KR20170074034A - CXCL14 Biomarker for Diagnosing Liver Fibrosis - Google Patents

Abstract

The present invention relates to a biomarker for diagnosing liver fibrosis or liver cirrhosis and a use thereof. More particularly, the present invention relates to a biomarker for diagnosing liver fibrosis or liver cirrhosis, and more particularly to a method for measuring the expression level of mRNA of CXCL 14 (CXC motif) ligand 14) Compositions and kits for diagnosing liver fibrosis or liver cirrhosis. By using the composition of the present invention as a biomarker, it is possible to diagnose liver fibrosis or liver cirrhosis by a noninvasive method, and it is possible to diagnose liver fibrosis or liver cirrhosis patients more easily, promptly and accurately.

Description

Biomarker for Diagnosis of Liver Fibrosis CXCL14 {CXCL14 Biomarker for Diagnosing Liver Fibrosis}

The present invention relates to a biomarker for diagnosing liver fibrosis or liver cirrhosis and a use thereof. More particularly, the present invention relates to a biomarker for diagnosing liver fibrosis or liver cirrhosis, and more particularly to a method for measuring the expression level of mRNA of CXCL 14 (CXC motif) ligand 14) Compositions and kits for diagnosing liver fibrosis or liver cirrhosis.

Liver fibrosis is a state of toxic substances or a part of biochemical adaptation that accompanies various infectious, immune, and metabolic diseases. It is a state in which damaged liver tissue is not restored normally but collagen accumulation occurs like fibrous tissue. The major component of the accumulation is collagen, especially type I collagen (Olaso E et al., Molecular regulation of hepatic fibrogenesis, etc.), which is characterized by excessive accumulation of extracellular matrix collagen, fibronectin and laminin and proteoglycans, , ≪ / RTI > J Hepatol 29: 836-847-, 1998). This liver fibrosis is an inevitable hepatic pathologic consequence of chronic liver injury. It is a liver tissue that is replaced by a fibrous tissue that can not perform its inherent functions, such as biomass metabolism or bile secretion, ) Inevitably appears. Liver fibrosis is reversible, composed of thin fibrils and known to be free of nodule formation, and may be able to recover to normal if the cause of liver damage is lost. However, if the fibrosis process continues repeatedly, the exchange and binding between the extracellular matrix (ECM) may increase, leading to irreversible liver cirrhosis resulting in thick fibrils and regenerative nodules.

It has also been reported that liver fibrosis and liver cirrhosis are important risk factors for liver cancer, with about 80% of liver cancer occurring after progression to liver cirrhosis and a 6-fold higher incidence of cancer depending on fibrosis progression . Thus, hepatic fibrosis is the root cause of complications that frequently occur in hepatic dysfunction and end-stage liver disease, further increasing the risk of developing liver cancer.

As described above, according to the progress of hepatic fibrosis or liver cirrhosis in relation to liver damage, the diagnosis of the disease severity and prognosis changes and plays an important role in the prevention of liver cancer. Therefore, Diagnosis is very important.

Currently, histologic examination of liver biopsy specimens is used as a standard method for diagnosing liver fibrosis or liver cirrhosis, which has the disadvantage of invasive methods. There are several limitations to performing biopsy in patients who continue to increase in liver disease. Liver biopsy has side effects that can lead to pain, bleeding, and, in rare cases, death, It is not appropriate to monitor changes in liver fibrosis by biopsy in patients who are receiving it.

Therefore, it is urgent to develop a reliable, institution-specific diagnostic method for liver fibrosis or liver cirrhosis that can replace invasive liver biopsy.

Under these circumstances, the present inventors measured the expression level of CXCL14 in mouse and liver cirrhosis-inducing mice induced by hepatic fibrosis and found that mRNA of CXCL14 gene or its protein is used as a biomarker for diagnosing hepatic fibrosis or liver hardening non-invasively The present invention has been completed.

SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide a novel biomarker for non-invasively diagnosing liver fibrosis or liver cirrhosis.

More specifically, it is an object of the present invention to provide a composition for diagnosing liver fibrosis or liver cirrhosis comprising an agent for measuring the expression level of mRNA of CXCL14 gene or a protein thereof.

It is another object of the present invention to provide a kit for diagnosing liver fibrosis or liver cirrhosis comprising the composition of the present invention.

It is another object of the present invention to provide a method for detecting liver fibrosis or liver cirrhosis diagnostic marker CXCL14 from a patient's sample in order to provide information necessary for diagnosis of liver fibrosis or liver cirrhosis.

To achieve the above object, one aspect of the present invention provides a composition for diagnosing liver fibrosis or liver cirrhosis comprising an agent for measuring the expression level of mRNA of CXCL 14 (CXC motif) ligand 14) gene or a protein thereof .

Preferably, the agent for measuring the CXCL14 protein may be an antibody specific for the CXCL14 protein.

More preferably, the antibody may be a monoclonal antibody or a polyclonal antibody specific for the CXCL14 protein.

Preferably, the agent for measuring the mRNA expression level of the CXCL14 gene may be an antisense oligonucleotide complementary to the mRNA of the CXCL14 gene, a primer or a probe.

In another aspect of the present invention, there is provided a kit for liver fibrosis or liver cure diagnosis comprising the composition of the present invention.

In another aspect of the present invention, there is provided a method for detecting liver fibrosis or hepatocyte diagnostic marker CXCL14 from a sample of a patient to provide information necessary for diagnosis of liver fibrosis or liver cirrhosis.

Preferably, the sample may be selected from the group consisting of the patient's tissues, cells, blood, serum, plasma, saliva, and urine.

Preferably, the method further comprises the step of measuring the expression level of the mRNA of the CXCL14 gene or a protein thereof from the sample of the patient, and the degree of expression of the mRNA of the CXCL14 gene or the expression of the mRNA of the CXCL14 gene or the mRNA of the CXCL14 gene in the normal control sample With the degree of expression of the protein.

Also, preferably, the method for measuring the expression level of the mRNA of the CXCL14 gene may be a reverse transcription polymerase chain reaction, a competitive reverse transcription polymerase chain reaction, a real-time reverse transcription polymerase chain reaction, an RNase protection assay, a Northern blot or a DNA chip have.

Preferably, the method for measuring the expression level of the CXCL14 protein includes Western blotting, ELISA, radioimmunoassay, radioimmunoprecipitation, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation, Assay, complement fixation assay, FACS (fluorescence activated cell sorter) or protein chip.

As another embodiment of the present invention, there is provided a method for screening a therapeutic agent for liver fibrosis or liver cirrhosis comprising the step of treating a candidate agent for hepatic fibrosis or liver curing treatment and then detecting a change in the expression level of mRNA of the CXCL14 gene or a protein thereof to provide.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a composition for diagnosing liver fibrosis or liver cirrhosis by measuring the expression level of mRNA of CXCL14 gene or a protein thereof from a patient sample based on the decrease in the expression of CXCL14 in a liver fibrosis model and a liver cirrhosis patient, And methods.

In a specific example of the present invention, the expression level of CXCL14 gene was measured and compared in hepatic fibrosis patients and normal liver tissues or serum samples in order to examine whether the expression level of CXCL14 gene is decreased in patients with liver fibrosis. As a result, CXCL14 expression was significantly decreased in liver fibrosis patients compared with normal subjects, and CXCL14 expression was significantly decreased with progression of liver fibrosis in patients with liver fibrosis. Therefore, the CXCL14 gene or the CXCL14 protein can be used as a biomarker for the diagnosis of liver fibrosis or liver cirrhosis, and the CXCL14 gene or the CXCL14 protein can be used as a biomarker for diagnosing the degree of fibrosis progression (fibrosis grade) Proved to be available.

As used herein, the term "diagnosis " means identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to ascertain whether liver fibrosis or liver cirrhosis is present. Specifically, it can be confirmed that the CXCL14 gene or the CXCL14 protein is less expressed in the patient's sample than in the normal control to confirm liver fibrosis or liver cirrhosis. In addition, the CXCL14 gene or the CXCL14 protein can be detected in the sample of chronic hepatitis patients And whether or not liver fibrosis or liver hardening is progressing.

The CXCL14 gene is known to be involved in homeostasis as a gene involved in inflammatory reactions (immunoregulatory and inflammatory processes). The CXCL14 gene and its protein information can be found in the National Center for Biotechnology Information (NCBI). More specifically, the gene sequence of mouse CXCL14 is registered as NM_019568.2, and the protein sequence is registered as NP_062514.2. The gene sequence of human CXCL14 is NM_004887.4, and the protein sequence is registered as NP_004878.2. However, the function of the protein expressed by the CXCL14 gene has not yet been elucidated. In particular, the relationship between CXCL14 and liver fibrosis or liver cirrhosis is not known at all.

The degree of expression of CXCL14 can be determined by confirming the expression level of the protein encoded by the mRNA or gene of the CXCL14 gene.

In the present invention, "measurement of mRNA expression level of CXCL14 gene" is a process of confirming the presence and expression level of CXCL14 gene in a biological sample in order to diagnose hepatic fibrosis or liver cirrhosis. By measuring the amount of mRNA of CXCL14 gene, have. RT-PCR (Real-time RT-PCR), RNase protection assay (RT-PCR), and RT-PCR (Reverse Transcription Polymerase Chain Reaction) But are not limited to, RPA (RNase protection assay), Northern blot, and DNA chip.

The agent for measuring the degree of mRNA expression of the CXCL14 gene means a substance that can be used for detecting the mRNA of the CXCL14 gene in a sample of a patient. For example, it may be a primer, a probe, an antisense oligonucleotide, or the like that can complementarily bind to the mRNA of the CXCL14 gene. Preferably, the antisense nucleotide, primer or probe specifically binds to the mRNA nucleotide sequence of the CXCL14 gene and does not specifically bind to the nucleotide sequence of the other nucleic acid material.

Herein, complementary binding means that the antisense oligonucleotide is sufficiently complementary to hybridize selectively to the mRNA target of the CXCL14 gene under a predetermined hybridization or annealing condition, preferably physiological conditions, Quot; means " including both substantially complementary and perfectly complementary, and is preferably completely complementary.

For example, the agent used to detect the mRNA biomarker of the CXCL14 gene of the present invention may be an antisense oligonucleotide. The term "antisense oligonucleotide" means a nucleic acid-based molecule having a complementary sequence to the mRNA sequence of the target CXCL14 gene and capable of forming a duplex with the mRNA of the CXCL14 gene. Can be used to detect the marker.

As another example, the agent used to detect the mRNA biomarker of the CXCL14 gene of the present invention is a primer pair or a probe, and the base sequence of the CXCL14 gene is known. A primer or a probe that specifically amplifies a specific region can be designed.

The term "primer" refers to a nucleic acid sequence having a short free 3 'hydroxyl group, capable of forming a template pair with a complementary template and serving as a starting point for template strand copying ≪ RTI ID = 0.0 > 7 < / RTI > Primers are usually synthesized but can also be used in naturally occurring nucleic acids. The sequence of the primer does not necessarily have to be exactly the same as the sequence of the template, but is sufficiently complementary that it can hybridize with the template. The primers can be prepared by polymerisation (i. E., DNA synthesis in the presence of a reagent for DNA polymerase or reverse transcriptase and four different nucleoside triphosphates) at the appropriate buffer solution and temperature. can do. In the present invention, PCR amplification is performed using a sense of CXCL14 nucleotide sequence and an antisense primer to diagnose liver fibrosis or liver cirrhosis. The PCR conditions, the lengths of the sense and antisense primers can be modified based on what is known in the art. Preferably, the primer of the present invention can be a primer capable of amplifying the mRNA of the CXCL14 gene.

In another example, the agent used to detect the mRNA biomarker of the CXCL14 gene of the present invention may be a probe. The term "probe" means a nucleic acid fragment such as RNA or DNA corresponding to a short period of a few nucleotides or a few hundred nucleotides that can specifically bind to mRNA, and is labeled to confirm the presence or absence of a specific mRNA . The probe may be prepared in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, or an RNA probe. In the present invention, hybridization using a probe complementary to the CXCL14 polynucleotide can be used to diagnose liver fibrosis or liver cirrhosis through hybridization. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art.

The primers or probes of the present invention can be chemically synthesized using a phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may incorporate additional features that do not alter the basic properties. Examples of additional features that may be incorporated include, but are not limited to, methylation, capping, substitution of one or more nucleic acids with homologues, and modification between nucleic acids.

In the present invention, "measurement of protein expression level" is a process of confirming the presence and expression level of a protein expressed in a CXCL14 gene in a biological sample in order to diagnose liver fibrosis or liver cirrhosis. The amount of protein can be confirmed by using an antibody. Analysis methods include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, and rocket but are not limited to, immunoassays, rocket immunoelectrophoresis, tissue immuno staining, immunoprecipitation assays, complement fixation assays, fluorescence activated cell sorters (FACS), and protein chips.

The agent for measuring the CXCL14 protein level herein is preferably an antibody. "Antibody" means a specific protein molecule, as is known in the art, directed against an antigenic site. For the purpose of the present invention, an antibody refers to an antibody that specifically binds to a CXCL14 protein, which is a marker of the present invention. The CXCL14 gene is cloned into an expression vector according to a conventional method, To obtain the CXCL14 protein, which can be prepared from the obtained CXCL14 protein by conventional methods. Also included are partial peptides that can be made from the CXCL14 protein, and the partial peptides of the invention include at least 7 amino acids, preferably 9 amino acids, and more preferably 12 amino acids. The form of the antibody of the present invention is not particularly limited, and a polyclonal antibody, a monoclonal antibody or a part thereof having antigen binding ability is included in the antibody of the present invention and includes all immunoglobulin antibodies. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies.

Antibodies used in the detection of liver fibrosis or liver cirrhosis diagnostic markers of the present invention are not limited to complete forms having two full length light chains and two full length heavy chains as well as functional fragments of antibody molecules . A functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

The present invention also provides a composition for diagnosing liver fibrosis or liver cirrhosis, which comprises a preparation for measuring the expression level of mRNA of CXCL14 gene or its protein, in the form of a kit.

The kit of the present invention can confirm the expression level of mRNA or CXCL14 protein of CXCL14 gene which is a marker of liver fibrosis or liver cirrhosis. The kit of the present invention may further comprise one or more other component compositions suitable for the assay, as well as antibodies recognizing primers, probes, antisense oligonucleotides or optionally CXCL14 proteins for measuring the degree of expression of the CXCL14 gene mRNA or CXCL14 protein , Solutions or devices.

As a specific example, the kit for measuring the degree of mRNA expression of the CXCL14 gene in the present invention may be a kit containing essential elements necessary for conducting RT-PCR. The RT-PCR kit contains, in addition to each primer pair specific for the mRNA of the CXCL14 gene, a test tube or other suitable container, reaction buffer (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), Taq polymer Enzymes such as lyase and reverse transcriptase, DNase, RNAse inhibitor, DEPC-water, sterile water, and the like. It may also contain a primer pair specific to the gene used as a quantitative control. Also preferably, the kit of the present invention may be a diagnostic kit containing essential elements necessary for performing a DNA chip (DNA chip). The DNA chip kit may include a substrate on which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, a reagent for preparing a fluorescent-labeled probe, a preparation, an enzyme, and the like. In addition, the substrate may contain a cDNA corresponding to a quantitative control gene or a fragment thereof.

As another specific example, in the present invention, a kit for measuring the degree of expression of CXCL14 protein includes a substrate, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, a chromogenic substrate, etc. for immunological detection of the antibody can do. 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 may be used as the fluorescent substance and FITC or RITC may be used as the fluorescent substance. The coloring substrate solution may be ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) o-phenylenediamine), TMB (3,3 ', 5,5'-tetramethylbenzidine) may be used.

In another aspect, the present invention provides a method for detecting liver fibrosis or liver cirrhosis diagnostic marker CXCL14 from a patient's sample in order to provide information necessary for diagnosis of liver fibrosis or liver cirrhosis.

More specifically, the degree of expression of the CXCL14 gene can be detected at the mRNA level or the protein level, and the mRNA or protein can be separated from the patient's sample using a known process.

The term "patient sample" in the present invention includes, but is not limited to, tissues, cells, blood, serum, plasma, saliva, urine or the like in which the degree of expression of CXCL14, an liver fibrosis or hepatocyte marker gene, .

Through these detection methods, the degree of expression of CXCL14 in patients suspected to have liver fibrosis or liver cirrhosis can be compared with the level of expression of CXCL14 in the normal control group to diagnose the actual liver fibrosis or liver cirrhosis of the patient. CXCL14 expression was measured in patients who were presumed to be caused by liver fibrosis or liver cirrhosis, and the level of CXCL14 expression was measured in normal controls. The expression levels of CXCL14 were measured in liver fibrosis or liver cirrhosis Less predicted expression in presumptive patients can lead to the diagnosis of liver fibrosis or liver cirrhosis by liver fibrosis or liver cirrhosis.

Alternatively, in order to determine whether a patient with chronic hepatitis developed due to liver cirrhosis, a change in the expression level of CXCL14 from a sample of chronic hepatitis patients was measured, and when a significant decrease in CXCL14 expression was measured, the corresponding chronic hepatitis patient developed liver cirrhosis .

Examples of an assay method for measuring the mRNA expression level of the CXCL14 gene include a reverse transcription polymerase chain reaction, a competitive reverse transcription polymerase chain reaction, a real-time reverse transcription polymerase chain reaction, an RNase protection assay, a Northern blot or a DNA chip, no. Through these detection methods, it is possible to compare the degree of mRNA expression of CXCL14 gene in the normal control group and the degree of mRNA expression of the CXCL14 gene in patients suspected of having liver fibrosis or hepatitis, and whether the expression level of the CXCL14 gene is significantly decreased The liver fibrosis or liver cirrhosis of patients suspected to have liver fibrosis or liver cirrhosis can be diagnosed.

The mRNA expression level of the CXCL14 gene is preferably determined using a reverse transcriptase polymerase reaction method or a DNA chip using a primer specific to the mRNA of the CXCL14 gene, which is an liver fibrosis or hepatocyte marker.

The reverse transcription polymerase chain reaction was followed by electrophoresis to determine the band pattern and band thickness, and the presence or absence of mRNA expression of the CXCL14 gene was verified and compared with that of the control group. Thus, non-invasive liver fibrosis or liver cirrhosis It is easy to diagnose.

Analytical methods for measuring protein levels include Western blotting, ELISA, radioimmunoassay, radioimmunoassay, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immuno staining, immunoprecipitation assays, complement fixation Assay methods, FACS or protein chips, but are not limited thereto. Through these analytical methods, it is possible to compare the amount of antigen-antibody complex formed in the normal control group with the amount of antigen-antibody complex formed in patients with suspected hepatic fibrosis or hepatic cirrhosis, and whether or not a significant expression amount of CXCL14 protein is decreased , It is possible to diagnose whether hepatic fibrosis or liver cirrhosis is actually suspected in patients with liver fibrosis or liver cirrhosis.

As used herein, the term "antigen-antibody complex" refers to a combination of an antibody specific for CXCL14 protein, which is a hepatic fibrosis or hepatocyte marker, and an amount of the antigen- antibody complex formed through the size of a signal of a detection label It is measurable quantitatively.

Such detection labels may be selected from the group consisting of enzymes, minerals, ligands, emitters, microparticles, redox molecules, and radioisotopes, but are not necessarily limited thereto. When an enzyme is used as the detection label, available enzymes include? -Glucuronidase,? -D-glucosidase,? -D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholine Glucoamylase, terazo, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructoketase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphoenolpyruvate decar ≪ / RTI > beta-lactamase, and the like. The minerals include, but are not limited to, fluorescein, isothiocyanate, rhodamine, picoeriterine, picocyanin, allophycocyanin, o-phthaldehyde, fluororescamine and the like. Ligands include, but are not limited to, biotin derivatives. Emitters include, but are not limited to, acridinium esters, luciferin, luciferase, and the like. Fine particles include, but are not limited to, colloidal gold, colored latex, and the like. Redox molecules include ferrocene, ruthenium complex compounds, Biology hydrogen, quinone, Ti ions, Cs ions, diimide, 1,4-benzoquinone, _{hydroquinone, K 4 W (CN) 8} , [Os (bpy) 3] 2 + , [RU (bpy) ₃ ] 2 ⁺ , [MO (CN) ₈ ] 4 ^-, and the like. Radioisotope includes include ^{3 H, 14 C, 32 P} , 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, 186 Re are not limited to .

In one embodiment, the degree of protein expression is measured by ELISA. ELISAs include direct ELISA using labeled antibodies that recognize the antigen attached to the solid support, indirect ELISA using labeled antibodies that recognize the capture antibody in a complex of antibodies recognizing the antigen attached to the solid support, A direct sandwich ELISA using another labeled antibody that recognizes an antigen in the complex of antibody and antigen, a method of reacting with another antibody recognizing an antigen in a complex of an antibody and an antigen attached to a solid support, Indirect sandwich ELISA using a secondary antibody, and various ELISA methods. More preferably, the antibody is attached to a solid support, the sample is reacted, and the labeled antibody recognizing the antigen of the antigen-antibody complex is adhered to produce an enzyme, or an antibody that recognizes the antigen of the antigen-antibody complex Is detected by a sandwich ELISA method in which a labeled secondary antibody is attached and the enzyme is developed. Confirmation of complex formation of CXCL14 protein, which is an hepatic fibrosis or hepatocyte marker, and antibody can confirm the onset of liver fibrosis or liver cirrhosis.

Preferably, Western blotting is used with one or more antibodies against the CXCL14 protein. The whole protein is separated from the sample, and the protein is separated according to size by electrophoresis, and then transferred to the nitrocellulose membrane to react with the antibody. The amount of CXCL14 protein produced by expression of the gene can be confirmed by confirming the amount of the generated antigen-antibody complex using the labeled antibody to confirm whether or not liver fibrosis or liver hardening has occurred. The detection method is performed by examining the amount of CXCL14 gene expression in the control group and the amount of expression of the CXCL14 gene in a patient suspected of liver fibrosis or liver cirrhosis. The mRNA or protein level of the CXCL14 gene can be expressed as the absolute (e.g., / / ml) or relative (e.g., the relative intensity of the signal) difference of the CXCL14 protein.

Preferably, one or more antibodies against the CXCL14 protein is arranged at a predetermined position on the substrate, and the protein chip is immobilized at a high density. A method of analyzing a sample using a protein chip is a method of separating a protein from a sample, hybridizing the separated protein with a protein chip to form an antigen-antibody complex, reading the protein, Liver fibrosis or liver cirrhosis.

In another embodiment, the present invention provides a method of treating liver fibrosis or liver cirrhosis, comprising administering to a subject an amount of CXCL14 gene that is expected to treat liver fibrosis or liver cirrhosis, or the level of expression of CXCL14 protein encoded by the CXCL14 gene, Or a method for screening a therapeutic agent for liver cirrhosis.

Specifically, it can be useful for screening therapeutic agents for hepatic fibrosis or liver cirrhosis by comparing the increase or decrease in the expression of CXCL14 in the presence or absence of candidate agents for hepatic fibrosis or hepatocyte treatment. Substances that indirectly or directly reduce the concentration of CXCL14 can be selected as agents for the treatment of liver fibrosis or liver cirrhosis. In other words, the degree of expression of CXCL14 in liver fibrosis or hepatocyte cells in the absence of candidate substances for hepatic fibrosis or hepatic cirrhosis is measured, and the degree of expression of the marker CXCL14 of the present invention in the presence of a candidate agent for hepatic fibrosis or hepatic cirrhosis , And then comparing the quantities of CXCL14 expression in the presence of candidates for hepatic fibrosis or hepatic cirrhosis treatment to the level of CXCL14 expression in the absence of candidates for hepatic fibrosis or hepatic cirrhosis. It can be predicted as a remedy.

The present invention provides a technique for diagnosing liver fibrosis or liver cirrhosis using a non-invasive method using the mRNA of the CXCL14 gene or a protein thereof as a biomarker, and it can be used to diagnose liver fibrosis or liver cirrhosis more easily, Diagnosis is possible.

FIG. 1 shows the results of confirming the change of mRNA expression level of CXCL14 gene according to progress of liver fibrosis in hepatic fibrosis patient and normal liver tissue.
FIG. 2 shows the results of ELISA for the change of CXCL14 protein expression level according to progress of liver fibrosis in the serum of patients with liver fibrosis and normal persons.
FIG. 3 shows the results of ELISA analysis of changes in the level of CXCL14 protein expression in serum of patients by grading (Mild, Advanced) according to degree of fibrosis in patients with liver fibrosis.
FIG. 4 shows the results of confirming the expression of CXCL14 protein according to the degree of portal inflammation in patients with liver fibrosis.
FIG. 5 shows the results of confirming the expression of CXCL14 protein according to the degree of piecemeal necrosis in patients with liver fibrosis.

Hereinafter, preferred embodiments of the present invention will be described in detail so that those skilled in the art can easily carry out the present invention. The embodiments of the present invention can be modified into various other forms, and the scope of the present invention is not limited to the embodiments described below.

Example 1. A patient sample

In this experiment, liver tissue of 15 patients with liver fibrosis (F1, F3, F4) and 5 without fibrosis (F0) were received at Seoul National University Hospital of the Catholic University of Korea and used in this experiment. According to the degree of fibrosis F0, F1, F3, and F4 patients, respectively, were used.

To test the expression of CXCL14 in a larger number of samples, sera from 78 chronic hepatitis B patients (56 males and 22 females) were used in the study. There were 5 stage 0, 36 stage 1, 29 stage 3, and 8 stage 4 patients with chronic hepatitis B who received serum. .

Example 2. Selection of candidate genes for liver fibrosis biomarker

Hepatocytes were isolated using a collagen two-step perfusion method in the DEN-induced rat model, and RNA was extracted from the isolated hepatocytes using TRISOL. The extracted RNA was subjected to RNA quality control using a Bianalyer, and only RNAs with a RIN (RNA integrity number) value of 8.0 or higher were used in the microarray. Rat gene expression A 4x44k microarray kit was used to perform a microarray to obtain a differentially expressed gene. Through the literature review, possible genes as biomarkers for diagnosis of liver fibrosis were selected. Among them, the expression of CXCL14 gene I have studied in more detail.

Example 3. Measurement of mRNA expression level in liver tissue of CXCL14 gene

In order to confirm the expression level of CXCL14 gene in liver fibrosis patients, eleven patients (stage 1, stage 3, stage 4) and 5 patients without fibrosis (stage 0) Changes in mRNA expression were measured according to degree of fibrosis. Specifically, RNA was extracted using TRIZOL, and then RNA was synthesized by using ImProm II RTase. Based on the synthesized cDNA, the expression level of CXCL14 was measured by PCR with primers targeting CXCL14 (CXCL14 Forward: 5'-GCGCAGGGTCTACGAAGAAT-3 ', CXCL14 Reverse: 5'-CAGGACCTCTAAGCGGAAGC-3'). The results of the PCR are shown in FIG. 1A, and the band density of the PCR was measured and converted into a graph, which is shown in FIG. 1B for comparison and analysis.

As a result, it was confirmed that the expression of CXCL14 mRNA decreased as the degree of liver fibrosis increased as shown in Fig.

Example 4. Measurement of Protein Expression Level in Serum of CXCL14 Gene (ELISA)

Serum samples from 43 patients with hepatic fibrosis and 5 without fibrosis (Stage 0: 5, 1:20, 3:18, 4: 5) were tested for the expression of CXCL14 protein in patients with liver fibrosis The expression of CXCL14 protein was measured by the degree of liver fibrosis. Specifically, blood was collected from each person and centrifuged at 12,000 rpm for 15 minutes using a centrifuge to obtain serum alone and used as an experimental material. Capture Ab of CXCL14 was diluted in PBS and coated on a Nunc 96 well maxisorp plate overnight. The following day, the concentration of CXCL14 in the serum was measured by ELISA using centrifuged serum.

The degree of liver fibrosis was assessed by the degree of liver fibrosis used in the Knodell histologic activity index. The liver fibrosis grade was divided into four grades: grade 4 (stage 0: no fibrosis, stage 1: periportal dilatation, stage 3: bridging fibrosis, stage 4: liver cirrhosis), serum CXCL14 concentration, Correlation were analyzed using Spearman's correlation test.

As a result, as shown in FIG. 2, the expression of CXCL14 protein was significantly decreased as the degree of hepatic fibrosis increased (p value ≤ 0.05).

Example 5 Measurement of CXCL14 Protein Expression According to the Progression of Liver Fibrosis

In order to confirm the change of expression of CXCL14 according to the degree of progression of hepatic fibrosis, patients with liver fibrosis were treated with mild fibrosis (grade 0 and grade 1) and grade 3 and grade 4 with advanced fibrosis ), And the concentration of CXCL14 in the serum between the two groups was compared using the Mann-Whitney's test.

As a result, as shown in FIG. 3, the expression of CXCL14 protein was significantly decreased in the severe patient group compared to the mild patient group (p value ≤ 0.05).

Example 6. Expression of CXCL14 according to degree of portal inflammation

Patients with liver fibrosis were graded according to the degree of portal inflammation and the level of CXCL14 expression was compared in each patient group. Specifically, one of the four components of the Knodell histologic activity index is portal inflammation, with four stages (0: no inflammation, 1: mild inflammation, 3: moderate inflammation, 4 : Severe inflammation). Subsequently, the correlation between serum CXCL14 concentration and portal inflammation level was analyzed using Spearman's correlation test.

As a result, as shown in FIG. 4, the expression of CXCL14 protein was significantly decreased as the degree of inflammation of the liver increased (p value ≤ 0.05).

Example 7. Expression of CXCL14 according to degree of piecemeal necrosis

Patients with liver fibrosis were graded according to piecemeal necrosis and CXCL14 expression levels were compared in each patient group. In particular, one of the four components of the Knodell histologic activity index is the degree of fragmentation necrosis, which depends on the degree of necrosis in 7 stages (0: no, 1: mild necrosis, 3: moderate necrosis, 4: (Nodular necrosis), severe necrosis, 5: moderate necrosis + bridging necrosis, 6: severe necrosis + bridging necrosis, (Spearman's correlation test).

As a result, as shown in FIG. 5, the expression of CXCL14 protein was significantly decreased as the degree of fragmentation necrosis increased (p value ≤ 0.05).

<110> THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> CXCL14 Biomarker for Diagnosing Liver Fibrosis <130> PB15-12811 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CXCL14 Forward <400> 1 gcgcagggtc tacgaagaat 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CXCL14 Reverse <400> 2 caggacctct aagcggaagc 20

Claims (10)

A composition for diagnosing liver fibrosis or liver cirrhosis comprising an agent for measuring the expression level of mRNA of CXCL14 (Chemokine (CXC motif) ligand 14) gene or a protein thereof.
The method according to claim 1,
Wherein the agent for measuring the CXCL14 protein is an antibody specific for the CXCL14 protein.
3. The method of claim 2,
Wherein said antibody is a monoclonal antibody or polyclonal antibody specific for CXCL14 protein.
The method according to claim 1,
Wherein the agent for measuring the mRNA expression level of the CXCL14 gene is an antisense oligonucleotide complementary to the mRNA of the CXCL14 gene, a primer or a probe.
A kit for the diagnosis of liver fibrosis or liver cirrhosis comprising the composition of any one of claims 1 to 4.
A method for detecting liver fibrosis or hepatocyte diagnostic marker CXCL14 from liver tissue samples of a patient to provide information necessary for diagnosis of liver fibrosis or liver cirrhosis.
The method according to claim 6,
A step of measuring the expression level of the mRNA or the protein of the CXCL14 gene from the liver tissue sample of the patient and comparing the expression level of the mRNA or the protein of the CXCL14 gene with the mRNA of the CXCL14 gene or the expression of the protein thereof in the control sample The method comprising the steps of:
8. The method of claim 7,
Wherein the method for measuring the mRNA expression level of the CXCL14 gene is a reverse transcription polymerase chain reaction, a competitive reverse transcription polymerase chain reaction, a real-time reverse transcription polymerase chain reaction, an RNase protection assay, a Northern blot or a DNA chip.
8. The method of claim 7,
Methods for measuring the expression level of the CXCL14 protein include Western blotting, ELISA, radioimmunoassay, radioimmunoprecipitation, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assays, complement fixation Assay, FACS (fluorescence activated cell sorter) or protein chip.
A method for screening a therapeutic agent for hepatic fibrosis or liver cirrhosis comprising the step of detecting a change in the expression level of mRNA of the CXCL14 gene or a protein thereof after treating a candidate agent for hepatic fibrosis or liver cure.

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