JP2009178057A - Method and kit for detecting combined therapeutic effect of interferon and ribavirin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method by which the effectiveness of combined therapy of interferon and ribavirin can be determined before starting the therapy or at an early stage after starting the therapy. <P>SOLUTION: The method for detecting the combined therapeutic effect of the interferon and the ribavirin uses the difference in expression levels of a specific biomaterial discovered by the following comparison as an index exhibiting the effectiveness of the combined therapy of the interferon and the ribavirin by comparing the expression levels of the specific biomaterial in each cell before and after administration of the interferon and the ribavirin to detect the combined therapeutic effects of the interferon and the ribavirin in a testee of a carrier of hepatitis C virus. The specific biomaterial is at least one selected from the group consisting of ANXA3, PCNA, LMNB1, PAK1, 4EBP1, KARS, PSME2, ENO1, STAT1, PHB, SP-100, and PPP1CB. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for detecting the effectiveness of combined treatment of IFN (interferon) and ribavirin for hepatitis C caused by hepatitis C virus, and a detection kit therefor.

  Hepatitis C is a disease caused by infection with hepatitis C virus (HCV). HCV is a 50-nm-diameter spherical outer shell (envelope) virus that belongs to the Flaviviridae hepacivirus genus and has a genome size of about 9.5 kb (about 9500 bases). Have a gene. It was discovered by Chiron in 1988 from chimpanzee plasma, which appears to contain NANB virus, which was the cause of post-transfusion hepatitis, which did not decrease even after HBV screening for blood transfusion was performed.

  Hepatitis C genotypes are diverse, and genotypes are currently classified as type 1a (I), 1b (II), 2a (III), 2b (IV), 3a, 3b, 4, 5a, 6a. ing. Although the replication mechanism in such HCV virus-infected cells is hardly known, recently, a relationship with PAK1 (P21-activated kinase 1) present in the cells has been reported (Non-patent Document 1). ).

  HCV rarely becomes fulminant due to initial infection, but after the onset of acute hepatitis, there are many cases in which alanine aminotransferase (ALT) levels remain high, and viruses continue to be positive and transition to chronic hepatitis. It can be an asymptomatic carrier. It is reported that about 70% of first-infected persons become carriers and become chronic.

  In addition, chronic hepatitis C rarely heals spontaneously, and 30-50% of patients develop cirrhosis between 20-30 years after infection. In addition, 60-80% of cirrhosis patients develop liver cancer.

  Conventionally, interferon (IFN) has been used as a treatment method for chronic hepatitis C, but the response rate was only about 30%. For this reason, in recent years, combined treatment of IFN and ribavirin (RBV), which is one of antiviral drugs, has been carried out, and the therapeutic effect has been improved even in intractable cases. There is also a combined treatment of polyethylene glycolated IFN and RBV in which polyethylene glycol is bound to increase the blood half-life of IFN.

  However, even in the above combination treatment, the ratio of the most effective cases is about 50 to 60%, and the effect is not recognized for about half of the patients. On the other hand, for example, peginterferon alfa-2b and ribavirin combination therapy has a drug administration period of 24 to 48 weeks, and side effects such as fever, headache, malaise, loss of appetite, itching of the body, hair loss, thrombocytopenia, etc. As reported, combined treatment of IFN and RBV is particularly burdensome for elderly people and patients without physical strength. Therefore, if it is possible to determine whether IFN and RBV combination treatment is effective before treatment or at the beginning of treatment, treatment with side effects can be avoided for patients who cannot be treated with combination treatment. Other treatment methods can be applied to such patients.

  As a method for determining the effectiveness of combined treatment of IFN and RBV for the treatment of hepatitis C, the core of hepatitis C virus contained in a sample collected from a patient infected with genotype 1b hepatitis C virus It has been proposed to check whether the 70th arginine in the amino acid sequence in the region is mutated to another amino acid and whether the 91st amino acid is methionine (Patent Document 1). However, in this method, it is necessary to carry out the identification of the mutated amino acid in the virus, and the method cannot be applied if the type of the virus that is infected is other than 1b. Moreover, since it is necessary to identify the amino acid of the virus, the complexity is inevitable.

  On the other hand, as a result of applying IFN and RBV to human hepatoma-derived Huh-7 cells not infected with virus, the expression levels of STAT1, MxA-1, PPP1CB, SP-100, PHB and PTBP1 proteins increased. (Non-Patent Documents 2 and 3).

JP 2007-43985 A H. Ishida, et al., J. Biol. Chem. 282 (16), 11836-11848 (2007) Annual Meeting of the Japanese Society for Agricultural Chemistry 2007 (Tokyo) [Tokyo] Abstracts of Lectures, p86, Title No. 2A22P12 2006 Annual Meeting of the Chemical Society of Japan Kyushu Branch Program Abstracts, p47, Presentation Number A-20

  There is no report yet on the means for quickly detecting the effectiveness of the combination therapy of IFN and RBV for the treatment of hepatitis C, and it is simple and quick to determine the effectiveness of the combination therapy of IFN and RBV. The establishment of an accurate detection method is highly desired.

  It is an object of the present invention to provide a method that can determine the effectiveness of a combination treatment of IFN and RBV before the start of treatment or at an early stage after the start of treatment.

  Therefore, the present inventors have conducted intensive studies and found that changes in the expression level of specific biological substances appear in the cells of subjects in whom the combined treatment effect of IFN and RBV is effective, and using these specific biological substances as markers. The present inventors have found a method for detecting the effectiveness of the combined therapeutic effect of IFN and RBV, and have reached the present invention.

That is, the present invention is a method for detecting in vitro the combined therapeutic effect of interferon and ribavirin in a subject of hepatitis C virus carrier,
Measuring and comparing the expression level of a specific biological substance in each cell before and after administration of interferon and ribavirin,
Here, the difference in the expression level of the specific biological substance found by the comparison is used as an index indicating the effectiveness of the combined treatment of interferon and ribavirin,
Provided is a detection method, wherein the specific biological material is at least one selected from the group consisting of ANXA3, PCNA, LMNB1, PAK1, 4EBP1, KARS, PSME2, ENO1, STAT1, PHB, SP-100, and PPP1CB To do.

  In the detection method, the step of measuring and comparing the expression level of the specific biological substance in each cell before and after the administration of the interferon and ribavirin is a specific biological substance in the cell collected from the subject before the administration of the interferon and ribavirin And the expression level of the specific biological substance in the cells collected from the subject after administration of interferon and ribavirin.

  In the detection method, the step of measuring and comparing the expression level of the specific biological substance in each cell before and after the administration of the interferon and ribavirin is a specific biological substance in the cell collected from the subject before the administration of the interferon and ribavirin And the expression level of a specific biological substance contained in the cell after administering interferon and ribavirin to the cell in vitro.

  The cells can be liver cells or peripheral blood derived lymphocytes.

  The specific biological material is at least one selected from the group consisting of ANXA3, PCNA, LMNB1, PAK1, 4EBP1, KARS, PSME2, STAT1, PHB, and SP-100, and the expression level of the specific biological material is It can be measured by quantifying the mRNA transcript of the gene encoding the specific biological material.

  An increase in the cells after administration of interferon and ribavirin at the expression level of the specific biological substance can be an index indicating the effectiveness of the combined treatment of interferon and ribavirin.

  The expression level of the specific biological material can be measured by quantifying the specific biological material, that is, the protein itself.

  The specific biological material is at least one selected from the group consisting of ANXA3, PCNA, PAK1, 4EBP1, KARS, PSME2, ENO1, STAT1, PHB, SP-100, and PPP1CB. An increase in the expression level in cells after administration of interferon and ribavirin can be an indicator of the effectiveness of the combined treatment with interferon and ribavirin.

  The specific biological material is LMNB1, and the expression level of the protein of the specific biological material is decreased in the cells after the administration of interferon and ribavirin than in the cells before the administration of interferon and ribavirin. It can be an indicator of the effectiveness of combination therapy.

  The type of HCV may be HCV1b type.

  The interferon can be a pegylated interferon.

  The present invention also provides a kit for detecting the combined therapeutic effect of interferon and ribavirin in a hepatitis C virus carrier, comprising SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, At least one of oligonucleotides comprising at least 10 nucleotides in the base sequence shown in 18, 20, 22, 24, 26, 28, 30, 32, or 34, or at least 10 nucleotides in the base sequence complementary thereto. A kit for detecting the combined therapeutic effect of interferon and ribavirin in a hepatitis C virus carrier is provided.

  The present invention also relates to a kit for detecting the combined therapeutic effect of interferon and ribavirin in a hepatitis C virus carrier, comprising ANXA3, PCNA, LMNB1, PAK1, 4EBP1, KARS, PSME2, ENO1, STAT1, PHB, SP A detection kit comprising at least one protein selected from the group consisting of −100 and PPP1CB as a biomarker is provided.

  Until now, the effect of the combination therapy, which has not been known unless IFN and RBV are administered for several weeks, can be achieved by the method of the present invention by the expression level of a specific in vivo protein and / or the gene encoding the in vivo protein. By measuring and comparing the expression level, it becomes possible to detect it quickly, simply and accurately, and the burden on the administration patient is greatly reduced.

  In addition, administration of these expensive medicines can avoid an economic burden on patients and society, which can greatly contribute to the treatment of chronic hepatitis C.

  In the present invention, when referred to as a hepatitis C virus carrier, the genotype is any of type 1a (I), 1b (II), 2a (III), 2b (IV), 3a, 3b, 4, 5a, or 6a. Indicates a subject infected with a virus. Since there are many cases of genotype 1b chronic hepatitis C in Japan, it often represents a subject infected with this type of virus, but there is no particular limitation here.

  The treatment method for which the effectiveness is determined by the method of the present invention is a combination treatment of IFN and RBV (hereinafter sometimes simply referred to as “combination treatment”). Here, the type of IFN is not particularly limited, and may be any IFN used for the treatment of chronic hepatitis C. Usually, IFNα, particularly IFNα2b and IFNα2a are used, but not limited thereto. In addition, in order to increase the blood half-life of IFN, PEG-IFN in which polyethylene glycol is bound to IFN is also used, and “interferon” in the present specification and claims is unless otherwise specified. Or, unless the context clearly indicates otherwise, it is also meant to include IFN derivatives used for the treatment of chronic hepatitis C, such as PEG-IFN. In addition, according to the current treatment guideline for genotype 1b chronic hepatitis C, the combination therapy of PEG-IFNα2b and RBV is performed for 48 weeks when the serum HCV RNA concentration is a high viral load of 100 kIU / mL or more. In non-combination therapy, IFN (non-PEGylated) is said to be administered for 2 years, and the serum HCV RNA concentration is 100 to 500 kIU / mL, which is moderate among high virus cases. In addition, PEG-IFNα2a can be administered alone. On the other hand, when the serum HCV RNA concentration is a low viral load of less than 100 kIU / mL, IFN (non-PEGylated) is administered for 24 weeks or PEG-IFNα2a is administered for 24-48 weeks. Further, in the case of re-administration, in the case of high viral load, it is the same as the above-mentioned initial administration, and in the case of low viral load, combined treatment of IFNα2b (non-PEGylated) and RBV for 24 weeks or PEG-IFNα2a alone 48 weeks of administration or 48 weeks of single administration of IFN (non-PEGylated). In addition, the combination treatment whose effectiveness is determined by the method of the present invention is not necessarily limited to the combination treatment according to the guideline, and may be another combination treatment of IFN and RBV.

  The cells used in the method of the present invention are not limited as long as they are collected from a patient infected with HCV, and may be a body fluid such as blood or a cell derived from a liver biopsy specimen. Among these, cells derived from blood that can be easily collected and have good sensitivity (including blood components such as serum and plasma in addition to whole blood) are preferable, and lymphocytes derived from peripheral blood are particularly preferable.

  In the method of the present invention, the expression level of a specific biological substance contained in cells before and after the combined treatment of IFN and RBV is measured. Here, the “specific biological substance” means annexin A3, nuclear proliferation antigen (PCNA), lamin B1 (LMNB1), serine / threonine protein kinase PAK1, translation initiation factor 4EBP1, lysyl tRNA synthetase, proteasome activator complex subunit 2 , Α-enolase, transcription factor STAT1, prohibitin, nuclear autoantigen Sp-100, and serine / threonine protein phosphatase PP-1β catalytic subunit PPP1CB. In the present specification, the respective symbols representing the genes encoding these substances, ANXA3, PCNA, LMNB1, PAK1, 4EBP1, KARS, PSME2, ENO1, STAT1, PHB, SP-100, and PPP1CB, are identified respectively. In some cases, substances may be used, and these names are used interchangeably for proteins, genes, and mRNAs.

  These will be described in more detail. In other words, Annexin A3 is one of a family of proteins that binds to calcium and phospholipids, and is known as a protein that plays a role in cell growth and signal transduction pathways, and has the potential for angiogenesis (Biochem BiophysRes Commun. 2005 Dec 2; 337 (4): 1283-7. Epub 2005 Oct 10) and anticoagulant effects are also known. The gene encoding this protein is denoted ANXA3. Here, annexin A3 typically has an amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and typically has a base sequence shown in SEQ ID NO: 2 in the sequence listing as a gene sequence encoding it. I can give you.

  Proliferating Cell Nuclear Antigen (PCNA), a proliferation-related antigen formerly called cyclin, is an intracellular polypeptide antigen found in both normal and transformed cells. PCNA synthesis levels correlate with the degree of cell proliferation and DNA synthesis during the cell cycle. PCNA has been identified as an auxiliary protein of DNA polymerase δ, and is known to bind strongly to a DNA replication site. Here, PCNA typically has an amino acid sequence shown in SEQ ID NO: 3 in the sequence listing, and a typical example of a gene sequence encoding it is the base sequence shown in SEQ ID NO: 4 in the sequence listing. be able to.

  Lamin B1 (LMNB1) consists of a two-dimensional matrix of proteins present adjacent to the inner nuclear membrane. The Lamin family creates a matrix and is highly conserved during evolution. During mitosis, the lamin protein is phosphorylated and the lamina matrix is reversibly degraded. Lamin protein is thought to be involved in nuclear stability, chromatin structure and gene expression. Vertebrate lamin consists of two types, A and B. This gene directs one of the two B-type proteins, B1. Here, Lamin B1 typically has the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing, and typically has the base sequence shown in SEQ ID NO: 6 in the sequence listing as a gene sequence encoding it. I can give you.

  The serine / threonine protein kinase PAK1 is one of the PAK proteins, a family of serine / threonine p21-activating kinases that are important agonists that link Rho (GTPase) to cytoskeleton reorganization and signal transduction to the nucleus. is there. It plays an important role in many intracellular functions, including control of cell growth, differentiation, migration, and cell death in mammalian cells. The PI3K / mTOR pathway involving PAK1 is known as a pathway that regulates cell growth and mRNA translation, and has been reported to be activated by growth factors such as insulin and EGF (epidermal growth factor). Yes. In this pathway, PAK1 itself is activated by being phosphorylated. Here, “PAK1” includes PAK1 before phosphorylation and phosphorylated active PAK1. Here, PAK1 typically has the amino acid sequence shown in SEQ ID NO: 7 in the sequence listing, and the gene sequence encoding it typically includes the base sequence shown in SEQ ID NO: 8 in the sequence listing. be able to. In the amino acid sequence shown in SEQ ID NO: 7, the sites that can be phosphorylated include serine at position 21, serine at position 57, threonine at position 84, serine at position 144, serine at position 149, serine at position 199. , Serine at position 204, threonine at position 212, serine at position 220, serine at position 223, threonine at position 225, and threonine at position 423.

  Eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) was found as a protein that is rapidly phosphorylated by insulin, and it has since become clear that this protein plays an important role in the initiation of mRNA translation. It was. Here, 4EBP1 typically has the amino acid sequence shown in SEQ ID NO: 9 in the sequence listing, and the gene sequence encoding it typically includes the base sequence shown in SEQ ID NO: 10 in the sequence listing. be able to.

  Lysyl tRNA synthetase (KARS) is an enzyme responsible for synthesizing aminoacyl tRNA that is a substrate in translation of the genetic code in the ribosome, and plays a role in causing the corresponding amino acid to be carried by tRNA. Lysyl-tRNA synthetase belongs to the class II family of tRNA synthetases and is a homodimer localized in the cytoplasm and shown to be a target for autoantibodies in human autoimmune diseases polymyositis or dermatomyositis. ing. In addition, it has been reported that KARS overexpression decreases the protease activity of HIV. HIV protease produces viral enzymes and structural proteins by cleaving viral precursor proteins in HIV infected cells, inducing virus maturation, and the virus becomes infectious. There is no report on the relationship between HCV and KARS. Here, KARS typically has the amino acid sequence shown in SEQ ID NO: 11 or 13 in the sequence listing, and the gene sequence encoding it is typically shown in SEQ ID NO: 12 or 14 in the sequence listing. The base sequence can be raised.

  Proteasome activator complex subunit 2 (PSME2) is a subunit of the proteasome known as a multifunctional (proteolytic enzyme) complex. The proteasome has a very regular structure consisting of two complexes (20S core and 19S regulator). The 20S core is composed of 4 cyclic structures of 28 non-identical subunits (2 rings are composed of 7 α subunits and 2 rings are composed of 7 β subunits). The 19S regulator consists of a base (having 6 ATPase subunits and 2 non-ATPase subunits) and a lid (having up to 10 non-ATPase subunits). Proteasomes are distributed at high concentrations in eukaryotic cells and cleave peptides in an ATP / ubiquitin-dependent process of the non-lysosomal pathway. The original function of the modified proteasome (immunoproteasome) is the processing of MHC class I peptides. The immune proteasome has a selective regulator that replaces the 19S regulator, called 11S regulator or PA28. Three subunits (α, β, γ) of 11S regulator have been identified, and the PSME2 gene encodes one of the two subunits induced by interferon, the β subunit. Three β and three α subunits combine to form a heterohexameric ring. Here, PSME2 typically has the amino acid sequence shown in SEQ ID NO: 15 in the sequence listing, and the gene sequence encoding it typically includes the base sequence shown in SEQ ID NO: 16 in the sequence listing. be able to.

  α-Enolase is a multi-catalytic enzyme that plays a role in various processes such as glycolysis and growth regulation. One of the three enolases (isoenzymes) found in mammals, the homodimeric soluble enzyme α Enolase, the gene that encodes it, also encodes τ crystallin, a shorter monomeric lens protein. The two proteins are made from the same mRNA. Full-length proteins (isoenzymes) are found in the cytoplasm. The shorter protein is produced from a selective translation start, is localized in the nucleus, and binds to a sequence element in the c-myc promoter. ENO1 itself has not been reported to be associated with antiviral activity. On the other hand, it is proved that this MBE-1 isoform of ENO1 (97% homology) induces apoptosis of cells infected with viruses other than HCV. Here, α-enolase typically has an amino acid sequence shown in SEQ ID NO: 17 or 19 in the sequence listing, and typically a gene sequence encoding it has SEQ ID NO: 18 or 20 in the sequence listing. The base sequence shown in FIG.

  The transcription factor STAT1 is a protein involved in the JAK-STAT signal pathway that works when type IFN induces an antiviral state. In the JAK-STAT signal pathway, after IFN binds to the receptor, the signal is transmitted and the expression of antiviral proteins Mx1, PKR, 2 ', 5'-OAS, p53, etc. is induced, and the antiviral state is induced in the cell. Be guided. In response to cytokines and growth factors, STAT family proteins are phosphorylated by the phosphorylase associated with the receptor and then form homodimers or heterodimers. These dimers move to the cell nucleus where they act as transcriptional activators. In addition to interferon, this protein is known to be activated by the action of various ligands such as EGF, PDGF, and IL6. Various genes that mediate the expression of this protein are thought to be important for stimulation from other cells and cell survival in response to pathogens. Here, STAT1 typically has the amino acid sequence shown in SEQ ID NO: 21 or 23 in the sequence listing, and typically a sequence in the sequence listing as a gene sequence encoding it. The base sequence shown in No. 22 or 24 can be mentioned.

  Prohibitin (PHB) is an evolutionarily conserved gene that is ubiquitously expressed and appears to be a negative regulator of cell growth and has the potential to be a tumor suppressor. PHB mutations are associated with sporadic breast cancer. Prohibitin is expressed as two transcripts with 3 'untranslated regions of different lengths. The longer transcript is present at higher levels in growing tissues and cells, suggesting that this longer '3 untranslated region may function as a trans-acting regulatory RNA. It has also been reported that the protein functions as a tumor suppressor by suppressing the activity of the transcription factor E2F1, thereby increasing the transcriptional activity of p53. p53 has been reported not only to have an antitumor effect, but also to induce apoptosis in virus-infected cells other than HCV, and functions as an antiviral protein. PHB typically has the amino acid sequence shown in SEQ ID NO: 25 in the sequence listing, and a typical example of a gene sequence encoding it is the base sequence shown in SEQ ID NO: 26 in the sequence listing. .

  The nuclear autoantigen Sp-100 is a nuclear antigen, and the appearance of autoantibodies against the protein is related to diseases such as primary biliary cirrhosis, but the relationship with HCV has been known in the past. It is not done. In addition, it has been reported that HSV-1 immediate early protein ICP0, which is derived from herpes simplex virus (HSV-1) and has a function of creating an environment suitable for virus propagation, is related to antiviral pathways other than HCV. It is a suggested protein. Sp-100 typically has the amino acid sequence shown in SEQ ID NO: 27, 29, or 31 in the sequence listing, and the gene sequence encoding it typically includes SEQ ID NO: 28, 30, Or the base sequence shown in 32 can be mention | raise | lifted.

  The serine / threonine protein phosphatase PP-1β catalytic subunit PPP1CB is one of the three catalytic subunits of protein (dephosphorylating enzyme) 1 (PP1). PP1 is a serine / threonine-specific protein phosphatase and is known to be involved in the regulation of various cellular processes such as cell division, glycogen metabolism, muscle contractility, protein synthesis, and transcription of HIV-1 virus. Studies with mice suggest that PP1 acts as a suppressor of learning and memory. At least three alternative splicing that directs two different isoforms has been observed. PPP1CB typically has the amino acid sequence shown in SEQ ID NO: 33 in the sequence listing, and a typical example of a gene sequence encoding it is the base sequence shown in SEQ ID NO: 34 in the sequence listing. .

  In the present specification, “before and after administration of IFN and RBV” means that before and after both drugs are actually administered to a subject in the same manner as the treatment of IFN and RBV, and cells collected from the subject before administration. Means both before and after administration of both drugs in vitro.

  As used herein, “collected before administration of interferon and ribavirin” includes collection from a subject who has never actually experienced either administration of both agents, but in addition, at least one week before collection Within a month, and preferably within one month, includes a broad meaning including collection from a subject who is not affected by either interferon or ribavirin.

  In the present invention, both drugs are administered to cells collected from a subject before administration of interferon and ribavirin at a ratio proportional to the mass in an amount known as a therapeutically effective amount in vitro. Is not particularly limited, and a typical example is mixing cells and drugs and culturing for 4 to 72 hours.

  “Collected from the subject after administration of interferon and ribavirin” means that an amount generally known as a therapeutically effective amount was collected 4 to 72 hours after administration to a hepatitis C virus carrier, A single collection may be used, but several collections at different times are possible. If several collections are made at different times, the difference in expression level may be the difference at any point in time. For example, in a case where the expression level rapidly increases at a certain time point after 4 hours to 72 hours and then returns to a normal level as time passes, in the present specification, the expression level is different. Become. Here, the amount of the drug when administering both drugs in vivo is not particularly limited, but may be about 2 to 200 IU IFN and 1 μg to 20 μg RBV per 1 ml of blood.

This is also true when comparing in vitro expression levels after administration of interferon and ribavirin and before administration. That is, cells collected from a non-drug-administered hepatitis C virus carrier can be exposed to both drugs in vitro and the expression levels compared after 4 to 72 hours. The amount of drug when administering both drugs in vitro is not particularly limited, but can be on the order of 0.2-200 IU IFN and 1 μg-20 μg RBV for 3 × 10 ⁵ cells per ml.

  The expression levels of these specific biological substances can be measured by quantifying these proteins themselves in the subject cells before and after the combined treatment with IFN and RBV. The measurement method is not particularly limited, and various protein quantification methods known to those skilled in the art can be used. For example, proteome analysis incorporating gel electrophoresis including two-dimensional electrophoresis, shotgun method using LC-MS, western blotting using an antibody against a specific biological substance, immunoassay, and the like.

  The expression level of these specific biological substances can also be measured by quantifying mRNA transcripts of genes encoding these proteins in subject cells before and after combined treatment with IFN and RBV. The method for quantifying the mRNA transcript is not particularly limited, and one or a combination of various quantification methods known to those skilled in the art can be used. These methods include, for example, hybridization, Northern blotting, polymerase chain reaction, and a method using a DNA array.

  In the present invention, as a result of measuring the expression level of a specific biological material in cells after administration of interferon and ribavirin and the expression level of the specific biological material in cells before administration of interferon and ribavirin, the specific biological material found If there is a difference in the expression level, the effectiveness of combined treatment of interferon and ribavirin will be shown.

  A specific standard for “difference in expression level” can be appropriately set in any measurement means known to those skilled in the art. Although not particularly limited, for example, in cells collected from the subject before and after the administration of interferon and ribavirin, at least one of the expression levels is about 1.4 to 2 times or more compared to the expression level of one of them. If so, it can be said that there is a clear difference.

  According to the present invention, whether or not the expression level of a gene and / or protein in a specimen is significantly increased or decreased at the initial stage of a combination treatment is examined, and the effectiveness of the combination effect is determined using the result. it can. That is, when the expression level of the gene or protein in the specimen does not significantly increase or decrease at the initial stage of the combination therapy, it is highly probable that the combination therapy is invalid, and it is possible to avoid useless therapy with a low possibility of success.

  Here, in order to carry out such a detection method more easily, it is also possible to create the following kit.

  That is, as a kit for detecting the combined therapeutic effect of interferon and ribavirin in a hepatitis C virus carrier, the above ANXA3, PCNA, LMNB1, PAK1, 4EBP1, KARS, PSME2, ENO1, STAT1, PHB, SP-100, Or interferon comprising at least one, preferably two or more of oligonucleotides comprising at least 10 consecutive nucleotides contained in the base sequence derived from PPP1CB, or at least 10 nucleotides complementary to them. A kit for detecting the combined therapeutic effect of ribavirin is provided.

  These are typically the bases shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34 in the Sequence Listing. Detecting the combined therapeutic effect of interferon and ribavirin in a hepatitis C virus carrier comprising at least one of the oligonucleotides comprising at least 10 consecutive nucleotides contained in the sequence or at least 10 nucleotides complementary to the nucleotide sequence It is a kit to do. Using the oligonucleotide contained in such a kit as a probe, for example, by analyzing the mRNA in the cell using the Northern blotting technique, the change in the amount of the target RNA contained in the cell before and after administration can be easily examined. Is possible. Other components included in the kit are buffers, instructions for comparing expression levels, and the like.

  Furthermore, detection can be efficiently performed by preparing a DNA array in which a plurality of these oligonucleotides are immobilized on a solid support.

  In another embodiment, a kit for detecting the combined therapeutic effect of interferon and ribavirin in a hepatitis C virus carrier, typically, SEQ ID NO: 2, 4, 6, 8, 10 , 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34, or at least 10 nucleotides in the base sequence complementary thereto, or at least 10 nucleotides complementary thereto Selected from the group consisting of a pair of an oligonucleotide primer comprising: and an oligonucleotide primer comprising the same sequence and at least 10 consecutive nucleotides contained in another region, or at least 10 nucleotides of a complementary nucleotide sequence thereto Comprising at least one oligonucleotide primer pair Kit for detecting the combined therapeutic effects of interferon and ribavirin in hepatitis virus carriers is provided. That is, a kit for detecting the combined therapeutic effect of interferon and ribavirin in a hepatitis C virus carrier, comprising a pair of primers that can be appropriately selected from the above base sequences is provided. Using each one pair or two or more pairs contained in such a kit as a primer pair for RT-PCR, the expression level of the amount of mRNA contained in the cell or the amount of cDNA is measured, It is possible to compare.

  The present invention also provides a kit for detecting the combined therapeutic effect of interferon and ribavirin in a hepatitis C virus carrier, comprising ANXA3, PCNA, LMNB1, PAK1, 4EBP1, KARS, PSME2, ENO1, STAT1, PHB, SP A detection kit comprising at least one protein selected from the group consisting of −100 and PPP1CB as a biomarker is provided. Such a kit is particularly useful when an antibody of a protein used as a biomarker is induced in a living body. In fact, for some of the markers listed here, autoantibodies are known, and these kits can detect these autoantibodies in a small amount efficiently, and can be used in combination with interferon and ribavirin. The effect can be detected.

  Hereinafter, the present invention will be described more specifically based on examples. However, this example shows only specific examples of the present invention, and the present invention is not limited to the following examples.

(Detection of HCV replicon replication inhibitory effect of IFN and RBV in vitro)
HCV replicon cells were used to analyze the induction of antiviral status by the combined use of IFN and RBV. The HCV replicon is obtained by removing a part of the structural region and non-structural region of HCV and inserting an IRES (internal ribosome entry site) and a neomycin resistance gene into the portion. RNA is synthesized in vitro using this structure as a template, introduced into the liver cancer cell line Huh-7, and then selectively cultured with neomycin. As a result, a neomycin resistance gene is expressed from the replicated HCV replicon RNA, and only the cells in which the replicon is expressed become viable and grow to form colonies. In replicon-replicating cells, HCV replicon RNA is replicated at a very high level, and the replicating RNA and the expressed HCV protein can be detected stably and stably.

  For the culture of HCV replicon cells, Dulbecco's modified Eagle medium (manufactured by GIBCO) with moderate addition of FCS was used. In particular, a medium supplemented with G418 was used for subculture. Cells were cultured in DMEM medium using a 37 ° C. incubator equilibrated with 5% CO 2 gas.

For IFN, IFN-α2b, human, recombinant (manufactured by Procpec-TanyTechnoGene) was dissolved in sterile water to make 5.2 × 10 ⁶ IU / ml. RBV (manufactured by Sigma) was dissolved in sterilized water to make 100 mg / ml.

The HCV replicon retains the luciferase translation region, and the amount of replicon replication in HCV replicon cells can be measured by luciferase activity. The luciferase assay was performed using a Steady Glo ^™ luciferase assay system (Promega). In addition, since it was necessary to evaluate the toxicity to cells when HCV replicon cells were treated with IFN-α2b, which is a therapeutic agent for hepatitis C, alone or in combination, Cell Counting Kit-8 (WST-8 Cytotoxicity was evaluated using Dojin Chemical).

Table 1 shows a part of the results obtained by treating IFN-α2b and RBV alone or in combination with HCV replicon cells for 24 hours and examining the influence on HCV replicon replication. 4.3 When IU / ml IFN-α2b alone or 10 μg / ml RBV alone was administered to cells, replicon replication was inhibited by 50%. When IFN-α2b and RBV were used in combination, the combination index (CI) was 0.5 or less, and a synergistic effect of both drugs was observed. In addition, the toxicity to the cells by the treatment of both these drugs was not observed.

(Proteome analysis when INF and RBV are co-administered to HCV-infected cells in vitro)
Co-administration of IFN-α2b and RBV was attempted with HCV infected cells. HCV replicon cells are prepared to a density of 3 × 10 ⁵ cells / ml in DMEM medium containing 5% FCS (inactivated) and pre-cultured in a 37 ° C. incubator (5% CO ₂ condition) for 24 hours. It was. After 24 hours, the medium was removed, and DMEM medium (containing 5% FCS) supplemented with 100 IU / ml IFN-α2b and 3 μg / ml RBV, either alone or in combination, was newly added.

  The treatment time for each treatment is 24 hours from the start of the experiment.

  At the time of cell collection, the medium was first removed, the cell surface was washed once with PBS, about 5 ml of 1 mM EDTA-PBS was added, and the cells were scraped with a cell scraper (manufactured by NUNC). The collected cells were centrifuged at 200 × g for 5 minutes, the supernatant was removed, and the suspension was resuspended in PBS that had been cooled (three times). When suspended for the third time, the number of cells was counted with a hemocytometer. did. After the final centrifugation, the supernatant was removed with a Pasteur, and PBS was removed as completely as possible.

  Next, the obtained cells were subjected to two-dimensional electrophoresis. For electrophoresis, the following two types of buffers were used. Swelling buffer (cell lysis buffer) is 7M Urea (Wako), 2M Thiourea (Wako), 3% CHAPS (Dojin Chemical), 1% TritonX-100 (Wako), 0.2 % Ampholyte, 0.0002% Bromophenol blue (BPB; manufactured by Wako) was dissolved, dispensed, and stored frozen. About 30 minutes before use, thawing was performed, and immediately before use, DTT (for SH group oxidation protection; manufactured by Wako) was added to a final concentration of 75 mM. Equilibration buffer is cryopreserved after dissolving 6M Urea, 0.375M Tris-HCl (pH 8.8), 2% Sodium Dodecyl Sulfate (SDS; manufactured by Wako), 20% glycerol (manufactured by Wako), and 0.0002% BPB. I kept it. About 30 minutes before use, thawing was performed, and 2% DTT was added for equilibration buffer I, and 2.5% Iodoacetoamide (IAA; manufactured by Wako) was added for equilibration buffer II immediately before use.

100 μl of swelling buffer was added to the pellet obtained from 1 × 10 ⁶ cells of the collected cell pellet (containing a final concentration of 75 mM DTT). After dissolving with the swelling buffer, the solution was completely dissolved by applying it to an ultrasonic cleaner for 10 minutes. After ultrasonication, centrifugation was performed at 15,000 × g for 10 minutes (under 4 ° C.). The supernatant after centrifugation was transferred to another microtube, and desalted and impurities were removed with a 2-D clean-up kit (Amersham Bioscience). The 2-D clean-up kit was used according to the product protocol. The clean-up pellet was redissolved with a swelling buffer and centrifuged at 15,000 × g for 5 minutes as a sample. After clean-up, the protein concentration of the sample redissolved with the swelling buffer was measured using the RC DC protein assay kit (manufactured by Bio-Rad). And applied to the first-dimensional electrophoresis apparatus.

  In the first dimension of the two-dimensional electrophoresis, an IPG strip pH3-10 NL 7 cm (manufactured by Bio-Rad) was used, and electrophoresis was performed with an electrophoresis apparatus Protean IEF Cell (manufactured by Bio-Rad). First, 135 μl or more of swelling buffer was added to each lane of the swelling tray, and the gel surface of the IPG strip was mounted face down. Each lane was overlaid with mineral oil and allowed to stand for 11 to 15 hours in a horizontal place to swell the gel. During the swelling, the room temperature was kept at 22 ° C.

  After completion of the swelling, the strip was taken out from the swelling tray. The gel surface of the strip was set upward on the cup loading tray for the sample cup, and the movable electrode was set on the anode side and the cathode side so as to ride on the end of the strip gel surface. A sample cup was placed on the acidic side of the strip and 100 μl of sample solution was applied.

The focusing conditions for the first dimensional isoelectric focusing were set as follows.
Step 1 (Step of removing excess salt at low voltage): 250V, 30min, Rapid ramping mode Step 2 (Step of increasing voltage from desalting voltage to focusing voltage): 4,000V, 1h, Slow ramping mode Step 3 (Protein Focusing on pI value of 4,000V, 10,000Vhour (V x hour), rapid ramping mode
Step4 (Step to prevent diffusion of focused protein): 500V, 2 hours, rapid ramping mode

  After the first dimensional run is completed, remove the strip from the cup loading tray, then apply 2.5 ml of equilibration buffer I to the swelling tray, place the strip with the gel side up and shake for 20 minutes. That ’s it. Next, the strip was placed in a swelling tray to which 2.5 ml of equilibration buffer II had been applied with the gel surface facing up, and the mixture was shaken for 20 minutes while being shielded from light with aluminum foil.

  Second-dimensional SDS-PAGE electrophoresis was performed under the conditions of 26 mA and 200 V according to a conventional method.

  The gel after electrophoresis was visualized by Coomassie staining. Coomassie staining was performed according to a conventional method, and after staining, the staining solution was discarded, and decolorization solution (5% methanol, 7% acetic acid) was added to remove the color. After decolorization, it was replaced with ultrapure water and shaken for a while, and the gel image was captured with a TIFF format gray scale. Next, it was subjected to in-gel digestion and MS analysis.

  2-D Analysis Software PDQuest (Version 7.3; manufactured by Bio-Rad) was used for differential analysis between the gel images. First, spot detection and spot matching between gels were automatically performed, and normalization was performed based on the sum of expression intensity of all detected spots. All spots were quantified based on the expression intensity, and the experimental results for three series were statistically analyzed.

In each treatment, a large number of spots in which the expression level was changed by 2 times or more were detected as compared with the untreated control gel. These spots were classified into the following two types.
(1) Increased expression protein: protein spot with increased expression compared to control gel (2) Reduced expression protein: protein spot with decreased expression compared to control gel

  In-gel digestion was performed around spots where significant changes in expression levels were seen compared to controls. In consideration of the amount of protein necessary for MS analysis, gel pieces for in-gel digestion were cut out from a plurality of gels stained by Coomassie staining.

  The gel after decolorization was washed several times with Milli-Q water, and the spot was picked from the gel with a pipette tip. Transfer the excised gel piece to a 1.5 ml microtube (use 96-well plate for many samples), add 400 μl of decolorization solution (50% acetonitrile, 25 mM ammonium bicarbonate) to remove the CBB dye, After shaking for 10 minutes at room temperature, the decolorizing solution was removed with a pipette. This operation was repeated until the blue color disappeared from the gel piece.

  Next, in order to dehydrate the gel piece, 200 μl of acetonitrile was added, the gel piece was contracted, and centrifuged to precipitate the gel piece, and the acetonitrile was completely removed by a centrifugal evaporator. After dehydration, in order to carry out reductive alkylation, 100 μl of reducing solution (100 mM DTT, 25 mM ammonium bicarbonate) is added, left to react at 56 ° C. for 1 hour, and then 100 μl of 25 mM weight is removed to remove DTT. Ammonium carbonate was added and shaken at room temperature for 10 minutes.

  After the reduction, 100 μl of an alkylating solution (55 mM iodoacetamide, 25 mM ammonium bicarbonate) was added, and the SH group was alkylated by allowing to stand at room temperature for 45 minutes protected from light. The alkylation solution was removed and 100 μl of wash buffer (25 mM ammonium bicarbonate) was added and shaken at room temperature for 10 minutes. In order to perform in-gel digestion, 400 μl of dehydrating solution (50% CAN, 25 mM ammonium bicarbonate) was added and shaken at room temperature for 10 minutes to dehydrate the gel. 200 μl of acetonitrile was added to shrink the gel piece, and the gel piece was flushed with a desktop centrifuge to precipitate the gel piece. Then, the acetonitrile was removed, and the remaining acetonitrile was removed with a centrifugal evaporator.

  15-30 μl of trypsin solution (10 ng / μl, 50 mM ammonium bicarbonate) was added to the dehydrated gel pieces and allowed to react overnight at 37 ° C. After 40 μl of 50% acetonitrile / 5% TFA solution was added to the digested gel piece and vortexed, it was shaken for 15 minutes. Centrifugation was performed at 5000 rpm to precipitate the gel piece and collect the supernatant. After repeating this operation about twice, the sample was concentrated with a centrifugal evaporator until the supernatant became 10 μl or less to prepare a sample.

  Prior to application to the target, a matrix was prepared. The matrix was thinly applied to the sample part and the calibrant part of AnchorChip, and 1 μl of the concentrated sample was applied. The sample placed on AnchorChip is concentrated simultaneously with drying, and the spot diameter is reduced to 0.2 mm. The sample was desalted by placing 0.1% TFA on top of the sample and sucking it off. Similarly, in the case of the calibrant, the calibrant was desalted by applying 0.1 μTFA after applying 0.5 μl of the calibrant and sucking it. After desalting, it was sufficiently air-dried.

  The peptide mass fingerprinting (PMF) method was used as the protein identification method. Auto FLEX TOF / TOF (BRUKER DALTONICS) was used for MALDI-TOF MS analysis.

Laser irradiation was performed about twice per location, and when a good spectrum was obtained, the spectrum was integrated, and measurement and integration were performed until the number of Shots reached around 200 while changing the laser irradiation position. Note that calibration was performed before measurement of the sample. For calibration, Peptide calibration standard (Angiotensin II [M + H] ⁺ average: 1,047.20, Angiotensin I [M + H] ⁺ average: 1,297.51, Substance P [M + H] ⁺ average: 1,348.66, Bombesin [M + H ] ⁺ average: 1,620.88, ACTH clip 1-17 [M + H] ⁺ average: 2,094.46, ACTH clip 18-39 [M + H] ⁺ average: 2,466.73, Somatostatin 28 [M + H] ⁺ average: 3,149.61; BRUKER DALTONICS Was applied to a target and measured.

  For the analysis of the sample, the External Calibration. FAMS method was selected and analyzed. After the integration chart was obtained, the data was saved with the Flex control and the spectrum was transferred to Flex Analysis. Using the Mass List Find button on the Flex Analysis screen, peak picking was performed over the entire range, the data was transferred to Biotools, and a database search was performed.

  From the identification result of the spot which performed MS analysis by PMF method, the protein whose expression level changed when IFN-α2b and RBV treatment were used together was found. As compared with the case of treatment with IFN alone, spots in which the expression level was changed by the combined treatment of IFN-α2b and RBV were shown.

  That is, FIGS. 1A to 1D show the types of proteins in which the expression level is observed to be changed by 2 times or more when IFN-α2b and RBV are administered to HCV replicon cells in vitro and the changes in the expression level.

  Among the proteins whose expression levels are changed when IFN-α2b and RBV are used in combination, STAT1 and Mx1 involved in the JAK-STAT signal pathway that acts when type I IFN induces an antiviral state are also included. It was.

  Moreover, the expression level of both proteins increased more than twice when IFN-α2b alone was used, and when used together with RBV, a significant increase was observed compared to the expression level of IFN-α2b alone.

  For proteins other than STAT1 and Mx1, PHB has been extracted next. PHB was added approximately twice as much as IFN-α2b alone treatment, and a significant increase was observed in combination with RBV compared to IFN-α2b alone. It was shown to be a protein.

  In Sp-100, IFN-α2b alone treatment did not show an increase of about 2 times, but it was in an increasing trend, and in combination with RBV, a significant increase was observed compared with IFN-α2b alone. Therefore, it was shown that it becomes a marker protein for antiviral state induction.

  Next, PAK1 located downstream of the PI3K / mTOR pathway has been extracted. PAK1 did not increase by a factor of about 2 when IFN-α2b alone was treated, but it was on the increase compared to control, and significantly increased compared with IFN-α2b alone when used in combination with RBV. Was recognized. From these results, it was shown that the activation of PAK1 by the PI3K / mTOR pathway may be one of the reasons why the therapeutic effect of hepatitis C is enhanced by the combined use of both drugs.

  A change in the expression level of ENO1 was also observed in the combined treatment. Further, looking at changes in the expression level of KARS, the expression level significantly increased after 24 hours when IFN-α2b and RBV were used in combination, compared with the case of IFN-α2b alone.

  In addition, expression of ANXA3, PSME2, PCNA, 4EBP1, and PPP1CB was significantly increased by the combined use of both drugs. On the other hand, expression of LMNB1 was significantly reduced by the combined use of both drugs.

  All of the above proteins have a significant increase or decrease in expression when IFN-α2b and RBV are used in combination, compared with the case where IFN-α2b is administered alone. It can be seen that it can be used as a marker for detecting.

(Comprehensive gene expression analysis of peripheral blood-derived lymphocytes when IFN and RBV are co-administered to an HCV carrier)
Total RNA was extracted from HCV carrier peripheral blood-derived lymphocytes administered with both PEG-IFNα2b and RBV drugs, and gene expression analysis was performed using a DNA microarray.

1. Blood collection of subjects with excellent and ineffective cases The blood of four patients who were infected with HCV and received combination therapy was subjected to the experiment. These patients received 1.5 μg / kg PEG-IFNα2b subcutaneously once weekly and continued daily oral RBV administration of 600-800 mg / day for 48 weeks. The dose of RBV was adjusted according to body weight (600 mg / day when the body weight is 60 kg or less, 800 mg / day when the body weight exceeds 60 kg). Patients whose serum HCV RNA is below the detection limit by qualitative PCR after 24 weeks from the end of combination therapy are effective cases (virological response cases, VR), quantitative PCR at the end of combination therapy A patient who was positive for HCV RNA in serum by qualitative PCR (described below) and / or qualitative PCR was regarded as an ineffective case (virological ineffective case, NVR). As a result, it was possible to divide into two groups, one who was effective in combination treatment and two who were ineffective.

  The HCV RNA concentration in serum was first determined by quantitative PCR (Amplic HCV monitor version 2.0, manufactured by Roche Diagnostics). The detection limit of this measurement method is about 0.5K IU / ml. When HCV RNA was not measured by quantitative PCR, it was measured by qualitative PCR (Amplicon HCV version 2.0, manufactured by Roche Diagnostics) with a lower detection limit.

  From 4 patients, blood was collected in advance before treatment (day 0: 0), and peripheral blood lymphocytes were isolated therefrom using Ficol-Paque PLUS manufactured by GE Healthcare. In addition, these four patients received 1.5 μg / kg PEG-IFNα2b subcutaneously once weekly, and daily RBV oral administration of 600-800 mg / day for 48 weeks. The blood was collected on the day following the first subcutaneous administration (day 1: day 1) and the day after the second administration (day 8: day 8), and peripheral blood lymphocytes were isolated.

  Total RNA was extracted from the obtained peripheral blood lymphocytes (concentration) using TRIZOL® Reagent: 15596-026 manufactured by Invitrogen, according to the manufacturer's instructions. The extracted total RNA was synthesized with cDNA using Affymetrix GeneChip® Two-Cycle Target Labeling and Control Reagents: 900494 according to the manufacturer's instructions and then synthesized with cRNA. Subsequently, cDNA synthesis was performed again, and cRNA was synthesized while performing biotin labeling. This was used for DNA microarray analysis.

  The array used is Human Genome U133 Plus2.0 array: 520019 manufactured by Affymetrix. Biotin-labeled cRNA was hybridized to DNA immobilized on the array according to the manufacturer's instructions at 45 ° C. for 16 hours.

  Thereafter, the microarray was washed with Fludics Station 450 manufactured by Affymetrix, immersed in a fluorescent dye solution containing phycoerythrin-streptavidin: S866 reagent manufactured by Molecular Probes, and biotinylated anti-streptavidin antibody manufactured by Vector Laboratories ( Goat): Dipped in a biotinylated antibody solution containing BA-0500. It was again immersed in a fluorescent dye solution containing phycoerythrin-streptavidin for staining. The fluorescence intensity was measured with a GeneChip (R) scanner 3000 manufactured by Affymetrix.

  The measured data was subjected to gene expression analysis using analysis software GeneSpringGX manufactured by TOMY DIGITAL BIOLOGY. The results are shown in FIGS.

  As apparent from FIGS. 2A to 2E, for ANXA3, PAK1, 4EBP1, SP-100, and PSME2, a significant increase in expression was observed on day 1 or 8, and LMNB1, KARS, PCNA, PHB As for STAT1, an increasing tendency was observed.

(Analysis by immune reaction when IFN and RBV are administered in combination to HCV-infected cells in vitro)
In the same manner as in Example 2, IFN-α2b (10 IU / ml) and ribavirin (3 μg / ml) were administered in combination to cultured HCV replicon cells, and subjected to analysis 24 hours later. First, a cell extract was prepared from the cultured cells using Cell Culture Lysis Reagent CCLR (5-fold dilution with PBS; Promega) and subjected to Western blotting.

  In Western blotting, phospho-PAK1 (Thr423) PAK2 (Thr402) antibody (Cell Signal Technology) is used to detect phosphorylated PAK1 (p-PAK1), and PAK1 antibody (Cell Signaling Technology) is used to detect PAK1. GAPDH antibody (Santa Cruz Biotechnology) was used to detect GAPDH required for normalization.

  Of the results obtained by standardizing the detected band intensity of p-PAK1 and PAK with the band intensity of GAPDH, the result of p-PAK is shown in FIG.

  In the case of treatment with IFN-α2b alone, phosphorylation of PAK1 was increased by about 1.6 times compared with the case of no treatment. Further, when IFN-α2b and RBV were used in combination, the increase was about 3.5 times, which was a significant increase compared to the case where IFN-α2b was treated alone.

It is a figure which shows the result of proteosome analysis about the change of an expression level when IFN and RBV are administered to a replicon cell. It is a figure which shows the result of proteosome analysis about the change of an expression level when IFN and RBV are administered to a replicon cell. It is a figure which shows the result of proteosome analysis about the change of an expression level when IFN and RBV are administered to a replicon cell. It is a figure which shows the result of proteosome analysis about the change of an expression level when IFN and RBV are administered to a replicon cell. It is a figure which shows the result of the comprehensive gene expression analysis of the peripheral blood origin lymphocyte at the time of administering together with IFN and RBV to the HCV carrier. It is a figure which shows the result of the comprehensive gene expression analysis of the peripheral blood origin lymphocyte at the time of administering together with IFN and RBV to the HCV carrier. It is a figure which shows the result of the comprehensive gene expression analysis of the peripheral blood origin lymphocyte at the time of administering together with IFN and RBV to the HCV carrier. It is a figure which shows the result of the comprehensive gene expression analysis of the peripheral blood origin lymphocyte at the time of administering together with IFN and RBV to the HCV carrier. It is a figure which shows the result of the comprehensive gene expression analysis of the peripheral blood origin lymphocyte at the time of administering together with IFN and RBV to the HCV carrier. It is a figure which shows the result of having analyzed the expression level change of p-PAK1 at the time of administering IFN and RBV to a replicon cell by immune reaction.

Claims (13)

A method for detecting in vitro the combined therapeutic effect of interferon and ribavirin in a subject with a hepatitis C virus carrier comprising:
Measuring and comparing the expression level of a specific biological substance in each cell before and after administration of interferon and ribavirin,
Here, the difference in the expression level of the specific biological substance found by the comparison is used as an index indicating the effectiveness of the combined treatment of interferon and ribavirin,
The detection method, wherein the specific biological material is at least one selected from the group consisting of ANXA3, PCNA, LMNB1, PAK1, 4EBP1, KARS, PSME2, ENO1, STAT1, PHB, SP-100, and PPP1CB. The detection method according to claim 1,
The step of measuring and comparing the expression level of the specific biological substance in each cell before and after the administration of the interferon and ribavirin comprises the expression level of the specific biological substance in the cell collected from the subject before the administration of the interferon and ribavirin and the interferon And a method of measuring and comparing the expression level of the specific biological substance in cells collected from the subject after administration of ribavirin. The detection method according to claim 1,
The step of measuring and comparing the expression level of the specific biological material in each cell before and after the administration of the interferon and ribavirin, the expression level of the specific biological material in the cell collected from the subject before the administration of interferon and ribavirin, A detection method, which is a step of measuring and comparing the expression level of a specific biological substance contained in a cell after administering interferon and ribavirin to the cell in vitro.   The detection method according to any one of claims 1 to 3, wherein the cells are liver cells or peripheral blood-derived lymphocytes.   The specific biological material is at least one selected from the group consisting of ANXA3, PCNA, LMNB1, PAK1, 4EBP1, KARS, PSME2, STAT1, PHB, and SP-100, and the expression level of the specific biological material is The detection method according to any one of claims 1 to 4, which is measured by quantifying an mRNA transcript of a gene encoding the specific biological substance.   The expression level of the specific biological substance is increased in cells after administration of interferon and ribavirin than in cells before administration, which is an index indicating the effectiveness of the combined treatment of interferon and ribavirin. Item 6. The detection method according to Item 5.   The detection method according to any one of claims 1 to 4, wherein the expression level of the specific biological material is measured by quantifying the specific biological material.   The specific biological material is at least one selected from the group consisting of ANXA3, PCNA, PAK1, 4EBP1, KARS, PSME2, ENO1, STAT1, PHB, SP-100, and PPP1CB, and the expression level of the specific biological material The method according to claim 7, wherein the increase in the cells after administration of interferon and ribavirin is an index indicating the effectiveness of the combined treatment of interferon and ribavirin. .   Combined treatment of interferon and ribavirin is that the specific biological material is LMNB1 and the expression level of the specific biological material is decreased in cells after administration of interferon and ribavirin than in cells before administration. The detection method according to claim 7, which serves as an index indicating the effectiveness of.   The detection method according to any one of claims 1 to 9, wherein the HCV type is HCV1b type.   The detection method according to any one of claims 1 to 10, wherein the interferon is pegylated interferon. A kit for detecting the combined therapeutic effect of interferon and ribavirin in a hepatitis C virus carrier,
At least 10 continuous sequences included in the base sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 in the sequence listing A kit for detecting a combined therapeutic effect of interferon and ribavirin in a hepatitis C virus carrier, comprising at least one of a nucleotide or an oligonucleotide comprising at least 10 nucleotides of a base sequence complementary thereto. A kit for detecting the combined therapeutic effect of interferon and ribavirin in a hepatitis C virus carrier,
A detection kit comprising at least one protein selected from the group consisting of ANXA3, PCNA, LMNB1, PAK1, 4EBP1, KARS, PSME2, ENO1, STAT1, PHB, SP-100, and PPP1CB as a biomarker.

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