and acetylated α-tubulin expression in liver sections of mice subjected to combination treatment with SB431542 (10 mg kg ^-1 body weight, intraperitoneal injection) and CCl _{4 (each group n = 3), and thick} As a result of IHC analysis of picrosirius red stain for collagen expression, the result of quantifying α-SMA and acetylated α-tubulin-positive cells in the liver tissue of the mouse treated as described above is shown. . Statistical significance of the difference between each treatment group and the control treatment group (*** p <0.005). Scale bar: 200 μm.
1C shows Western blotting results for detection of acetylated α-tubulin, tatyrosine α-tubulin and α-SMA in liver tissue samples.
1D is a survival analysis using a Kaplan-Meier survival curve for the expression levels of Atat1 (probe ID; 79969) and Hdac6 (probe ID; 10013) in hepatocellular carcinoma (HCC) patients (n=364). It shows one result.

In an embodiment of the present invention, the present inventors have confirmed that the acetylation of microtubules is specifically increased in the initial cirrhosis stage, thereby completing the present invention.

Accordingly, the present invention provides a method for providing information for early cirrhosis diagnosis, including the following steps.

(a) obtaining a biological sample of the subject; And

(b) measuring the level of microtubule acetylation in the biological sample.

In an embodiment of the present invention, the microtubule acetylation may be measured by detecting acetylated α-tubulin, but is not limited thereto.

In another embodiment of the present invention, the measurement is Western blot, ELISA, radioimmunoassay, radiation immunodiffusion, Ouchterlony immunodiffusion, Rocket immunoelectrophoresis, immunostaining, immunoprecipitation assay. , Complement fixation analysis, mass spectrometry, FACS, and may be measured by one or more methods selected from the group consisting of a protein chip, but is not limited thereto.

In addition, the present invention provides a composition for diagnosing early cirrhosis, including an agent for measuring microtubule acetylation.

In one embodiment of the present invention, the agent may be an antibody or an aptamer, but is not limited thereto.

In addition, the present invention provides a kit for early cirrhosis diagnosis comprising the composition.

In the present invention, "acetylated α-tubulin" is a kind of protein constituting the primary cilia. For the purposes of the present invention, the acetylated tubulin is a protein contained in a sample of a subject (a patient with cirrhosis or hepatic fibrosis or a suspected patient), and by detecting the protein in a sample, the level of acetylation in microtubules is measured, and initial cirrhosis Can be diagnosed.

In the present invention, “liver cirrhosis” is the last stage of chronic liver disease, resulting in fibrous degeneration in which scars accumulate in the liver as a healing response to damage to hepatocytes. Liver cirrhosis refers to decreased liver function as fibrous degeneration progresses. The causes of cirrhosis are liver cirrhosis due to hepatitis A, liver cirrhosis due to chronic hepatitis B, liver cirrhosis due to hepatitis C, liver cirrhosis due to liver disease due to autoimmune diseases, liver cirrhosis due to non-alcoholic fatty liver. , Liver cirrhosis due to hemochromatosis due to iron metabolic disorder. Liver cirrhosis can be judged as a sign of portal hypertension if ascites or swelling of the lower extremities, or enlargement of the spleen are observed on physical examination or abdominal ultrasound examination, or esophageal varices are observed on endoscopy. It is very rare that a biopsy is performed for the diagnosis of cirrhosis. Among them, the main reason for causing fatty liver occurs due to excessive alcohol consumption for a long period of time, and appears to be high in frequency in patients with cirrhosis.

The present invention may be for diagnosis of “initial cirrhosis” among cirrhosis, but is not limited thereto.

In normal liver tissues, portal fibroblasts and hepatic stellate cells (HSCs) are at rest, and the level of α-SMA is quite low. Hepatitis virus and alcohol-induced inflammatory response of hepatocytes increases the secretion of transforming growth factor (TGF-β1) secreted from immune cells and activates portal fibroblasts and HSCs to express α-SMA protein. To induce differentiation into myofibroblasts. The term "initial cirrhosis" refers to a state in which cirrhosis has not progressed while being exposed to the cause of cirrhosis, that is, a step in which fibrosis of collagen begins to occur. Therefore, a very small amount of α-SMA protein-positive myofibroblasts were observed in tissues of early cirrhosis. Therefore, an increase in the expression level of α-SMA protein is a biological marker that can predict the onset of cirrhosis.

In the present invention, when the microtubule acetylation level is higher than that of the control group, it may be predicted to be early cirrhosis, and the control group may be a normal person or a subject having a low level of α-SMA.

In the present invention, "expression" means that a protein or nucleic acid is produced in a cell. 'Protein' is used interchangeably with'polypeptide' or'peptide' and, for example, refers to a polymer of amino acid residues as commonly found in proteins in a natural state. 'Polynucleotide' or'nucleic acid' refers to deoxyribonucleotides (DNA) or ribonucleotides (RNA) in the form of single- or double-stranded. Unless otherwise limited, known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to those of naturally occurring nucleotides are also included. 'mRNA' refers to RNA that transfers genetic information (gene-specific nucleotide sequence) to ribosomes that specify amino acid sequences from specific genes in the process of protein synthesis.

The term "detection" or "measurement" as used in the present invention means to quantify the concentration of a detected or measured object. In addition, the term "diagnosis" as used herein refers to determining the susceptibility of a subject to a specific disease or disease, determining whether the subject currently has a specific disease or disorder, or Or determining the prognosis of the diseased subject, or therametrics (eg, monitoring the condition of the subject to provide information on treatment efficacy).

The meaning of'increased expression (or high expression)' of a protein means that what was not expressed is expressed (i.e., that which has not been detected is detected) or that which is relatively overexpressed (i.e., that the amount of detection is increased) than the normal level. Means. The meaning of the opposite term thereof can be understood by those skilled in the art as having the opposite meaning according to the above definition.

Meanwhile, the agent for measuring the protein expression level in the present invention may be an antibody or aptamer that specifically binds to the protein.

In the present invention, "antibody" refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein, and includes all of polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. In addition, as long as it has antigen-antibody binding, a part of the whole antibody is also included in the antibody of the present invention, and all kinds of immunoglobulin antibodies that specifically bind to the protein of the present invention are included. For example, a complete antibody having two full-length light chains and two full-length heavy chains, as well as functional fragments of antibody molecules, i.e., Fab, F(ab'), F(ab') with antigen-binding function 2 and Fv, and the like. Further, the antibody of the present invention includes special antibodies such as humanized antibodies and chimeric antibodies, and recombinant antibodies as long as it can specifically bind to the protein of the present invention.

In the present invention, the term “aptamer” refers to a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) having a stable tertiary structure as a substance capable of specifically binding to an analyte to be detected in a sample. , Specifically, the presence of the target protein in the sample can be confirmed. The production of aptamer is synthesized by determining the sequence of an oligonucleotide having high binding ability and selective to the target protein to be identified according to a general aptamer production method, and then applying the 5'or 3'end of the oligonucleotide. In order to bind to the functional group of the timer chip, it may be _{made by modifying it with -SH, -COOH, -OH or NH 2} , but is not limited thereto.

The composition of the present invention containing the protein-specific antibody may additionally include an agent required for a method of detecting a known protein, and a subject ( In the present invention, it is possible to measure the expression level of a protein in a subject suspected of cirrhosis, a subject to be checked for cirrhosis, or a biological sample obtained from the subject).

The method of detecting the antibody of the method of the present invention can be any known method for detecting the presence of an antibody. Such methods may be heterogeneous or homogeneous, sequential or simultaneous, competitive or non-competitive. U.S. Patent Nos. 5,563,036, 5,627,080, 5,633,141, 5,679,525, 5,691,147, 5,698,411, 5,747,352, 5,811,526, 5,851,778 and 5,976,822 apply the format and application of several different assays. Explain. The detection method may be in a quantitative configuration for assaying the concentration or amount of an autoantibody, or a qualitative configuration for testing the presence or absence of an autoantibody.

In the detection of the present invention, more preferably, a secondary antibody conjugated with a detectable label (eg, anti-human IgG antibody) conjugate is added to the anti-microtubule acetylation of the secondary antibody conjugate and the immunocomplex of the present invention. After inducing binding between antibodies to form a sandwich-type antigen-antibody complex, a sandwich assay method may be used to identify a detectable label conjugated to a secondary antibody bound to the sandwich-type antigen-antibody complex.

Secondary antibodies include natural immunoglobulins isolated from non-human primates (eg, anti-human IgG murine antibody, anti-human IgG goat antibody, etc.) and recombinant, synthetically produced immunoglobulin fragments.

The label of the secondary antibody is preferably a conventional coloring agent that performs a color reaction, and HRP (Horseradish peroxidase), basic dephosphorylation enzyme (Alkaline phosphatase), colloid gold (Coloid gold), FITC (Poly L-lysine-fluorescein isothiocyanate) ), a fluorescent substance such as Rhodamine-B-isothiocyanate (RITC) and a label such as a dye (Dye) may be used.

The sandwich assay method is described in Schuurs et al. U.S. Patent Nos. 3,791,932 and 4,016,043, and Pankratz, et al., U.S. Patent No. 5,876,935, and identification of a detectable label bound to the secondary antibody is performed by a conventional ELISA (Enzyme-linked immunosorbent assay). ), RIA (Radioimmnoassay), Sandwich assay, Western blot on polyacrylamide gel, immunoblot analysis, or immunohistochemical staining.

The diagnostic kit of the present invention may include an antibody that recognizes a target protein as a marker or a primer, a probe that recognizes an mRNA as a marker, as well as one or more other constituent compositions, solutions, or devices suitable for an analysis method.

In order to confirm the presence or concentration of acetylated tubulin protein for confirming the acetylation of microtubules or microtubule acetylation, the sample was treated with a coloring agent or a detection agent, and then the initial liver cirrhosis diagnosis kit according to the present invention It is also possible to use a substrate.

The kit may include an agent that specifically detects acetylated tubulin for confirming acetylation of microtubules or acetylation of microtubules.

In the kit of the present invention, the detection agent (eg, antibody or fluorescent antibody) or color developer (eg, dye) may be accommodated or adsorbed on a substrate, and when the substrate is immersed in a urine sample, the substrate is dissolved and the detection agent is Alternatively, a coloring agent may be released into the sample to bind or react with an acetylated tubulin protein to confirm acetylation of microtubules or acetylation of microtubules, from which the microtubules are determined through the color, absorbance, or fluorescence level of the sample. The acetylated tubulin protein can be identified or quantified to confirm the acetylation of or microtubule acetylation.

The detection agent may also be included in a substrate for adsorbing or accommodating an acetylated tubulin protein for confirming acetylation or microtubule acetylation of microtubules isolated from the sample, or may exist separately from the adsorption or separation substrate.

The detection agent contained in the substrate specifically binds to an acetylated tubulin protein to confirm acetylation of microtubules or acetylation of microtubules, and acetylates microtubules without passing or reacting other substances in the urine sample. It can play a role of detecting.

In addition, such a detection agent may be an agent that measures the level of protein.

In the present invention, the term "an agent for measuring the level of a protein" means an agent used in a method of measuring the level of a target protein contained in a sample. Examples of this include western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immune diffusion, rocket immunoelectrophoresis. , Tissue immunostaining, immunoprecipitation assay (Immunoprecipitation Assay), complement fixation assay (Complement Fixation Assay), FACS and protein chip assay (protein chip assay), etc. It may be, but is not particularly limited thereto.

In addition, a secondary antibody capable of detecting the antibody or aptamer may be additionally included, and those labeled with a fluorescent substance or radioactive isotope may be used.

In the present invention, the term “analysis” may preferably mean “measurement”, the qualitative analysis may mean measuring and confirming the presence or absence of a target substance, and the quantitative analysis It may mean measuring and confirming a change in the level of presence (level of expression) or quantity. In the present invention, the analysis or measurement may be performed without limitation, including both a qualitative method and a quantitative method, and preferably, a quantitative measurement may be performed.

The biological sample can be used without limitation as long as it is collected from a subject for which initial cirrhosis is to be predicted.For example, cells or tissues obtained by biopsy, blood, whole blood, serum, plasma, saliva, cerebrospinal fluid, various secretions, urine, It may be feces, etc. Preferably, it may be to use a biological liquid sample (body fluid sample), for example tissue, cells, blood, plasma, serum, saliva, nasal fluid, sputum, joint capsule fluid, amniotic fluid, ascites, cervical secretion, vaginal secretion, urine And it may be to use cerebrospinal fluid. The present invention is of great technical significance in that it is possible to diagnose through bodily fluid samples (for example, plasma) other than the direct liver lesion tissue in which cirrhosis has occurred, and thus the effectiveness, rapidity, convenience, and safety of diagnosis are increased. In the present invention, the sample may most preferably be a tissue, particularly a liver tissue, and the tissue may be a formalin-fixed paraffin-embedded (FFPE) sample. The biological sample may be a gene sample, and the gene sample includes DNA, mRNA, or cDNA synthesized from mRNA.

The sample can be pretreated prior to use for detection or diagnosis. For example, it may include homogenization, filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like. The sample may be prepared to increase the detection sensitivity of the protein marker. For example, a serum sample obtained from a patient is anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, It can be pretreated using a method such as sequential extraction or gel electrophoresis.

The measurement of the protein expression level is not particularly limited as long as it is by a protein expression measurement method known in the art.For example, Western blot, ELISA, radioimmunoassay, radioimmune diffusion method, Ouchterlony immune diffusion method (Ouchterlony immunodiffusion), rocket immunoelectrophoresis, immunostaining, immunoprecipitation assay, complement fixation assay, mass spectrometry, FACS, and protein chip.

The gene expression level measurement is a group consisting of PCR, RNase protection assay, northern blotting, southern blotting, in situ hybridization, and DNA chip according to a gene expression measurement method known in the art. It may be one or more selected from.

Terms used in the present invention have selected general terms that are currently widely used as possible while taking functions of the present invention into consideration, but this may vary according to the intention or precedent of a technician working in the field, the emergence of new technologies, and the like. In addition, in certain cases, there are terms arbitrarily selected by the applicant, and in this case, the meaning of the terms will be described in detail in the description of the corresponding invention. Therefore, the terms used in the present invention should be defined based on the meaning of the term and the overall contents of the present invention, not a simple name of the term.

Throughout the specification of the present invention, when a certain part "includes" a certain constituent element, it means that other constituent elements may be further included rather than excluding other constituent elements unless otherwise stated. The terms "about", "substantially", etc. of the degree used throughout the specification of the present invention are used at or close to the numerical value when manufacturing and material tolerances specific to the stated meaning are presented, and the present invention To aid in understanding of, accurate or absolute figures are used to prevent unreasonable use of the stated disclosure by unscrupulous infringers.

In the entire specification of the present invention, the term "combination of these" included in the expression of the Makushi format refers to one or more mixtures or combinations selected from the group consisting of the constituent elements described in the expression of the Makushi format. It means to include at least one selected from the group consisting of constituent elements.

Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the examples.

Example 1. Experimental method

1. Antibodies and reagents

α-SMA (Sigma-Aldrich; #A5228), acetylated-α-tubulin (Cell Signaling Tech., Beverly, MA, USA; #5335), detyrosinated-α-tubulin (Millipore, Billerica, MA, USA, #MAB3201) , α-tubulin (Sigma-Aldrich, #T9026), GFP (Santa Cruz Biotech.; #sc-9996), pMLC (Ser 19; Abcam, Cambridge, MA, USA, #ab64162), YAP (Santa Cruz Biotech., #sc-101199), phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (Cell Signaling Tech.; #8828), Smad2/3 (Santa Cruz Biotech., #sc-133098) and dynein (Millipore, Billerica, MA, USA, #MAB1618). Antibodies against TGF-β1 (R&D systems, MN, USA; #240B) were used, as well as EHNA (Tocris Bioscience, UK, #1261) and nocodazole (Sigma-Aldrich, Taufkirchen, Germany, #M1404) as growth factors or inhibitors. ) Was used.

2. Animal models and procedures

Six-week-old male C57BL/6N mice were purchased from DBL (Chungcheongbuk-do, Republic of Korea). Mice were injected with control (corn oil) or CCl ₄ (0.5 ml kg-1 body weight, intraperitoneal injection) twice a week for 2 weeks or 4 weeks. SB431542 (10 mg kg-1 body weight, intraperitoneal injection) was injected into mice twice a week for 4 weeks 1 day before _{CCl 4 injection.} Mice were sacrificed on day 2 after the final CCl _{4 injection.} All procedures related to mice used in this study were approved by the Chung-Ang University Bioethics Committee (IRB).

3. Immunohistochemistry

Liver tissue obtained from mice was fixed with 4% paraformaldehyde and cryoprotected by increasing the concentration of sucrose solution (10, 20 and 30%) until the tissue settled. Subsequently, the tissue was embedded in an OCT compound (Tissue-Tek) and sectioned into 15 μm. Sections were ^{blocked with MOM TM} blocking solution and normal goat serum (Vector Biolaboratories, Brulingame, CA, USA) for 1 hour at room temperature. The tissue was then incubated with antibodies specific for α-SMA and Ac-α-tubulin. After removing the unbound primary antibody with PBS, the tissue was incubated with the secondary antibody in 2% normal goat serum for 1 hour at room temperature. Thereafter, the tissue was washed with PBS and mounted with Fluoromount (Southern Biotechnology Associates, Birmingham, AL, USA). Fluorescence-positive regions were analyzed using imaging software NIS-Elements advanced research (Nikon).

4. Western Blotting

Cells were treated with 1% Nonidet P-40 (NP-40), 1% sodium dodecyl sulfate (SDS), 150 mM NaCl, 6 mM Na ₂ HPO ₄ , 4 mM NaH ₂ PO ₄ , 2 mM ethylene diamine tetraacetic acid (EDTA). ), 50 mM NaF, 1 mM Na ₃ VO ₄ , 1 mM 1,4-dithiothreitol (DTT) and 1 mM phenyl methyl sulfonyl fluoride (PMSF) containing lysis buffer to lyse cells, binding protein The membrane with this was incubated at 4° C. for 12 to 16 hours with the indicated primary antibody. Subsequently, the membrane was further incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, PA, USA) for 1-2 hours at RT. Signal enhanced chemiluminescence (Bio-Rad, Hercules, CA, USA) reagent was used, and the band density was measured using Quantity One® system (Bio-Rad).

5. Statistical analysis

For comparison between three or more groups, statistical analysis was performed by one-way ANOVA, and PRISM 8 software was used. To determine the significance of one-way variance analysis, the sum of squares between groups and the sum of squares within groups are divided by the respective degrees of freedom. The sum of squares of the mean (Mean square / MSA, MSW) was calculated. Thereafter, the ratio of MSA/MSW was calculated to calculate the F value, indicating significance in the data with a P value of 0.05 or less. Statistical results were post-tested using Tukey's method. The data shown in each graph were expressed as the total mean (SD) ± standard error of three or more independent experimental values unless otherwise specified, and a p-value less than 0.05 was considered statistically significant.

Example 2. Microtubule Acetylation number CCl ₄ Confirmation of the effect on the progression of induced liver fibrosis

_{After intraperitoneal (ip) injection of CCl 4} into mice for 2 weeks, the amount of acetylated microtubules in the liver tissue of _{CCl 4} treated mice significantly increased compared to the liver of control (corn oil) treated mice. Confirmed.

As shown in Fig. 1a, vascular smooth muscle cells (VSMC), which are cells that control the elasticity and contractile force of the blood vessel wall, have a green color (α-SMA), which can be observed in the inner wall of blood vessels of normal liver tissue. , It was confirmed that the expression of α-SMA was not increased in the mice treated with carbon tetrachloride for 2 weeks except around the blood vessels. However, even at week 2, the number of acetylated α-tubulin-positive cells significantly increased, and it was confirmed that these cells were dispersed in a radial arrangement.

In addition, as shown in FIG. 1B, it _{was confirmed that this CCl 4} -induced increase in acetylated microtubules disappeared by treatment with SB431542, a TGF-β receptor (TGFR) specific inhibitor.

In addition, as shown in Figure 1c, as a result of Western blotting using the sample, it was confirmed that the expression of acetylated microtubules and α-SMA up-regulated by _{CCl 4 was reduced in mice treated with the TGFR inhibitor SB431542.} , This suggests that TGF-β1 is a potent agent that promotes the formation of acetylated microtubules in early hepatic fibrosis.

That is, it was confirmed that the specific formation of acetylated microtubules was promoted in the early stages of liver fibrosis.

Hepatocellular carcinoma (HCC) patients have cirrhosis, which is confirmed to be caused by chronic liver disease for a long time. Accordingly, the present inventors further confirmed the relationship between the survival rate and microtubule acetylation in 364 hepatocellular carcinoma patients with cirrhosis.

As shown in Figure 1d, the results of performing a survival analysis according to the expression level of Atat1 (probe ID; 79969) and Hdac6 (probe ID; 10013) in hepatocellular carcinoma (HCC) patients (n=364), hepatocytes It was confirmed that there was a clinical relationship between a high level of microtubule acetylation and a low survival rate in patients with carcinoma.

As described above, by confirming that the acetylation of microtubules is increased in the early stages of liver fibrosis, it is possible to provide information necessary for diagnosing liver fibrosis, that is, the initial stages of cirrhosis. In addition, it is manufactured with a kit provided by the present inventors and can be used to quickly diagnose the initial stage of cirrhosis from a patient sample.

The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that other specific forms can be easily modified without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and are not limiting.

Claims (8)

(a) obtaining a biological sample of the subject;
(b) measuring the level of microtubule acetylation in the biological sample; And
(c) When the microtubule acetylation level is higher than that of the control group, an information providing method for diagnosing early cirrhosis comprising the step of predicting that it is an initial cirrhosis
The biological sample is a cell or tissue,
The cirrhosis is characterized in that excluding cirrhosis caused by alcohol, information providing method for initial cirrhosis diagnosis.
The method of claim 1,
The microtubule acetylation is characterized by being measured by detecting acetylated α-tubulin. Method for providing information for early cirrhosis diagnosis.
delete The method of claim 1,
The measurements were Western blot, ELISA, radioimmunoassay, radioimmunoassay, Ouchterlony immunodiffusion, Rocket immunoelectrophoresis, immunostaining, immunoprecipitation assay, complement fixation assay, mass spectrometry ( Mass spectrometry), FACS and protein chip, characterized in that measured by one or more selected from the group consisting of, information providing method for early cirrhosis diagnosis.
As a composition for diagnosing early cirrhosis, including an agent for measuring microtubule acetylation, but taking cells or tissues as a sample,
The cirrhosis is characterized in that excluding cirrhosis caused by alcohol, the composition for early cirrhosis diagnosis.
The method of claim 5,
The microtubule acetylation is characterized in that measured by detecting acetylated α-tubulin, the composition for early cirrhosis diagnosis.
The method of claim 5,
The formulation is characterized in that the antibody or aptamer, the composition for early cirrhosis diagnosis.
Cells or tissues as a sample, but comprising the composition of claim 5, as a kit for early cirrhosis diagnosis,
The cirrhosis kit, characterized in that excluding cirrhosis caused by alcohol.

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