WO2017065206A1 - Marker for non-alcoholic steatohepatitis and use thereof - Google Patents

Marker for non-alcoholic steatohepatitis and use thereof Download PDF

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WO2017065206A1
WO2017065206A1 PCT/JP2016/080346 JP2016080346W WO2017065206A1 WO 2017065206 A1 WO2017065206 A1 WO 2017065206A1 JP 2016080346 W JP2016080346 W JP 2016080346W WO 2017065206 A1 WO2017065206 A1 WO 2017065206A1
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pcpe
protein
gene
nash
adipose tissue
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徹 南野
逸平 清水
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国立大学法人新潟大学
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    • C—CHEMISTRY; METALLURGY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

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  • the present invention relates to a non-alcoholic steatohepatitis marker and use thereof.
  • This application claims priority on October 13, 2015 based on Japanese Patent Application No. 2015-202418 for which it applied to Japan, and uses the content for it here.
  • NAFLD non-alcoholic fatty liver disease
  • NASH non-alcoholic steatohepatitis
  • NAFLD is a disease in which fatty liver disorder similar to alcoholic liver disorder is observed despite no history of alcohol consumption. About 90% of NAFLD is non-progressive simple fatty liver with good prognosis, and the remaining about 10% is classified as NASH that can progress to cirrhosis and hepatocellular carcinoma (see Non-Patent Document 1, for example). reference).
  • an object of the present invention is to provide a new NASH marker.
  • the present invention includes the following aspects.
  • a NASH marker comprising a Procollagen C-endopeptidase enhancer (PCPE-1) gene or PCPE-1 protein.
  • a kit for NASH diagnosis comprising a specific binding substance for PCPE-1 protein, a primer set for amplifying PCPE-1 gene cDNA, or a probe that specifically hybridizes to mRNA of PCPE-1 gene .
  • a NASH detection method comprising a step of quantifying the expression level of a PCPE-1 gene or a PCPE-1 protein in a biological sample.
  • a new NASH marker can be provided.
  • 6 is a graph showing the results of real-time PCR in Experimental Example 2.
  • 6 is a photograph showing the results of Western blotting in Experimental Example 2.
  • 10 is a graph showing the results of quantification of mouse PCPE-1 protein of Experimental Example 3.
  • 6 is a photograph showing the results of Western blotting in Experimental Example 4.
  • 10 is a graph showing the results of quantification of PCPE-1 protein in Experimental Example 5. It is a graph which shows the fixed_quantity
  • NASH marker In one embodiment, the present invention provides a NASH (non-alcoholic steatohepatitis) marker consisting of the PCPE-1 gene or PCPE-1 protein.
  • the PCPE-1 gene is also called a PCOLCE gene and is a gene encoding the PCPE-1 protein.
  • the Genbank accession number of the human PCPE-1 gene is NM_002593
  • the Genbank accession number of the mouse PCPE-1 gene is NM_008788.
  • SEQ ID NO: 1 shows the nucleotide sequence of the cDNA of the human PCPE-1 gene
  • SEQ ID NO: 2 shows the amino acid sequence of the human PCPE-1 protein
  • SEQ ID NO: 3 shows the base sequence of the mouse PCPE-1 gene cDNA
  • SEQ ID NO: 4 shows the amino acid sequence of the mouse PCPE-1 protein.
  • PCPE-1 protein increases in the serum of NASH patients. Moreover, it was clarified that the expression level of PCPE-1 gene is increased specifically by brown adipose tissue by obesity stress.
  • the PCPE-1 gene or PCPE-1 protein can be used as a NASH marker.
  • the NASH marker of this embodiment makes it possible to easily diagnose NASH. Moreover, it becomes possible to make a diagnosis in a minimally invasive manner.
  • the present invention provides a specific binding substance for PCPE-1 protein, a primer set for amplifying PCPE-1 gene cDNA, or a probe that specifically hybridizes to mRNA of PCPE-1 gene.
  • a NASH diagnostic kit is provided.
  • specific binding substances include antibodies, antibody fragments, aptamers and the like.
  • the antibody may be produced, for example, by immunizing an animal such as a mouse with the PCPE-1 protein or a partial peptide thereof as an antigen. Alternatively, it can also be prepared by screening an antibody library such as a phage library.
  • Antibody fragments include Fv, Fab, scFv and the like.
  • the above antibody or antibody fragment may be polyclonal or monoclonal.
  • An aptamer is a substance having a specific binding ability to a labeling substance.
  • examples of aptamers include nucleic acid aptamers and peptide aptamers.
  • Nucleic acid aptamers having specific binding ability to the PCPE-1 protein can be selected by, for example, the systematic evolution of ligand by exponential enrichment (SELEX) method.
  • Peptide aptamers having a specific binding ability to PCPE-1 protein can be selected by, for example, the two-hybrid method using yeast.
  • the specific binding substance is not particularly limited as long as it can specifically bind to the PCPE-1 protein, and may be a commercially available one.
  • the specific binding substance may be fixed on a carrier to constitute a protein chip or the like.
  • PCPE-1 gene or protein in a biological sample can be detected using the NASH diagnostic kit of this embodiment. If the expression level of the PCPE-1 gene or protein is higher than that of healthy individuals, the subject from whom the biological sample is derived is likely to have NASH.
  • the primer set is not particularly limited as long as it can amplify the PCPE-1 gene cDNA of the animal species to be diagnosed.
  • the primer for amplifying the cDNA of the human PCPE-1 gene includes a set of a sense primer consisting of the base sequence shown in SEQ ID NO: 5 and an antisense primer consisting of the base sequence shown in SEQ ID NO: 6.
  • the probe that specifically hybridizes to mRNA is not particularly limited as long as it specifically hybridizes to the mRNA of the PCPE-1 gene.
  • the probe may be fixed on a carrier to constitute a DNA microarray or the like.
  • the NASH diagnostic kit of this embodiment may be the specific binding substance described above. In this case, NASH can be easily diagnosed with minimal invasiveness.
  • the NASH diagnostic kit of the present embodiment preferably includes the above primer set or probe.
  • the present invention provides a method for detecting NASH comprising the step of quantifying the expression level of a PCPE-1 gene or PCPE-1 protein in a biological sample. It can also be said that the detection method of the present embodiment is a NASH diagnosis method.
  • PCPE-1 gene expression levels are quantified by real-time PCR using a primer set for amplifying the PCPE-1 gene cDNA; a microarray in which probes that specifically hybridize to the mRNA of the PCPE-1 gene are immobilized Analysis of gene expression by Northern blotting using a probe that specifically hybridizes to the mRNA of the PCPE-1 gene.
  • the detection using a reversed-phase protein array is an analysis method for detecting a specific substance in a sample by fixing the sample to the solid phase in an array and reacting a specific binding substance to the specific substance.
  • the detection method of this embodiment is a method for determining whether or not a biopsy sample is derived from a NASH patient. That is, when the expression level of PCPE-1 gene or PCPE-1 protein in a biopsy sample is higher than the expression level of PCPE-1 gene or PCPE-1 protein in a biopsy sample derived from a healthy person, It can be determined that the test sample is from a NASH patient.
  • PCPE-1 protein in a biological sample can be rephrased as the abundance of PCPE-1 protein in the biological sample.
  • PCPE-1 protein is considered to be transcribed, translated and secreted in brown adipose tissue and reach the liver through the bloodstream.
  • a standard may be provided when detecting NASH.
  • Examples of such a standard include a biological sample derived from a healthy person. When the expression level of the PCPE-1 gene or the PCPE-1 protein is higher than that of a healthy person, the subject is more likely to have NASH.
  • Example 1 Search for proteins secreted from brown adipose tissue
  • the inventors analyzed proteins secreted from brown adipose tissue. As a result, a NASH marker was found. The contents of this experimental example will be described in more detail below.
  • the adipose tissue includes brown adipose tissue and white adipose tissue.
  • Brown adipose tissue is known to be a thermogenic organ, but little is known about its function as an endocrine organ.
  • the inventors searched for proteins secreted by brown adipose tissue by DNA microarray analysis.
  • NCBI National Biotechnology Information Center
  • FIG. 1 is a graph showing the results of real-time PCR.
  • “**” indicates that there is a significant difference at a risk rate of less than 1%.
  • PCPE-1 protein was detected by Western blotting.
  • an anti-PCPE-1 antibody (model “MAB2239”, R & D Systems) was used.
  • GPDH glyceraldehyde 3-phosphate dehydrogenase
  • an anti-GAPDH antibody (model “# 3683S”, Cell Signaling Technology) was used.
  • FIG. 2 is a photograph showing the results of Western blotting. As a result, it has been clarified that the expression level of PCPE-1 protein is increased specifically by brown adipose tissue by obesity stress.
  • PCPE-1 level in plasma increased due to obesity stress The amount of PCPE-1 protein in the plasma of an obese mouse model in which a 4-week-old mouse was fed with a high fat diet for 8 months and a control mouse fed with a normal diet were quantified by ELISA.
  • a commercially available kit (model “DL-PCPE1-Mu”, DLDEVELOP) was used for quantification of the PCPE-1 protein.
  • FIG. 3 is a graph showing the results of quantification of mouse PCPE-1 protein.
  • “**” indicates that there is a significant difference at a risk rate of less than 1%.
  • FIG. 4 is a photograph showing the results of Western blotting. As a result, it became clear that obesity stress increases the abundance of PCPE-1 protein in the liver. As shown in Experimental Example 2, no increase in PCPE-1 gene expression in the liver was observed even when obesity stress was applied. Therefore, it was considered that the PCPE-1 protein detected in this experimental example was expressed in a place other than the liver.
  • FIG. 5 is a graph showing the quantitative results of PCPE-1 protein.
  • “*” indicates that there is a significant difference at a risk rate of less than 5%.
  • the abundance of PCPE-1 protein was increased in the serum of NASH patients. This result indicates that the PCPE-1 protein is a NASH marker.
  • a commercially available kit (model “ACE-EC1-E205-EX”, Cosmo Bio) was used for quantification of type I collagen.
  • FIG. 6 is a graph showing the results of quantification of type I collagen secretion.
  • “*” indicates that there is a significant difference when the risk rate is less than 5%
  • “**” indicates that there is a significant difference when the risk rate is less than 1%.
  • a new NASH marker can be provided.

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Abstract

Provided is a marker for non-alcoholic steatohepatitis, comprising a procollagen C-endopeptidase enhancer (PCPE-1) gene or a PCPE-1 protein.

Description

非アルコール性脂肪性肝炎マーカー及びその使用Nonalcoholic steatohepatitis marker and use thereof
　本発明は、非アルコール性脂肪性肝炎マーカー及びその使用に関する。本願は、２０１５年１０月１３日に、日本に出願された特願２０１５－２０２４１８号に基づき優先権を主張し、その内容をここに援用する。 The present invention relates to a non-alcoholic steatohepatitis marker and use thereof. This application claims priority on October 13, 2015 based on Japanese Patent Application No. 2015-202418 for which it applied to Japan, and uses the content for it here.
　近年、食生活の欧米化と運動不足による肥満人口の増加に伴い、非アルコール性脂肪性肝疾患（non-alcoholic fatty liver disease、ＮＡＦＬＤ）及び非アルコール性脂肪性肝炎（non-alcoholic steatohepatitis、ＮＡＳＨ）の患者が増加している。 In recent years, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) have been accompanied by an increase in the obese population due to westernization of diet and lack of exercise. The number of patients is increasing.
　ＮＡＦＬＤとは、飲酒歴がないにもかかわらずアルコール性肝障害に類似した脂肪性肝障害が認められる疾患である。ＮＡＦＬＤの約９０％は非進行で予後良好な単純性脂肪肝であるが、残りの約１０％は、肝硬変、肝細胞癌へと進行し得るＮＡＳＨに分類される（例えば、非特許文献１を参照）。 NAFLD is a disease in which fatty liver disorder similar to alcoholic liver disorder is observed despite no history of alcohol consumption. About 90% of NAFLD is non-progressive simple fatty liver with good prognosis, and the remaining about 10% is classified as NASH that can progress to cirrhosis and hepatocellular carcinoma (see Non-Patent Document 1, for example). reference).
　ＮＡＳＨの病態に陥ると、肝臓の線維化が進行し、肝硬変へと進展する。しかしながら、肝臓の線維化が進行する機序はほとんどわかっていない。現在、ＮＡＳＨの診断方法としては、肝臓生検による侵襲的手法しか存在しない。このため、ＮＡＳＨを容易に診断できるマーカーの開発が求められている。そこで、本発明は、新たなＮＡＳＨマーカーを提供することを目的とする。 When falling into the pathological condition of NASH, fibrosis of the liver progresses and progresses to cirrhosis. However, little is known about the mechanism by which liver fibrosis proceeds. Currently, there are only invasive techniques using liver biopsy as diagnostic methods for NASH. For this reason, development of the marker which can diagnose NASH easily is calculated | required. Therefore, an object of the present invention is to provide a new NASH marker.
　本発明は以下の態様を含む。
（１）Ｐｒｏｃｏｌｌａｇｅｎ　Ｃ－ｅｎｄｏｐｅｐｔｉｄａｓｅ　ｅｎｈａｎｃｅｒ（ＰＣＰＥ－１）遺伝子又はＰＣＰＥ－１タンパク質からなる、ＮＡＳＨマーカー。
（２）ＰＣＰＥ－１タンパク質に対する特異的結合物質、ＰＣＰＥ－１遺伝子のｃＤＮＡを増幅するためのプライマーセット、又はＰＣＰＥ－１遺伝子のｍＲＮＡに特異的にハイブリダイズするプローブ、を備える、ＮＡＳＨ診断用キット。
（３）生体試料中の、ＰＣＰＥ－１遺伝子又はＰＣＰＥ－１タンパク質の発現量を定量する工程を備える、ＮＡＳＨの検出方法。 The present invention includes the following aspects.
(1) A NASH marker comprising a Procollagen C-endopeptidase enhancer (PCPE-1) gene or PCPE-1 protein.
(2) A kit for NASH diagnosis, comprising a specific binding substance for PCPE-1 protein, a primer set for amplifying PCPE-1 gene cDNA, or a probe that specifically hybridizes to mRNA of PCPE-1 gene .
(3) A NASH detection method comprising a step of quantifying the expression level of a PCPE-1 gene or a PCPE-1 protein in a biological sample.
　本発明により、新たなＮＡＳＨマーカーを提供することができる。 According to the present invention, a new NASH marker can be provided.
実験例２のリアルタイムＰＣＲの結果を示すグラフである。6 is a graph showing the results of real-time PCR in Experimental Example 2. 実験例２のウエスタンブロッティングの結果を示す写真である。6 is a photograph showing the results of Western blotting in Experimental Example 2. 実験例３のマウスＰＣＰＥ－１タンパク質の定量結果を示すグラフである。10 is a graph showing the results of quantification of mouse PCPE-1 protein of Experimental Example 3. 実験例４のウエスタンブロッティングの結果を示す写真である。6 is a photograph showing the results of Western blotting in Experimental Example 4. 実験例５のＰＣＰＥ－１タンパク質の定量結果を示すグラフである。10 is a graph showing the results of quantification of PCPE-1 protein in Experimental Example 5. 実験例６のＩ型コラーゲンの分泌量の定量結果を示すグラフである。It is a graph which shows the fixed_quantity | quantitative_assay result of the secretion amount of the type I collagen of Experimental example 6.
［ＮＡＳＨマーカー］
　１実施形態において、本発明は、ＰＣＰＥ－１遺伝子又はＰＣＰＥ－１タンパク質からなる、ＮＡＳＨ（非アルコール性脂肪性肝炎）マーカーを提供する。 [NASH marker]
In one embodiment, the present invention provides a NASH (non-alcoholic steatohepatitis) marker consisting of the PCPE-1 gene or PCPE-1 protein.
　ＰＣＰＥ－１遺伝子は、ＰＣＯＬＣＥ遺伝子とも呼ばれ、ＰＣＰＥ－１タンパク質をコードする遺伝子である。ヒトＰＣＰＥ－１遺伝子のＧｅｎｂａｎｋアクセッション番号はＮＭ＿００２５９３であり、マウスＰＣＰＥ－１遺伝子のＧｅｎｂａｎｋアクセッション番号はＮＭ＿００８７８８である。配列番号１にヒトＰＣＰＥ－１遺伝子のｃＤＮＡの塩基配列を示し、配列番号２にヒトＰＣＰＥ－１タンパク質のアミノ酸配列を示す。また、配列番号３にマウスＰＣＰＥ－１遺伝子のｃＤＮＡの塩基配列を示し、配列番号４にマウスＰＣＰＥ－１タンパク質のアミノ酸配列を示す。 The PCPE-1 gene is also called a PCOLCE gene and is a gene encoding the PCPE-1 protein. The Genbank accession number of the human PCPE-1 gene is NM_002593, and the Genbank accession number of the mouse PCPE-1 gene is NM_008788. SEQ ID NO: 1 shows the nucleotide sequence of the cDNA of the human PCPE-1 gene, and SEQ ID NO: 2 shows the amino acid sequence of the human PCPE-1 protein. SEQ ID NO: 3 shows the base sequence of the mouse PCPE-1 gene cDNA, and SEQ ID NO: 4 shows the amino acid sequence of the mouse PCPE-1 protein.
　実施例において後述するように、発明者らは、ＮＡＳＨ患者の血清中でＰＣＰＥ－１タンパク質の存在量が増加することを見出した。また、肥満ストレスにより、褐色脂肪組織特異的にＰＣＰＥ－１遺伝子の発現レベルが上昇することを明らかにした。 As will be described later in the Examples, the inventors have found that the abundance of PCPE-1 protein increases in the serum of NASH patients. Moreover, it was clarified that the expression level of PCPE-1 gene is increased specifically by brown adipose tissue by obesity stress.
　係る知見から、ＰＣＰＥ－１遺伝子又はＰＣＰＥ－１タンパク質をＮＡＳＨマーカーとして用いることができる。本実施形態のＮＡＳＨマーカーにより、ＮＡＳＨを簡便に診断することが可能になる。また、低侵襲に診断することが可能になる。 From this knowledge, the PCPE-1 gene or PCPE-1 protein can be used as a NASH marker. The NASH marker of this embodiment makes it possible to easily diagnose NASH. Moreover, it becomes possible to make a diagnosis in a minimally invasive manner.
　本実施形態のマーカーにより、ＮＡＳＨに罹患していることが疑われる患者に対しては、運動療法や食餌療法を積極的に行うことにより、肝硬変、肝細胞癌への進行を抑制又は防止することができる。 With the marker of this embodiment, for patients suspected of suffering from NASH, by actively performing exercise therapy and diet therapy, suppressing or preventing progression to cirrhosis and hepatocellular carcinoma Can do.
［ＮＡＳＨ診断用キット］
　１実施形態において、本発明は、ＰＣＰＥ－１タンパク質に対する特異的結合物質、ＰＣＰＥ－１遺伝子のｃＤＮＡを増幅するためのプライマーセット、又はＰＣＰＥ－１遺伝子のｍＲＮＡに特異的にハイブリダイズするプローブ、を備える、ＮＡＳＨ診断用キットを提供する。 [NASH diagnostic kit]
In one embodiment, the present invention provides a specific binding substance for PCPE-1 protein, a primer set for amplifying PCPE-1 gene cDNA, or a probe that specifically hybridizes to mRNA of PCPE-1 gene. A NASH diagnostic kit is provided.
　特異的結合物質としては、例えば、抗体、抗体断片、アプタマー等が挙げられる。抗体は、例えば、マウス等の動物にＰＣＰＥ－１タンパク質又はその部分ペプチドを抗原として免疫することにより作製してもよい。あるいは、ファージライブラリー等の抗体ライブラリーのスクリーニング等により作製することもできる。 Examples of specific binding substances include antibodies, antibody fragments, aptamers and the like. The antibody may be produced, for example, by immunizing an animal such as a mouse with the PCPE-1 protein or a partial peptide thereof as an antigen. Alternatively, it can also be prepared by screening an antibody library such as a phage library.
　抗体断片としては、Ｆｖ、Ｆａｂ、ｓｃＦｖ等が挙げられる。上記の抗体又は抗体断片は、ポリクローナルであってもよく、モノクローナルであってもよい。 Antibody fragments include Fv, Fab, scFv and the like. The above antibody or antibody fragment may be polyclonal or monoclonal.
　アプタマーとは、標識物質に対する特異的結合能を有する物質である。アプタマーとしては、核酸アプタマー、ペプチドアプタマー等が挙げられる。ＰＣＰＥ－１タンパク質に特異的結合能を有する核酸アプタマーは、例えば、systematic evolution of ligand by exponential enrichment（ＳＥＬＥＸ）法等により選別することができる。また、ＰＣＰＥ－１タンパク質に対する特異的結合能を有するペプチドアプタマーは、例えば酵母を用いたＴｗｏ－ｈｙｂｒｉｄ法等により選別することができる。 An aptamer is a substance having a specific binding ability to a labeling substance. Examples of aptamers include nucleic acid aptamers and peptide aptamers. Nucleic acid aptamers having specific binding ability to the PCPE-1 protein can be selected by, for example, the systematic evolution of ligand by exponential enrichment (SELEX) method. Peptide aptamers having a specific binding ability to PCPE-1 protein can be selected by, for example, the two-hybrid method using yeast.
　特異的結合物質は、ＰＣＰＥ－１タンパク質に特異的に結合することができれば特に制限されず、市販のものであってもよい。また、特異的結合物質は、担体上に固定されてプロテインチップ等を構成していてもよい。 The specific binding substance is not particularly limited as long as it can specifically bind to the PCPE-1 protein, and may be a commercially available one. The specific binding substance may be fixed on a carrier to constitute a protein chip or the like.
　本実施形態のＮＡＳＨ診断用キットを用いて、生体試料中のＰＣＰＥ－１遺伝子の発現又はタンパク質の発現を検出することができる。ＰＣＰＥ－１遺伝子又はタンパク質の発現量が健常人と比較して上昇していた場合には、生体試料が由来する被検者がＮＡＳＨに罹患している可能性が高い。 The expression of PCPE-1 gene or protein in a biological sample can be detected using the NASH diagnostic kit of this embodiment. If the expression level of the PCPE-1 gene or protein is higher than that of healthy individuals, the subject from whom the biological sample is derived is likely to have NASH.
　プライマーセットとしては、診断対象の動物種のＰＣＰＥ－１遺伝子のｃＤＮＡを増幅することができるものであれば特に限定されない。例えば、ヒトＰＣＰＥ－１遺伝子のｃＤＮＡを増幅するプライマーとしては、配列番号５に示す塩基配列からなるセンスプライマー及び配列番号６に示す塩基配列からなるアンチセンスプライマーのセット等が挙げられる。 The primer set is not particularly limited as long as it can amplify the PCPE-1 gene cDNA of the animal species to be diagnosed. For example, the primer for amplifying the cDNA of the human PCPE-1 gene includes a set of a sense primer consisting of the base sequence shown in SEQ ID NO: 5 and an antisense primer consisting of the base sequence shown in SEQ ID NO: 6.
　ｍＲＮＡに特異的にハイブリダイズするプローブとしては、ＰＣＰＥ－１遺伝子のｍＲＮＡに特異的にハイブリダイズするものであれば特に限定されない。プローブは、担体上に固定されてＤＮＡマイクロアレイ等を構成していてもよい。 The probe that specifically hybridizes to mRNA is not particularly limited as long as it specifically hybridizes to the mRNA of the PCPE-1 gene. The probe may be fixed on a carrier to constitute a DNA microarray or the like.
　生体試料としては、血清、血漿、尿、組織等が挙げられる。組織としては、褐色脂肪組織、肝臓生検等が挙げられる。生体試料が、血清、血漿、尿等である場合、本実施形態のＮＡＳＨ診断用キットは、上記の特異的結合物質であってもよい。この場合、低侵襲で簡便にＮＡＳＨの診断を行うことができる。また、生体試料が組織等である場合、本実施形態のＮＡＳＨ診断用キットは、上記のプライマーセット又はプローブを備えるものであることが好ましい。 Examples of biological samples include serum, plasma, urine, tissue and the like. Examples of the tissue include brown adipose tissue and liver biopsy. When the biological sample is serum, plasma, urine, or the like, the NASH diagnostic kit of this embodiment may be the specific binding substance described above. In this case, NASH can be easily diagnosed with minimal invasiveness. When the biological sample is a tissue or the like, the NASH diagnostic kit of the present embodiment preferably includes the above primer set or probe.
［ＮＡＳＨの検出方法］
　１実施形態において、本発明は、生体試料中の、ＰＣＰＥ－１遺伝子又はＰＣＰＥ－１タンパク質の発現量を定量する工程を備える、ＮＡＳＨの検出方法を提供する。本実施形態の検出方法は、ＮＡＳＨの診断方法であるということもできる。 [How to detect NASH]
In one embodiment, the present invention provides a method for detecting NASH comprising the step of quantifying the expression level of a PCPE-1 gene or PCPE-1 protein in a biological sample. It can also be said that the detection method of the present embodiment is a NASH diagnosis method.
　生体試料は、上述したものと同様である。ＰＣＰＥ－１遺伝子の発現量の定量方法としては、ＰＣＰＥ－１遺伝子のｃＤＮＡを増幅するためのプライマーセットを用いたリアルタイムＰＣＲ；ＰＣＰＥ－１遺伝子のｍＲＮＡに特異的にハイブリダイズするプローブを固定したマイクロアレイによる遺伝子発現解析；ＰＣＰＥ－１遺伝子のｍＲＮＡに特異的にハイブリダイズするプローブを用いたノーザンブロッティング等が挙げられる。 The biological sample is the same as described above. PCPE-1 gene expression levels are quantified by real-time PCR using a primer set for amplifying the PCPE-1 gene cDNA; a microarray in which probes that specifically hybridize to the mRNA of the PCPE-1 gene are immobilized Analysis of gene expression by Northern blotting using a probe that specifically hybridizes to the mRNA of the PCPE-1 gene.
　また、ＰＣＰＥ－１タンパク質の発現量の定量方法としては、ＰＣＰＥ－１タンパク質に対する特異的結合物質を用いたウエスタンブロッティング、ＥＬＩＳＡ、免疫染色、逆相タンパク質アレイを用いた検出等が挙げられる。なお、逆相タンパク質アレイを用いた検出とは、試料を固相にアレイ状に固定し、特定物質に対する特異的結合物質を反応させることにより、試料中の特定物質を検出する解析方法である。 As a method for quantifying the expression level of PCPE-1 protein, Western blotting using a specific binding substance for PCPE-1 protein, ELISA, immunostaining, detection using a reverse phase protein array, and the like can be mentioned. The detection using a reversed-phase protein array is an analysis method for detecting a specific substance in a sample by fixing the sample to the solid phase in an array and reacting a specific binding substance to the specific substance.
　本実施形態の検出方法は、生検試料がＮＡＳＨ患者由来のものであるか否かを判定する方法であるということもできる。すなわち、生検試料中のＰＣＰＥ－１遺伝子又はＰＣＰＥ－１タンパク質の発現量が、健常人由来の生検試料中のＰＣＰＥ－１遺伝子又はＰＣＰＥ－１タンパク質の発現量と比較して高い場合、生検試料はＮＡＳＨ患者由来のものであると判定することができる。 It can also be said that the detection method of this embodiment is a method for determining whether or not a biopsy sample is derived from a NASH patient. That is, when the expression level of PCPE-1 gene or PCPE-1 protein in a biopsy sample is higher than the expression level of PCPE-1 gene or PCPE-1 protein in a biopsy sample derived from a healthy person, It can be determined that the test sample is from a NASH patient.
　また、生体試料中のＰＣＰＥ－１タンパク質の発現量は、生体試料中のＰＣＰＥ－１タンパク質の存在量といい換えることもできる。実施例において後述するように、ＰＣＰＥ－１タンパク質は、褐色脂肪組織で転写、翻訳、分泌され、血流を通じて肝臓に到達するものと考えられる。 In addition, the expression level of PCPE-1 protein in a biological sample can be rephrased as the abundance of PCPE-1 protein in the biological sample. As will be described later in Examples, PCPE-1 protein is considered to be transcribed, translated and secreted in brown adipose tissue and reach the liver through the bloodstream.
　ＮＡＳＨを検出する場合には標準を設けてもよい。このような標準としては、例えば、健常人由来の生体試料等が挙げられる。健常人と比較して、ＰＣＰＥ－１遺伝子又はＰＣＰＥ－１タンパク質の発現量が高い場合、被検者はＮＡＳＨに罹患している可能性が高い。 A standard may be provided when detecting NASH. Examples of such a standard include a biological sample derived from a healthy person. When the expression level of the PCPE-1 gene or the PCPE-1 protein is higher than that of a healthy person, the subject is more likely to have NASH.
　次に実施例を示して本発明を更に詳細に説明するが、本発明は以下の実施例に限定されるものではない。 Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.
［実験例１］
（褐色脂肪組織から分泌されるタンパク質の探索）
　発明者らは、褐色脂肪組織から分泌されるタンパク質を解析した。その結果、ＮＡＳＨマーカーを見出した。本実験例の内容について、以下に、より詳細に説明する。 [Experimental Example 1]
(Search for proteins secreted from brown adipose tissue)
The inventors analyzed proteins secreted from brown adipose tissue. As a result, a NASH marker was found. The contents of this experimental example will be described in more detail below.
　脂肪組織には、褐色脂肪組織と白色脂肪組織が存在する。褐色脂肪組織は熱産生器官であることが知られているが、内分泌器官としての機能についてはほとんど知られていなかった。そこで、発明者らは、褐色脂肪組織の内分泌器官としての機能を検討するために、ＤＮＡマイクロアレイ解析により褐色脂肪組織が分泌するタンパク質を探索した。 The adipose tissue includes brown adipose tissue and white adipose tissue. Brown adipose tissue is known to be a thermogenic organ, but little is known about its function as an endocrine organ. In order to examine the function of brown adipose tissue as an endocrine organ, the inventors searched for proteins secreted by brown adipose tissue by DNA microarray analysis.
　具体的には、４週齢のマウスに高脂肪食を８ヶ月間与えた肥満マウスモデルの褐色脂肪組織、及び脂肪組織特異的に低酸素状態にしたマウスの褐色脂肪組織において、共通に発現する分泌タンパク質を探索した。脂肪組織特異的に低酸素状態にしたマウスの褐色脂肪組織を用いたのは、肥満マウスの脂肪組織は低酸素状態になることが知られているためである。つまり、肥満させる以外の方法で脂肪組織を低酸素状態にしたマウスの褐色脂肪組織を用いた。より具体的には、脂肪組織特異的にVascular Endothelial Growth Factor（ＶＥＧＦ）－Ａをノックアウトしたマウスを用いた。 Specifically, it is commonly expressed in the brown adipose tissue of an obese mouse model in which a high-fat diet is given to a 4-week-old mouse for 8 months, and in the brown adipose tissue of a mouse that is hypoxic specifically to adipose tissue. We searched for secreted proteins. The reason why the brown adipose tissue of a mouse that has been hypoxic specifically for adipose tissue is used is that it is known that the adipose tissue of an obese mouse becomes hypoxic. That is, the brown adipose tissue of a mouse in which the adipose tissue was hypoxic by a method other than obesity was used. More specifically, mice in which Vascular Endothelial Growth Factor (VEGF) -A was knocked out specifically for adipose tissue were used.
　肥満食を与えた肥満マウスモデルの褐色脂肪組織で発現が上方制御される遺伝子の情報としては、全米バイオテクノロジー情報センター（ＮＣＢＩ）の遺伝子発現情報データベース（Gene Expression Omnibus；http://www.ncbi.nlm.nih.gov/geo/）にアクセッション番号ＧＳＥ２８４４０として公開されている情報を使用した。 For information on genes whose expression is up-regulated in brown adipose tissue in obese mouse models fed obese diets, see the National Biotechnology Information Center (NCBI) Gene Expression Information Database (Gene Expression Omnibus; http: //www.ncbi). .nlm.nih.gov / geo /), information published as accession number GSE28440 was used.
　その結果、褐色脂肪組織が分泌するタンパク質として、２３種類のタンパク質が同定された。それらのタンパク質のうちの１つがＰＣＰＥ－１遺伝子であった。 As a result, 23 types of proteins were identified as proteins secreted by brown adipose tissue. One of those proteins was the PCPE-1 gene.
［実験例２］
（肥満ストレスで褐色脂肪組織におけるＰＣＰＥ－１の発現レベルが上昇した）
　４週齢のマウスに高脂肪食を８週間与えた肥満マウスモデルの各臓器におけるＰＣＰＥ－１遺伝子の発現量をリアルタイムＰＣＲにより測定した。対照として通常食を与えたマウスを使用した。マウスＰＣＰＥ－１遺伝子のｃＤＮＡを増幅するためのプライマーとしては、センスプライマー（配列番号７）及びアンチセンスプライマー（配列番号８）を使用した。また、臓器としては、褐色脂肪組織、白色脂肪組織、肝臓、心臓、腎臓、肺、筋、脾臓を用いた。 [Experiment 2]
(The obesity stress increased the expression level of PCPE-1 in brown adipose tissue)
The expression level of PCPE-1 gene in each organ of an obese mouse model in which a 4-week-old mouse was fed with a high fat diet for 8 weeks was measured by real-time PCR. As a control, a mouse fed a normal diet was used. As a primer for amplifying the mouse PCPE-1 gene cDNA, a sense primer (SEQ ID NO: 7) and an antisense primer (SEQ ID NO: 8) were used. As organs, brown adipose tissue, white adipose tissue, liver, heart, kidney, lung, muscle, and spleen were used.
　図１は、リアルタイムＰＣＲの結果を示すグラフである。図中、「＊＊」は、危険率１％未満で有意差があることを示す。その結果、肥満ストレスにより、褐色脂肪組織特異的にＰＣＰＥ－１遺伝子の発現レベルが上昇することが明らかとなった。 FIG. 1 is a graph showing the results of real-time PCR. In the figure, “**” indicates that there is a significant difference at a risk rate of less than 1%. As a result, it became clear that the expression level of PCPE-1 gene was increased specifically by brown adipose tissue by obesity stress.
　続いて、ＰＣＰＥ－１のタンパクレベルでの発現を検討した。４週齢のマウスに高脂肪食を８ヶ月間与えた肥満マウスモデル及び通常食を与えた対照のマウス由来の褐色脂肪組織からタンパク質を抽出し、ウエスタンブロッティング法によりＰＣＰＥ－１タンパク質を検出した。ＰＣＰＥ－１タンパク質の検出には、抗ＰＣＰＥ－１抗体（型式「ＭＡＢ２２３９」、Ｒ＆Ｄシステムズ社）を使用した。また、ローディングコントロールとして、グリセルアルデヒド３リン酸脱水素酵素（ＧＡＰＤＨ）タンパク質を検出した。ＧＡＰＤＨタンパク質の検出には、抗ＧＡＰＤＨ抗体（型式「＃３６８３Ｓ」、Ｃｅｌｌ　Ｓｉｇｎａｌｉｎｇ　Ｔｅｃｈｎｏｌｏｇｙ社）を使用した。 Subsequently, the expression of PCPE-1 at the protein level was examined. Proteins were extracted from brown adipose tissue derived from obese mouse models in which high-fat diets were given to 4-week-old mice for 8 months and control mice fed normal diets, and PCPE-1 protein was detected by Western blotting. For the detection of PCPE-1 protein, an anti-PCPE-1 antibody (model “MAB2239”, R & D Systems) was used. As a loading control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein was detected. For the detection of GAPDH protein, an anti-GAPDH antibody (model “# 3683S”, Cell Signaling Technology) was used.
　図２は、ウエスタンブロッティングの結果を示す写真である。その結果、肥満ストレスにより、褐色脂肪組織特異的にＰＣＰＥ－１タンパク質の発現レベルが上昇することが明らかとなった。 FIG. 2 is a photograph showing the results of Western blotting. As a result, it has been clarified that the expression level of PCPE-1 protein is increased specifically by brown adipose tissue by obesity stress.
［実験例３］
（肥満ストレスで血漿中のＰＣＰＥ－１レベルが上昇した）
　４週齢のマウスに高脂肪食を８ヶ月間与えた肥満マウスモデル及び通常食を与えた対照のマウスの血漿中のＰＣＰＥ－１タンパク質の量をＥＬＩＳＡ法により定量した。ＰＣＰＥ－１タンパク質の定量には、市販のキット（型式「ＤＬ－ＰＣＰＥ１－Ｍｕ」、ＤＬＤＥＶＥＬＯＰ社）を使用した。 [Experiment 3]
(PCPE-1 level in plasma increased due to obesity stress)
The amount of PCPE-1 protein in the plasma of an obese mouse model in which a 4-week-old mouse was fed with a high fat diet for 8 months and a control mouse fed with a normal diet were quantified by ELISA. A commercially available kit (model “DL-PCPE1-Mu”, DLDEVELOP) was used for quantification of the PCPE-1 protein.
　図３はマウスＰＣＰＥ－１タンパク質の定量結果を示すグラフである。図中、「＊＊」は、危険率１％未満で有意差があることを示す。その結果、肥満ストレスにより、血漿中のＰＣＰＥ－１タンパク質の存在量が上昇することが明らかとなった。 FIG. 3 is a graph showing the results of quantification of mouse PCPE-1 protein. In the figure, “**” indicates that there is a significant difference at a risk rate of less than 1%. As a result, it became clear that the abundance of PCPE-1 protein in plasma increases due to obesity stress.
［実験例４］
（肥満ストレスにより肝臓におけるＰＣＰＥ－１レベルが上昇した）
　４週齢のマウスに高脂肪食を８ヶ月間与えた肥満マウスモデルの肝臓における、ＰＣＰＥ－１タンパク質の存在を、ウエスタンブロッティング法により検出した。対照として通常食を与えたマウスの肝臓を使用した。ＰＣＰＥ－１タンパク質の検出には、抗ＰＣＰＥ－１抗体（型式「ＭＡＢ２２３９」、Ｒ＆Ｄシステムズ社）を使用した。また、ローディングコントロールとして、グリセルアルデヒド３リン酸脱水素酵素（ＧＡＰＤＨ）タンパク質を検出した。ＧＡＰＤＨタンパク質の検出には、抗ＧＡＰＤＨ抗体（型式「＃３６８３Ｓ」、Ｃｅｌｌ　Ｓｉｇｎａｌｉｎｇ　Ｔｅｃｈｎｏｌｏｇｙ社）を使用した。 [Experimental Example 4]
(Obesity stress increased PCPE-1 levels in the liver)
The presence of PCPE-1 protein in the liver of an obese mouse model in which a 4-week-old mouse was fed with a high fat diet for 8 months was detected by Western blotting. The liver of a mouse fed a normal diet was used as a control. For the detection of PCPE-1 protein, an anti-PCPE-1 antibody (model “MAB2239”, R & D Systems) was used. As a loading control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein was detected. For detection of the GAPDH protein, an anti-GAPDH antibody (type “# 3683S”, Cell Signaling Technology) was used.
　図４は、ウエスタンブロッティングの結果を示す写真である。その結果、肥満ストレスにより、肝臓におけるＰＣＰＥ－１タンパク質の存在量が上昇することが明らかとなった。実験例２で示されたように、肥満ストレスを与えても肝臓におけるＰＣＰＥ－１遺伝子の発現の上昇は認められない。したがって、本実験例で検出されたＰＣＰＥ－１タンパク質は肝臓以外の場所で発現されたものであると考えられた。 FIG. 4 is a photograph showing the results of Western blotting. As a result, it became clear that obesity stress increases the abundance of PCPE-1 protein in the liver. As shown in Experimental Example 2, no increase in PCPE-1 gene expression in the liver was observed even when obesity stress was applied. Therefore, it was considered that the PCPE-1 protein detected in this experimental example was expressed in a place other than the liver.
［実験例５］
（ＮＡＳＨ患者の血清中でＰＣＰＥ－１レベルが上昇した）
　ＮＡＳＨ患者の血清中のＰＣＰＥ－１タンパク質の量を定量した（ｎ＝４６）。また、同時に、健常人（ｎ＝２９）及び肥満患者(ＢＭＩ２５以上)（ｎ＝２２）の血清中のＰＣＰＥ－１タンパク質の量を定量した。ＰＣＰＥ－１タンパク質の定量には、市販のキット（型式「ＭＢＳ７２２８３６」、ＭｙＢｉｏＳｏｕｒｃｅ社）を使用した。 [Experimental Example 5]
(PCPE-1 levels increased in the serum of NASH patients)
The amount of PCPE-1 protein in the serum of NASH patients was quantified (n = 46). At the same time, the amount of PCPE-1 protein in the serum of healthy individuals (n = 29) and obese patients (BMI25 or higher) (n = 22) was quantified. A commercially available kit (model “MBS722228”, MyBioSource) was used for quantification of the PCPE-1 protein.
　図５はＰＣＰＥ－１タンパク質の定量結果を示すグラフである。図中、「＊」は、危険率５％未満で有意差があることを示す。その結果、ＮＡＳＨ患者の血清中では、ＰＣＰＥ－１タンパク質の存在量が上昇することが明らかとなった。この結果は、ＰＣＰＥ－１タンパク質がＮＡＳＨマーカーであることを示す。 FIG. 5 is a graph showing the quantitative results of PCPE-1 protein. In the figure, “*” indicates that there is a significant difference at a risk rate of less than 5%. As a result, it was revealed that the abundance of PCPE-1 protein was increased in the serum of NASH patients. This result indicates that the PCPE-1 protein is a NASH marker.
［実験例６］
（ＰＣＰＥ－１は肝星細胞によるＩ型コラーゲンの分泌を促進した）
　ＮＡＳＨ患者の肝臓においてＴｒａｎｓｆｏｒｍｉｎｇ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ（ＴＧＦ）－βの発現が上昇することが知られている。そこで、ヒト肝星細胞株ＬＸ－２を、終濃度１．０ｎｇ／ｍＬのＴＧＦ－β（型式「２４０－Ｂ－００２」、Ｒ＆Ｄ社）の存在下で培養した。上記の細胞の培地に、リン酸緩衝液（ＰＢＳ）（ｎ＝４）又は終濃度８．５μＭのＰＣＰＥ－１タンパク質（型式「２６２７－ＰＥ－０２０」、Ｒ＆Ｄ社）（ｎ＝４）を添加して２４時間培養し、Ｉ型コラーゲンの分泌量を定量した。対照として、ＴＧＦ－βの非存在下で培養したＬＸ－２細胞の培地に、リン酸緩衝液（ＰＢＳ）（ｎ＝６）又は終濃度８．５μＭのＰＣＰＥ－１タンパク質（ｎ＝８）を添加して２４時間培養し、Ｉ型コラーゲンの分泌量をＥＬＩＳＡ法により定量した。Ｉ型コラーゲンの定量には市販のキット（型式「ＡＣＥ－ＥＣ１－Ｅ２０５－ＥＸ」、コスモバイオ社）を使用した。 [Experimental Example 6]
(PCPE-1 promoted secretion of type I collagen by hepatic stellate cells)
It is known that the expression of transforming growth factor (TGF) -β increases in the liver of NASH patients. Therefore, human hepatic stellate cell line LX-2 was cultured in the presence of TGF-β (type “240-B-002”, R & D) at a final concentration of 1.0 ng / mL. Add phosphate buffer (PBS) (n = 4) or 8.5 μM PCPE-1 protein (type “2627-PE-020”, R & D) (n = 4) to the cell culture medium. And cultured for 24 hours, and the amount of type I collagen secreted was quantified. As a control, phosphate buffer (PBS) (n = 6) or a final concentration of 8.5 μM PCPE-1 protein (n = 8) was added to the medium of LX-2 cells cultured in the absence of TGF-β. After addition, the cells were cultured for 24 hours, and the amount of type I collagen secreted was quantified by ELISA. A commercially available kit (model “ACE-EC1-E205-EX”, Cosmo Bio) was used for quantification of type I collagen.
　図６は、Ｉ型コラーゲンの分泌量の定量結果を示すグラフである。図中、「＊」は、危険率５％未満で有意差があることを示し、「＊＊」は、危険率１％未満で有意差があることを示す。 FIG. 6 is a graph showing the results of quantification of type I collagen secretion. In the figure, “*” indicates that there is a significant difference when the risk rate is less than 5%, and “**” indicates that there is a significant difference when the risk rate is less than 1%.
　その結果、ＰＣＰＥ－１タンパク質の添加により、ＬＸ－２細胞によるＩ型コラーゲンの分泌量が増加することが明らかとなった。この結果は、ＰＣＰＥ－１タンパク質が、肝臓の線維化に関与することを示す。 As a result, it was revealed that the amount of type I collagen secreted by LX-2 cells was increased by the addition of PCPE-1 protein. This result indicates that PCPE-1 protein is involved in liver fibrosis.
　本発明により、新たなＮＡＳＨマーカーを提供することができる。 According to the present invention, a new NASH marker can be provided.

Claims (3)

  1. 　Ｐｒｏｃｏｌｌａｇｅｎ　Ｃ－ｅｎｄｏｐｅｐｔｉｄａｓｅ　ｅｎｈａｎｃｅｒ（ＰＣＰＥ－１）遺伝子又はＰＣＰＥ－１タンパク質からなる、非アルコール性脂肪性肝炎マーカー。 Non-alcoholic steatohepatitis marker consisting of Procollagen C-endopeptidase enhancer (PCPE-1) gene or PCPE-1 protein.
  2. 　ＰＣＰＥ－１タンパク質に対する特異的結合物質、
    　ＰＣＰＥ－１遺伝子のｃＤＮＡを増幅するためのプライマーセット、又は
    　ＰＣＰＥ－１遺伝子のｍＲＮＡに特異的にハイブリダイズするプローブ、
    　を備える、非アルコール性脂肪性肝炎診断用キット。     Specific binding substances for PCPE-1 protein,
    A primer set for amplifying the PCPE-1 gene cDNA, or a probe that specifically hybridizes to the mRNA of the PCPE-1 gene;
    A diagnostic kit for non-alcoholic steatohepatitis.
  3. 　生体試料中の、ＰＣＰＥ－１遺伝子又はＰＣＰＥ－１タンパク質の発現量を定量する工程を備える、非アルコール性脂肪性肝炎の検出方法。 A method for detecting nonalcoholic steatohepatitis, comprising a step of quantifying the expression level of a PCPE-1 gene or PCPE-1 protein in a biological sample.
PCT/JP2016/080346 2015-10-13 2016-10-13 Marker for non-alcoholic steatohepatitis and use thereof WO2017065206A1 (en)

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WO2023175277A1 (en) 2022-03-17 2023-09-21 Nvh Medicinal Ligands specific for the pcpe-1 glycoprotein and uses thereof

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019164749A1 (en) 2018-02-26 2019-08-29 Arizona Board Of Regents On Behalf Of The University Of Arizona Ezetimibe metabolite as a non-invasive biomarker for non-alcoholic steatohepatitis (nash)
EP3759500A4 (en) * 2018-02-26 2021-12-08 Arizona Board of Regents on behalf of the University of Arizona Ezetimibe metabolite as a non-invasive biomarker for non-alcoholic steatohepatitis (nash)
WO2023175277A1 (en) 2022-03-17 2023-09-21 Nvh Medicinal Ligands specific for the pcpe-1 glycoprotein and uses thereof
FR3133609A1 (en) 2022-03-17 2023-09-22 Nvh Medicinal PCPE-1 glycoprotein specific ligands and their uses

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