WO2010005077A1 - Disease-related protein for parkinson’s disease, and use thereof - Google Patents

Disease-related protein for parkinson’s disease, and use thereof Download PDF

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WO2010005077A1
WO2010005077A1 PCT/JP2009/062595 JP2009062595W WO2010005077A1 WO 2010005077 A1 WO2010005077 A1 WO 2010005077A1 JP 2009062595 W JP2009062595 W JP 2009062595W WO 2010005077 A1 WO2010005077 A1 WO 2010005077A1
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protein
parkinson
disease
value
patient
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PCT/JP2009/062595
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French (fr)
Japanese (ja)
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勲 金子
博司 北野
正幸 横田
雄一 後藤
美穂 村田
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財団法人ヒューマンサイエンス振興財団
独立行政法人医薬基盤研究所
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    • 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
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette

Definitions

  • the present invention relates to a disease-related protein of Parkinson's disease, its use in a method for determining a pathological condition of Parkinson's disease, a kit for determining a pathological condition, a screening method for a therapeutic drug, and the like.
  • Parkinson's disease is a progressive disease accompanied by movement disorders such as slow movements, muscle rigidity, tremors, and gait disturbances.
  • movement disorders such as slow movements, muscle rigidity, tremors, and gait disturbances.
  • One person per 100 people is 55 years or older, and 100 people are 65 years or older. It is said that two people will develop the disease.
  • Its pathological features are that selective degeneration / necrosis of dopaminergic neurons in the substantia nigra of the cerebrum occurs and that Lewy bodies are observed in dopaminergic neurons (Non-Patent Documents 1 to 4). Parkinson's disease is progressive, but Parkinson's syndrome can generally be diagnosed because it is not progressive.
  • the present invention is effective in determining the pathological condition of Parkinson's disease, quickly finds a serum protein that can reflect the therapeutic effect and can be used for the prognosis, and a therapeutic monitoring method and prognostic method for Parkinson's disease using them. It was an object to provide a kit and a screening method for a therapeutic drug for Parkinson's disease.
  • the present inventors have conducted intensive research in view of the above problems, and using cICAT methods (Hansen, K. C. et al, Mol. Cell Proteomics, 2, 299-314 (2003)) Comparative quantitative analysis of serum proteins of patients was performed. Specifically, cerebrospinal fluid and healthy human serum closer to the diseased tissue are analyzed by the cICAT method, and then the same cerebrospinal fluid and Parkinson's disease patient serum are similarly analyzed by the cICAT method. An analysis method enabling identification and comparative quantification of low-expressing serum proteins derived from lesion tissues that could not be detected was performed (Japanese Patent Application No. 2008-054990). As a result, a protein specifically present in Parkinson's disease patients was found. Therefore, the present inventors have found a serum protein that is an effective marker for determining the pathological condition of Parkinson's disease, reflects the therapeutic effect quickly, and can be used for prognosis, and has completed the present invention.
  • the present invention relates to: (1) A method for determining the pathological condition of Parkinson's disease, comprising examining the amount of a protein identified as a marker for Parkinson's disease by a method comprising the following steps (a) to (c): (A) Analyzing tissue-derived protein of Parkinson's disease patient and serum protein of healthy person, and comparative quantitative value for the identified protein: Analytical value of protein derived from tissue of Parkinson's disease patient / Analytical value of serum protein of healthy person ...
  • the protein identified as the marker is a protein having a value of 5 or more or 1/5 or less (analyzed value of serum protein of Parkinson's disease patient / analyzed value of serum protein of healthy subject) (1) The method described in; (3) Proteins identified as the markers are latrophilin 3 (gi
  • a marker protein for Parkinson's disease in a sample (especially serum) obtained from a patient, it is possible to quickly and accurately determine a disease state, for example, treatment monitoring, prognosis, etc. Furthermore, screening for therapeutic agents can also be performed.
  • FIG. 1 is a diagram showing comparative quantitative values of serum from healthy individuals (JNS) and cerebrospinal fluid (PD-CSF) from Parkinson's disease patients.
  • proteins are arranged in order from the protein with the highest H / L ratio.
  • FIG. 2 is a view showing comparative quantitative values of serum of a Parkinson's disease patient (PDS) and cerebrospinal fluid (PD-CSF) of a Parkinson's disease patient.
  • proteins are arranged in order from the protein with the highest H / L ratio.
  • proteins are arranged in order from the protein with the smallest Gi #.
  • the present invention provides a method for determining the pathology of Parkinson's disease.
  • This determination method includes examining the amount of a protein identified as a marker of Parkinson's disease by an identification procedure including the following steps (a) to (c).
  • an identification procedure including the following steps (a) to (c).
  • the “analyzed value of protein derived from tissue of Parkinson's disease patient” in the formula is a value obtained by analyzing a protein fraction derived from tissue of Parkinson's disease patient.
  • the “analyzed value of serum protein of healthy person” in the formula is a value obtained by analyzing a protein fraction derived from the serum of healthy person.
  • the same protein derived from the tissue of the Parkinson's disease patient as in (a) and the serum protein of the Parkinson's disease patient are analyzed, and a comparative quantitative value for the identified protein: (in the same Parkinson's disease patient as in (a)) Analyzed value of tissue-derived protein / analyzed value of serum protein of Parkinson's disease patient).
  • the “analyzed value of the protein derived from the tissue of the same Parkinson's disease patient as in (a)” was obtained by analyzing the protein fraction derived from the same tissue as that of the same Parkinson's disease patient as in the step (a).
  • “Analyzed value of serum protein of Parkinson's disease patient” indicates a value obtained by analyzing the serum protein fraction of Parkinson's disease patient. Further, in step (c), (analyzed value of protein derived from tissue of Parkinson's disease patient / analyzed value of serum protein of healthy subject) ⁇ (analyzed value of protein derived from tissue of Parkinson's disease patient / analysis of serum protein of Parkinson's disease patient) The value of (analyzed value of serum protein of Parkinson's disease patient / analyzed value of serum protein of healthy person) is obtained according to (value). This obtained value enables comparative quantification of proteins present in the serum of Parkinson's disease patients and healthy individuals. In other words, the greatest advantage of this method is that proteins in serum that are relatively different in abundance between healthy individuals and Parkinson's disease patients can be identified.
  • Parkinson's disease marker protein identification procedures (a) to (c) used in the method of the present invention will be described in detail.
  • the first step of the identification procedure is: (A) Analyzing tissue-derived protein of Parkinson's disease patient and serum protein of healthy person, and comparative quantitative value for the identified protein: Analytical value of protein derived from tissue of Parkinson's disease patient / Analytical value of serum protein of healthy person ... (A) It is the process of obtaining.
  • Parkinson's disease patients are humans according to the following explanation, but they may be pets such as dogs and cats, or livestock such as cows, horses and pigs. Therefore, the present invention can be applied not only to humans but also to veterinary medicine, for example. Parkinson's disease patients include not only those with Parkinson's disease (patients) but also those suspected of having Parkinson's disease.
  • analyzing a protein means identifying proteins in samples obtained from Parkinson's disease patients and healthy persons and examining their abundance. The abundance may be determined by a relative ratio.
  • the protein derived from the tissue of Parkinson's disease patient is a protein derived from a lesion site or a potential lesion site of Parkinson's disease patient.
  • the types of proteins to be identified increase and the accuracy of diagnosis improves.
  • the analysis value of the protein derived from the tissue of the Parkinson's disease patient is canceled out, and therefore the protein derived from the tissue of the Parkinson's disease patient is any Parkinson's disease patient (healthy person). Or a protein obtained from any site.
  • a tissue of a Parkinson's disease patient for example, a sample of a lesion site that is commercially available or available from other routes may be used. Therefore, even if it is difficult or impossible to collect a protein sample from a lesion site of a Parkinson's disease patient due to technical or ethical problems, the above identification procedure can be performed.
  • the serum of a healthy person may be obtained from any part / tissue of a healthy person, for example, from the peripheral blood of a healthy person. It is preferable that the serum of a healthy person is obtained from the same site / tissue as the serum of a Parkinson's disease patient. For example, when the serum of a healthy person is obtained from peripheral blood, it is preferable that the serum of Parkinson's disease patient is also obtained from the peripheral blood.
  • the protein analysis means / method in the above identification procedure can be used by appropriately selecting one known to those skilled in the art.
  • isotope labeling is used for protein analysis.
  • examples of the isotope labeling method include cICAT method and iTRAQ method.
  • the protein is labeled with an appropriate isotope-containing reagent (cICAT reagent).
  • cICAT reagent Prior to labeling the protein, using general techniques, albumin, IgG, IgA, IgM, ⁇ 2-macroglobulin, ⁇ 1-antitrypsin, transferrin, haptoglobin, apo, present in high concentrations in each protein fraction.
  • Proteins such as AI, Apo A-II, C3 and transthyretin may be removed.
  • a general removal method that can be used in this step may be performed using a commercially available antibody column such as an antibody column (Hu 14, 10 Column 100 x 100 mm) manufactured by Agilent, but is not limited thereto.
  • a protein fraction derived from a tissue of Parkinson's disease patient may be separately labeled with a heavy chain (H chain) reagent, and a serum protein (NHS) fraction of a Parkinson's disease patient may be separately labeled with a light chain (L chain) reagent ( Or vice versa).
  • the tissue-derived protein fraction of Parkinson's disease patient used in the identification procedure of Parkinson's disease marker protein may be derived from a pool of cerebrospinal fluid (CSF) of a plurality of Parkinson's disease patients.
  • the serum protein fraction of a healthy person may be derived from a pool of a plurality of healthy person's sera.
  • the protein may be purified by a general method among those skilled in the art. That is, the labeled protein may be treated with a standard protease that fragments a peptide such as trypsin. Thereafter, in the present invention, in the purification step of the labeled protein, a plurality of fractions may be obtained using general column chromatography such as SCX column chromatography (4.6 ⁇ 100 mm). Alternatively, the labeled protein may be recovered using a standard avidin column. Furthermore, each protein fraction may be treated with TFA to cleave the biotin moiety.
  • a standard protease that fragments a peptide such as trypsin.
  • a plurality of fractions may be obtained using general column chromatography such as SCX column chromatography (4.6 ⁇ 100 mm).
  • the labeled protein may be recovered using a standard avidin column.
  • each protein fraction may be treated with TFA to cleave the biotin moiety.
  • MS general mass spectrometry
  • a nano-LC (LC-Packings) / QSTAR XL (AB, ESI-Q / TOF) mass spectrometer may be used.
  • the comparative quantitative value of each protein according to formula (A) is obtained.
  • the comparative quantitative value of each protein according to formula (A) may be calculated using a generally available integrated database such as HiSpec (Hitachi).
  • the second step of the identification procedure is: (B) Analyzing the same tissue-derived protein of Parkinson's disease patient as in (a) and serum protein of Parkinson's disease patient, and comparative quantitative value for the identified protein: Analysis value of protein derived from tissue of Parkinson's disease patient / Analysis value of serum protein of Parkinson's disease patient ... (B) It is the process of obtaining.
  • a comparative quantitative value is obtained by dividing the analysis value of the protein derived from the tissue of the Parkinson's disease patient by the analysis value of the serum protein of the Parkinson's disease patient. Moreover, you may calculate the comparative quantitative value of each protein by Formula (B) using generally available integrated databases, such as HiSpec (Hitachi).
  • the serum of a Parkinson's disease patient may be obtained from any site / tissue of the patient.
  • serum may be obtained from the peripheral blood of Parkinson's disease patients.
  • the third step of the identification procedure is: (C) (analyzed value of protein derived from tissue of Parkinson's disease patient / analyzed value of serum protein of healthy person) / (analyzed value of protein derived from tissue of Parkinson's disease patient / analyzed value of serum protein of Parkinson's disease patient) (C ) According to (Analyzed value of serum protein in patients with Parkinson's disease / Analyzed value of serum protein in healthy individuals) Is a step of obtaining the value of.
  • the formula (C) is obtained by dividing the comparative quantitative value of each protein obtained from the formula (A) by the comparative quantitative value of each protein obtained from the formula (B) to obtain (analyzed value of serum protein of Parkinson's disease patient). ) / (Analyzed value of serum protein of healthy person).
  • the present invention also provides a protein identified as a marker protein for Parkinson's disease by the above identification procedure.
  • the standard for identifying a marker protein of Parkinson's disease is that the comparative quantitative value (value of the expression in the above step (c)) of 1 (analyzed value of serum protein of Parkinson's disease patient / analyzed value of serum protein of healthy person) is 1. It can be high or low.
  • the value is 2 or more, preferably 5 or more, more preferably 10 or more.
  • the value is 1/2 or less, preferably 1/5 or less, more preferably 1/10 or less.
  • the comparative quantitative value is a protein of 5 or more or 1/5 or less from the viewpoint of diagnostic accuracy.
  • a preferable marker protein of Parkinson's disease in the method for determining a disease state of the present invention among the proteins identified by the above identification procedure, (analyzed value of serum protein of Parkinson's disease patient / analyzed value of serum protein of healthy person) A protein having a comparative quantitative value of 5 or more may be mentioned.
  • latrophilin 3 latrophilin homolog 3 (cow); lectomedin 3) (gi
  • the amount of these proteins can be increased in the body of Parkinson's disease patients, particularly in serum, compared to healthy individuals.
  • a protein having the comparative analysis value of 1/5 or less can be mentioned.
  • lactotransferrin gi
  • decorin isoform a preproprotein small leucine-rich protein 1B
  • extracellular precursor gi
  • amyloid beta A4 precursor-like protein 2 ( gi
  • the amount of these proteins can be reduced in the body of Parkinson's disease patients, particularly in serum, compared to healthy individuals.
  • extracellular precursor (gi
  • O 2 ⁇ active oxygen
  • this enzyme is deficient, it is considered that cell damage caused by extracellular superoxide (O 2 ⁇ ) may progress (Li, Y. et al., Na. Genet. 11, 367-380 (1995) and Muller, F. et al., Free Radic. Biol. Med 40, 1993-2004 (2006)).
  • superoxide dismutase 1 soluble (gi
  • the method of determining the pathological condition of Parkinson's disease of the present invention includes examining the amount of marker protein in a sample obtained from a patient.
  • the marker protein to be used has been identified by the above procedure.
  • the patient to which the method for determining a disease state of the present invention can be applied includes pets such as dogs and cats, and domestic animals such as cows, horses and pigs, preferably primates, particularly preferably humans. Therefore, the disease state determination method of the present invention can be applied not only to humans but also to veterinary medicine, for example.
  • the patient may be a person suspected of having Parkinson's disease as well as a patient suffering from Parkinson's disease.
  • the type of sample from the patient is not particularly limited.
  • the sample may be a bodily fluid, and examples of the bodily fluid sample include whole blood, serum, urine, sweat, saliva, ascites, and the like is preferable.
  • Various sample collection methods are known and used in the field, and can be appropriately selected and used.
  • blood can be collected from an arm vein, and serum can be obtained by a normal separation method such as centrifugation.
  • a patient and / or a healthy person obtain a sample by the same collection method and measure the amount of the marker protein by the same measurement method.
  • the protein detection and quantification method is not particularly limited, and methods known in the art can be used. Examples thereof include affinity chromatography, two-dimensional electrophoresis, isotope labeling such as the above-described cICAT or iTRAQ, and immunological methods using an antibody specific to the marker protein. . Methods used for proteomic analysis such as two-dimensional electrophoresis and isotope labeling may be applied. Immunological methods may be used to detect and quantify the marker protein. An antibody against the marker protein can be prepared by a known method and analyzed by a known method such as an ELISA method or an immunostaining method.
  • the isotope labeling method is not only qualitative but also excellent in quantitativeness.
  • the cICAT method (Hansen, K.C. et al, Mol. Cell Proteomics, 2, 299-314 (2003)) is more preferable.
  • the cICAT method is used as an effective means for analyzing a disease-related protein by conducting a comprehensive expression difference analysis between a patient and a healthy person or a protein before and after treatment, and is also used effectively in the present invention.
  • isotope labeling is combined with mass spectrometry (MS).
  • MS mass spectrometry
  • mRNA transcribed from the nucleotide sequence encoding the protein may be detected and / or quantified.
  • the detection and quantification of mRNA may be performed using a general method, for example, a quantitative real-time RT-PCR method for amplifying the full length or a part of mRNA transcribed from the nucleotide sequence encoding the protein of the present invention.
  • the present invention is not limited to this as long as mRNA can be detected and quantified.
  • the amount of the marker protein in a sample obtained from a patient under treatment and the same patient before and / or after treatment and / or from a healthy person are obtained in the same manner.
  • the marker protein in the sample examined in the same manner may be compared.
  • the term “during treatment” refers to a period during which a patient is treated with L-DOPA or chemotherapy.
  • a sample is obtained from the patient at least once before, during and / or after treatment.
  • the sample is sampled from the patient at appropriate time intervals before, during and after treatment.
  • the time-dependent change of the amount of the marker protein in the sample of Parkinson's disease patient can be examined in more detail, and the pathological condition of Parkinson's disease can be analyzed in more detail.
  • the presence / absence and extent of the patient's response to the treatment, or the state of treatment of the patient can be examined in more detail, and future changes in the disease state can be predicted with higher accuracy.
  • the method for determining the pathological condition of Parkinson's disease of the present invention can also be used for treatment monitoring and prognosis of Parkinson's disease.
  • the present invention provides a kit for determining the pathological condition of Parkinson's disease, which essentially comprises means for measuring the amount of the protein identified as a marker of Parkinson's disease.
  • the kit essentially includes means for measuring the amount of the marker protein in the sample in the above-described method of the present invention.
  • various methods for measuring the amount of marker protein in a sample are known, and can be appropriately selected and used.
  • the kit of the present invention contains a marker protein-specific monoclonal antibody, an ELISA microtiter plate, an appropriate coloring reagent, and the like.
  • kit of the present invention is that the above-mentioned protein is measured using an isotope labeling method, particularly a cICAT method.
  • a kit may contain reagents and instruments necessary for isotope labeling.
  • the kit of the present invention is preferably used for analysis of the pathology of Parkinson's disease.
  • the present invention provides a screening method for a therapeutic agent for Parkinson's disease.
  • the method includes administering a candidate agent to an animal suffering from Parkinson's disease, and then examining the amount of the marker protein in a sample taken from the animal before and after administration of the candidate agent.
  • the amount of marker protein decreases or increases, and as it worsens, the amount of marker protein increases or decreases. That is, it is considered that the marker protein is involved in the onset and progression of Parkinson's disease. Therefore, whether the therapeutic effect of Parkinson's disease is good or not can be determined by examining how much the amount of the marker protein in the sample is increased or decreased by administration of the candidate drug.
  • the above-described marker protein inhibitors, antagonists, antibodies and the like are candidates as therapeutic or preventive agents for Parkinson's disease.
  • the therapeutic agent for Parkinson's disease obtained by screening by such a method is also encompassed in the present invention.
  • a general marker protein for Parkinson's disease may be used in combination with the above method and kit.
  • markers include, but are not limited to, proteins such as Parkin, DJ-1, ⁇ -synuclein, PINK1, UCHL-1, and LRRK2 / dardarin.
  • the present invention provides a method for identifying a marker for Parkinson's disease used for determination of the pathological condition of Parkinson's disease.
  • the method includes the following steps (a) to (c): (A) Analyzing tissue-derived protein of Parkinson's disease patient and serum protein of healthy person, and comparative quantitative value for the identified protein: Analytical value of protein derived from tissue of Parkinson's disease patient / Analytical value of serum protein of healthy person ...
  • a marker of Parkinson's disease used for the determination of the above-mentioned pathological condition of Parkinson's disease, 5 or more or 1/5 or less (analyzed value of serum protein of Parkinson's disease patient / analyzed value of serum protein of healthy person) It is preferable to identify a protein having a value as a marker for Parkinson's disease to be used for the determination of the pathology of Parkinson's disease.
  • the present invention in another embodiment, is used as a marker for Parkinson's disease for the determination of the pathology of Parkinson's disease, latrophilin 3 (gi
  • superoxide dismutase 3 extracellular precursor (gi
  • Parkinson's disease patient serum (PDS: pooled for 10 patients), Japanese healthy standard blood serum (JNS: pooled for 10 patients) and Parkinson's disease patient cerebrospinal fluid (PD-CSF: Proteins obtained by pooling CSF of 10 Parkinson's disease patients (as lesion tissue-derived proteins) were identified and subjected to comparative quantitative analysis by the cICAT method.
  • PDS Parkinson's disease patient serum
  • JNS Japanese healthy standard blood serum
  • PD-CSF Parkinson's disease patient cerebrospinal fluid
  • an antibody column (Hu14, column 10 ⁇ 100 mm) manufactured by Agilent, albumin, IgG, IgA, IgM, ⁇ 2-macroglobulin, ⁇ 1-antitrypsin, transferrin, haptoglobulin, Apo AI, Apo A-II, C3, PD-CSF (100 ⁇ g protein) and JNS (100 ⁇ g) from which transthyretin, ⁇ 1-acidic glycoprotein, ⁇ 2-acidic protein had been removed were labeled with cICAT-H chain reagent and cICAT-L chain reagent, respectively.
  • the obtained cICAT peptide was analyzed with nano-LC / Q-Star XL, and using Hitachi integrated database system (HiSpec), cerebrospinal fluid (PD-CSF, H chain labeled) and healthy subject serum (JNS, Comparison of each protein with L chain label) The amount value (H / L) was calculated.
  • HiSpec Hitachi integrated database system
  • PD-CSF cerebrospinal fluid
  • JNS healthy subject serum
  • the denominator and numerator were ⁇ and 0, comparative quantitative calculation was impossible.
  • PDS / JNS there were 104 types that could not be comparatively quantified, and 202 types that could be comparatively quantified.
  • FIG. 3 shows the comparative quantitative values of proteins (202 types) that can be comparatively quantified, rearranged in the order of Gi #.
  • Parkinson's syndrome patient serum (PSS: pooled for 10 patients), Japanese healthy standard serum (JNS: pooled for 10 persons) and Parkinson's disease patients Proteins of cerebrospinal fluid (PD-CSF: pooled CSF of 10 Parkinson's disease patients (as lesion tissue-derived proteins) were similarly subjected to identification and comparative quantification analysis by cICAT method. Comparative quantitative values (PSS / JNS) are also shown in Table 1 for reference. * NA in the table indicates that quantification is impossible due to peak congestion.
  • the present invention can be used in the field of research on Parkinson's disease, the manufacture of kits for determining their pathology, and the field of drug discovery and screening for new therapeutic drugs for Parkinson's disease.

Abstract

Disclosed are: a method for determining the condition of Parkinson’s disease, which involves determining the quantity of a protein that is identified as a marker for Parkinson’s disease by a method which involves obtaining a value of [the (analysis value for the protein in the serum from a Parkinson’s disease patient)/(analysis value for the protein in the serum from a normal person) ratio]; a kit for use in the method; a method for the screening of a therapeutic agent for Parkinson’s disease; and others.

Description

パーキンソン病の疾患関連たんぱく質およびその使用Disease-related proteins for Parkinson's disease and uses thereof
　本発明は、パーキンソン病の疾患関連たんぱく質、パーキンソン病の病態の判定方法におけるその使用、病態判定用キット、治療薬のスクリーニング方法等に関する。 The present invention relates to a disease-related protein of Parkinson's disease, its use in a method for determining a pathological condition of Parkinson's disease, a kit for determining a pathological condition, a screening method for a therapeutic drug, and the like.
　パーキンソン病は、動作の緩慢、筋の固縮、振戦、歩行障害等の運動障害を伴う進行性疾患であり、５５歳以上であると１００人に１人、６５歳以上であると１００人に２人の頻度で発症するとされている。その病理学的特徴は、大脳の黒質のドパミン産生神経細胞の選択的変性・壊死が起こることと、Ｌｅｗｙ小体がドパミン産生神経細胞に認められることである（非特許文献１～４）。また、パーキンソン病は進行性であるが、パーキンソン様症候群は一般的には進行性でないことからも診断ができる。しかし、何れの方法でも簡便でなく、健常人と各疾患の区別および両疾患の診断を可能にする簡便で正確な方法を見出すことが強く望まれていた。特に、パーキンソン病の診断を可能にし、病気の進行状況および治療効果の病態を迅速に反映できる血清中のマーカーたんぱく質を特定することおよびパーキンソン病の発症原因たんぱく質を特定することは極めて重要であると切望されていた。
Litvan, I. et al, Arch Neurol, 55,969-978(1998) Gelb, D.J. et al, Arch Neurol, 56,33-39(1999) Huges, A.J. et al, J. Neurol Neurosurg Psychiatry, 55,181-184(1992) Jankovic, J. et al, Arch Neurol, 57,369-372(2000) Parkinson's disease is a progressive disease accompanied by movement disorders such as slow movements, muscle rigidity, tremors, and gait disturbances. One person per 100 people is 55 years or older, and 100 people are 65 years or older. It is said that two people will develop the disease. Its pathological features are that selective degeneration / necrosis of dopaminergic neurons in the substantia nigra of the cerebrum occurs and that Lewy bodies are observed in dopaminergic neurons (Non-Patent Documents 1 to 4). Parkinson's disease is progressive, but Parkinson's syndrome can generally be diagnosed because it is not progressive. However, none of the methods is simple, and it has been strongly desired to find a simple and accurate method that enables a healthy person to be distinguished from each disease and to diagnose both diseases. In particular, it is extremely important to identify a marker protein in serum that can diagnose Parkinson's disease, and to quickly reflect the disease progress and therapeutic effect, and to identify the protein that causes Parkinson's disease. It was anxious.
Litvan, I. et al, Arch Neurol, 55,969-978 (1998) Gelb, DJ et al, Arch Neurol, 56, 33-39 (1999) Huges, AJ et al, J. Neurol Neurosurg Psychiatry, 55,181-184 (1992) Jankovic, J. et al, Arch Neurol, 57,369-372 (2000)
　本発明は、パーキンソン病の病態判定に有効であり、治療効果を迅速に反映し、予後にも用いることのできる血清たんぱく質を見出し、それらを用いたパーキンソン病の治療モニタリング法や予後方法、そのためのキット、ならびにパーキンソン病の治療薬のスクリーニング方法等を提供することを課題とした。 The present invention is effective in determining the pathological condition of Parkinson's disease, quickly finds a serum protein that can reflect the therapeutic effect and can be used for the prognosis, and a therapeutic monitoring method and prognostic method for Parkinson's disease using them. It was an object to provide a kit and a screening method for a therapeutic drug for Parkinson's disease.
　本発明者らは、上記課題に鑑みて鋭意研究を重ね、ｃＩＣＡＴ法（Hansen, K. C. et al, Mol. Cell Proteomics, 2, 299-314(2003)）を用いて健常人およびパーキンソン病患者の血清中たんぱく質の比較定量解析を行った。詳細には、病変組織により近い脳脊髄液と健常人血清をｃＩＣＡＴ法で解析し、次いで同じ脳脊髄液とパーキンソン病患者血清を同様にｃＩＣＡＴ法で解析し、両者を連結させることにより、それまで検出不可能であった病変組織に由来した低発現血清たんぱく質の同定および比較定量を可能にする解析法を行った（特願２００８－０５４９９０）。その結果、パーキンソン病患者に特異的に存在するたんぱく質を見出した。したがって、本発明者らは、パーキンソン病の病態判定に有効なマーカーであり、治療効果を迅速に反映し、予後にも用いることのできる血清たんぱく質を見出し、本発明を完成させるに至った。 The present inventors have conducted intensive research in view of the above problems, and using cICAT methods (Hansen, K. C. et al, Mol. Cell Proteomics, 2, 299-314 (2003)) Comparative quantitative analysis of serum proteins of patients was performed. Specifically, cerebrospinal fluid and healthy human serum closer to the diseased tissue are analyzed by the cICAT method, and then the same cerebrospinal fluid and Parkinson's disease patient serum are similarly analyzed by the cICAT method. An analysis method enabling identification and comparative quantification of low-expressing serum proteins derived from lesion tissues that could not be detected was performed (Japanese Patent Application No. 2008-054990). As a result, a protein specifically present in Parkinson's disease patients was found. Therefore, the present inventors have found a serum protein that is an effective marker for determining the pathological condition of Parkinson's disease, reflects the therapeutic effect quickly, and can be used for prognosis, and has completed the present invention.
　すなわち、本発明は下記に関するものである：
　（１）パーキンソン病の病態の判定方法であって、下記工程（ａ）～（ｃ）を含む方法によりパーキンソン病のマーカーと同定されたたんぱく質の量を調べることを含む方法：
　（ａ）パーキンソン病患者の組織由来たんぱく質および健常人の血清たんぱく質を解析し、同定たんぱく質に関する比較定量値：
　　パーキンソン病患者の組織由来たんぱく質の解析値／健常人の血清たんぱく質の解析値…（Ａ）
を得て；
　（ｂ）（ａ）と同一のパーキンソン病患者の組織由来たんぱく質およびパーキンソン病患者の血清たんぱく質を解析し、同定たんぱく質に関する比較定量値：
　　（ａ）と同一のパーキンソン病患者の組織由来たんぱく質の解析値／パーキンソン病患者の血清たんぱく質の解析値…（Ｂ）
を得て；次いで
　（ｃ）（パーキンソン病患者の組織由来たんぱく質の解析値／健常人の血清たんぱく質の解析値）÷（パーキンソン病患者の組織由来たんぱく質の解析値／パーキンソン病患者の血清たんぱく質の解析値）…（Ｃ）
に従って、（パーキンソン病患者の血清たんぱく質の解析値／健常人の血清たんぱく質の解析値）の値を得ること；
　（２）該マーカーと同定されたたんぱく質が、５以上または１／５以下の（パーキンソン病患者の血清たんぱく質の解析値／健常人の血清たんぱく質の解析値）の値のたんぱく質である、（１）に記載の方法；
　（３）該マーカーと同定されたたんぱく質が、latrophilin 3（gi|14149677）、carnosinase 1（gi|21071039）、lactotransferrin（gi|54607120）、decorin isoform a preproprotein（gi|4503271）、superoxide dismutase 3, extracellular precurcer（gi|118582275）およびamyloid beta (A4) precursor-like protein 2（gi|4502147）からなる群より選択される少なくとも１種のたんぱく質である、（１）または（２）に記載の方法；
　（４）該マーカーと同定されたたんぱく質が、superoxide dismutase 3, extracellular precurcer（gi|118582275）である、（１）または（２）に記載の方法；
　（５）該マーカーと同定されたたんぱく質の量が同位体標識法を用いて調べられる、（１）～（４）のいずれか１つに記載の方法；
　（６）同位体標識法がｃＩＣＡＴ法である、（５）に記載の方法；
　（７）病態の判定がパーキンソン病患者の治療モニタリングである、（１）～（６）のいずれか１つに記載の方法；
　（８）病態の判定がパーキンソン病患者の予後である、（１）～（７）のいずれか１つに記載の方法；
　（９）治療中のパーキンソン病患者から得た試料中の（１）～（４）のいずれかに記載のマーカーと同定されたたんぱく質の量と、治療前および／または治療後の同じ患者、ならびに／あるいは健常人から得た試料中の該たんぱく質の量とを比較することをさらに含む、（１）～（８）のいずれか１つに記載の方法；
　（１０）（１）～（９）のいずれか１つに記載の方法において、（１）～（４）のいずれかに記載のマーカーと同定されたたんぱく質の量を測定するための手段を必須として含む、パーキンソン病の病態の判定用キット；
　（１１）パーキンソン病の治療薬のスクリーニング方法であって、
　（ａ）パーキンソン病患者に候補薬剤を投与すること；次いで
　（ｂ）該候補薬剤の投与前および投与後に該患者から採取した試料中の（１）～（４）のいずれかに記載のマーカーと同定されたたんぱく質の量を調べることを含む方法;
　（１２）下記工程（ａ）～（ｃ）を含む、パーキンソン病の病態の判定に使用されるパーキンソン病のマーカーの同定方法：
　（ａ）パーキンソン病患者の組織由来たんぱく質および健常人の血清たんぱく質を解析し、同定たんぱく質に関する比較定量値：
　　パーキンソン病患者の組織由来たんぱく質の解析値／健常人の血清たんぱく質の解析値…（Ａ）
を得て；
　（ｂ）（ａ）と同一のパーキンソン病患者の組織由来たんぱく質およびパーキンソン病患者の血清たんぱく質を解析し、同定たんぱく質に関する比較定量値：
　　（ａ）と同一のパーキンソン病患者の組織由来たんぱく質の解析値／パーキンソン病患者の血清たんぱく質の解析値…（Ｂ）
を得て；次いで
　（ｃ）（パーキンソン病患者の組織由来たんぱく質の解析値／健常人の血清たんぱく質の解析値）÷（パーキンソン病患者の組織由来たんぱく質の解析値／パーキンソン病患者の血清たんぱく質の解析値）…（Ｃ）
に従って、（パーキンソン病患者の血清たんぱく質の解析値／健常人の血清たんぱく質の解析値）の値を得ること；
　（１３）５以上または１／５以下の（パーキンソン病患者の血清たんぱく質の解析値／健常人の血清たんぱく質の解析値）の値を有するたんぱく質を、パーキンソン病の病態の判定に使用されるパーキンソン病のマーカーと同定する、請求項１２記載の方法；
　（１４）パーキンソン病の病態の判定のためにパーキンソン病のマーカーとして使用される、latrophilin 3（gi|14149677）、carnosinase 1（gi|21071039）、lactotransferrin（gi|54607120）、decorin isoform a preproprotein（gi|4503271）、superoxide dismutase 3, extracellular precursor（gi|118582275）およびamyloid beta (A4) precursor-like protein 2（gi|4502147）からなる群より選択される少なくとも１種のたんぱく質；
　（１５）パーキンソン病の病態の判定のためにパーキンソン病のマーカーとして使用されるsuperoxide dismutase 3, extracellular precursor（gi|118582275）。 That is, the present invention relates to:
(1) A method for determining the pathological condition of Parkinson's disease, comprising examining the amount of a protein identified as a marker for Parkinson's disease by a method comprising the following steps (a) to (c):
(A) Analyzing tissue-derived protein of Parkinson's disease patient and serum protein of healthy person, and comparative quantitative value for the identified protein:
Analytical value of protein derived from tissue of Parkinson's disease patient / Analytical value of serum protein of healthy person ... (A)
To obtain;
(B) Analyzing the same tissue-derived protein of Parkinson's disease patient as in (a) and serum protein of Parkinson's disease patient, and comparative quantitative value for the identified protein:
Analysis value of protein derived from tissue of Parkinson's disease patient same as (a) / Analysis value of serum protein of Parkinson's disease patient ... (B)
(C) (analyzed value of protein derived from tissue of Parkinson's disease patient / analyzed value of serum protein of healthy subject) ÷ (analyzed value of protein derived from tissue of Parkinson's disease patient / analysis of serum protein of Parkinson's disease patient) Value) ... (C)
To obtain the value of (analyzed value of serum protein of Parkinson's disease patient / analyzed value of serum protein of healthy subject);
(2) The protein identified as the marker is a protein having a value of 5 or more or 1/5 or less (analyzed value of serum protein of Parkinson's disease patient / analyzed value of serum protein of healthy subject) (1) The method described in;
(3) Proteins identified as the markers are latrophilin 3 (gi | 14149677), carnosinase 1 (gi | 21071039), lactotransferrin (gi | 54607120), decorin isoform a preproprotein (gi | 4503271), superoxide dismutase 3, extracellular The method according to (1) or (2), which is at least one protein selected from the group consisting of precurcer (gi | 118582275) and amyloid beta (A4) precursor-like protein 2 (gi | 4502147);
(4) The method according to (1) or (2), wherein the protein identified as the marker is superoxide dismutase 3, extracellular precurcer (gi | 118582275);
(5) The method according to any one of (1) to (4), wherein the amount of the protein identified as the marker is examined using an isotope labeling method;
(6) The method according to (5), wherein the isotope labeling method is a cICAT method;
(7) The method according to any one of (1) to (6), wherein the determination of the disease state is treatment monitoring of Parkinson's disease patients;
(8) The method according to any one of (1) to (7), wherein the determination of a disease state is a prognosis of a Parkinson's disease patient;
(9) the amount of the protein identified as a marker according to any of (1) to (4) in a sample obtained from a patient with Parkinson's disease being treated, and the same patient before and / or after treatment, and And / or the method according to any one of (1) to (8), further comprising comparing the amount of the protein in a sample obtained from a healthy person;
(10) In the method according to any one of (1) to (9), a means for measuring the amount of the protein identified as the marker according to any one of (1) to (4) is essential A kit for determining the pathology of Parkinson's disease, comprising:
(11) A screening method for a therapeutic drug for Parkinson's disease,
(A) administering a candidate drug to a Parkinson's disease patient; then (b) the marker according to any one of (1) to (4) in a sample collected from the patient before and after the candidate drug is administered; A method comprising examining the amount of the identified protein;
(12) A method for identifying a marker for Parkinson's disease used for determination of the pathological condition of Parkinson's disease comprising the following steps (a) to (c):
(A) Analyzing tissue-derived protein of Parkinson's disease patient and serum protein of healthy person, and comparative quantitative value for the identified protein:
Analytical value of protein derived from tissue of Parkinson's disease patient / Analytical value of serum protein of healthy person ... (A)
To obtain;
(B) Analyzing the same tissue-derived protein of Parkinson's disease patient as in (a) and serum protein of Parkinson's disease patient, and comparative quantitative value for the identified protein:
Analysis value of protein derived from tissue of Parkinson's disease patient same as (a) / Analysis value of serum protein of Parkinson's disease patient ... (B)
(C) (analyzed value of protein derived from tissue of Parkinson's disease patient / analyzed value of serum protein of healthy subject) ÷ (analyzed value of protein derived from tissue of Parkinson's disease patient / analysis of serum protein of Parkinson's disease patient) Value) ... (C)
To obtain the value of (analyzed value of serum protein of Parkinson's disease patient / analyzed value of serum protein of healthy subject);
(13) A protein having a value of 5 or more or 1/5 or less (analyzed value of serum protein of Parkinson's disease patient / analyzed value of serum protein of healthy person) is used for the determination of Parkinson's disease 13. The method of claim 12, wherein the method is identified as
(14) Latrophilin 3 (gi | 14149677), carnosinase 1 (gi | 21071039), lactotransferrin (gi | 54607120), decorin isoform a preproprotein (gi) used as a marker for Parkinson's disease | 4503271), at least one protein selected from the group consisting of superoxide dismutase 3, extracellular precursor (gi | 118582275) and amyloid beta (A4) precursor-like protein 2 (gi | 4502147);
(15) superoxide dismutase 3, extracellular precursor (gi | 118582275) used as a marker of Parkinson's disease for determination of the pathological condition of Parkinson's disease.
　本発明によれば、患者から得た試料（特に血清）中のパーキンソン病のマーカーたんぱく質の量を測定することにより、病態の判定、例えば、治療モニタリング、予後などを迅速かつ正確に行うことができ、さらには治療薬のスクリーニングを行うこともできる。 According to the present invention, by determining the amount of a marker protein for Parkinson's disease in a sample (especially serum) obtained from a patient, it is possible to quickly and accurately determine a disease state, for example, treatment monitoring, prognosis, etc. Furthermore, screening for therapeutic agents can also be performed.
図１は、健常人血清（ＪＮＳ）とパーキンソン病患者の脳脊髄液（ＰＤ－ＣＳＦ）の比較定量値を示す図である。図ではＨ／Ｌ比の高いたんぱく質から順に並べてある。FIG. 1 is a diagram showing comparative quantitative values of serum from healthy individuals (JNS) and cerebrospinal fluid (PD-CSF) from Parkinson's disease patients. In the figure, proteins are arranged in order from the protein with the highest H / L ratio. 図２は、パーキンソン病患者の血清（ＰＤＳ）とパーキンソン病患者の脳脊髄液（ＰＤ－ＣＳＦ）の比較定量値を示す図である。図ではＨ／Ｌ比の高いたんぱく質から順に並べてある。FIG. 2 is a view showing comparative quantitative values of serum of a Parkinson's disease patient (PDS) and cerebrospinal fluid (PD-CSF) of a Parkinson's disease patient. In the figure, proteins are arranged in order from the protein with the highest H / L ratio. パーキンソン病患者の血清（ＰＤＳ）と日本人健常者の血清（ＪＮＳ）の比較定量値を示す図である。図ではＧｉ＃の小さいたんぱく質から順に並べてある。It is a figure which shows the comparative quantitative value of the serum (PDS) of a Parkinson's disease patient, and the serum (JNS) of a healthy Japanese person. In the figure, proteins are arranged in order from the protein with the smallest Gi #.
　本発明は、１の態様において、パーキンソン病の病態の判定方法を提供する。この判定方法は、下記の工程（ａ）～（ｃ）を含む同定手順によりパーキンソン病のマーカーと同定されたたんぱく質の量を調べることを含む。前記同定手順では、まず、工程（ａ）において、パーキンソン病患者の組織由来たんぱく質および健常人の血清たんぱく質を解析し、同定たんぱく質に関する比較定量値：（パーキンソン病患者の組織由来たんぱく質の解析値／健常人の血清たんぱく質の解析値）を得る。式中の「パーキンソン病患者の組織由来たんぱく質の解析値」は、パーキンソン病患者の組織に由来するたんぱく質分画を解析して得られた値である。さらに、式中の「健常人の血清たんぱく質の解析値」は、健常人の血清に由来するたんぱく質分画を解析して得られた値である。次いで、工程（ｂ）において、（ａ）と同一のパーキンソン病患者の組織由来たんぱく質およびパーキンソン病患者の血清たんぱく質を解析し、同定たんぱく質に関する比較定量値：（（ａ）と同一のパーキンソン病患者の組織由来たんぱく質の解析値／パーキンソン病患者の血清たんぱく質の解析値）を得る。ここで、「（ａ）と同一のパーキンソン病患者の組織由来たんぱく質の解析値」は、工程（ａ）と同一のパーキンソン病患者と同一の組織に由来するたんぱく質分画を解析して得られた値である。「パーキンソン病患者の血清たんぱく質の解析値」は、パーキンソン病患者の血清たんぱく質分画を解析して得られた値を示す。さらに、工程（ｃ）では、（パーキンソン病患者の組織由来たんぱく質の解析値／健常人の血清たんぱく質の解析値）÷（パーキンソン病患者の組織由来たんぱく質の解析値／パーキンソン病患者の血清たんぱく質の解析値）に従って、（パーキンソン病患者の血清たんぱく質の解析値／健常人の血清たんぱく質の解析値）の値を得る。この得られた値により、パーキンソン病患者と健常人の血清中に存在するたんぱく質を比較定量することが可能となる。すなわち、この方法による最大の利点は、血清中のたんぱく質において、健常人とパーキンソン病患者間で相対的に存在量が相違するたんぱく質を同定できることにある。 In one aspect, the present invention provides a method for determining the pathology of Parkinson's disease. This determination method includes examining the amount of a protein identified as a marker of Parkinson's disease by an identification procedure including the following steps (a) to (c). In the identification procedure, first, in step (a), a protein derived from a tissue of a Parkinson's disease patient and a serum protein of a healthy person are analyzed, and a comparative quantitative value regarding the identified protein: (analyzed value of a protein derived from a tissue of a Parkinson's disease / healthy Analytical value of human serum protein). The “analyzed value of protein derived from tissue of Parkinson's disease patient” in the formula is a value obtained by analyzing a protein fraction derived from tissue of Parkinson's disease patient. Further, the “analyzed value of serum protein of healthy person” in the formula is a value obtained by analyzing a protein fraction derived from the serum of healthy person. Next, in the step (b), the same protein derived from the tissue of the Parkinson's disease patient as in (a) and the serum protein of the Parkinson's disease patient are analyzed, and a comparative quantitative value for the identified protein: (in the same Parkinson's disease patient as in (a)) Analyzed value of tissue-derived protein / analyzed value of serum protein of Parkinson's disease patient). Here, the “analyzed value of the protein derived from the tissue of the same Parkinson's disease patient as in (a)” was obtained by analyzing the protein fraction derived from the same tissue as that of the same Parkinson's disease patient as in the step (a). Value. “Analyzed value of serum protein of Parkinson's disease patient” indicates a value obtained by analyzing the serum protein fraction of Parkinson's disease patient. Further, in step (c), (analyzed value of protein derived from tissue of Parkinson's disease patient / analyzed value of serum protein of healthy subject) ÷ (analyzed value of protein derived from tissue of Parkinson's disease patient / analysis of serum protein of Parkinson's disease patient) The value of (analyzed value of serum protein of Parkinson's disease patient / analyzed value of serum protein of healthy person) is obtained according to (value). This obtained value enables comparative quantification of proteins present in the serum of Parkinson's disease patients and healthy individuals. In other words, the greatest advantage of this method is that proteins in serum that are relatively different in abundance between healthy individuals and Parkinson's disease patients can be identified.
　次に、本発明の方法に用いるパーキンソン病マーカーたんぱく質の同定手順（ａ）～（ｃ）について詳しく述べる。 Next, Parkinson's disease marker protein identification procedures (a) to (c) used in the method of the present invention will be described in detail.
　上記同定手順の第１工程は、
　（ａ）パーキンソン病患者の組織由来たんぱく質および健常人の血清たんぱく質を解析し、同定たんぱく質に関する比較定量値：
　　　パーキンソン病患者の組織由来たんぱく質の解析値／健常人の血清たんぱく質の解析値…（Ａ）
を得る工程である。 The first step of the identification procedure is:
(A) Analyzing tissue-derived protein of Parkinson's disease patient and serum protein of healthy person, and comparative quantitative value for the identified protein:
Analytical value of protein derived from tissue of Parkinson's disease patient / Analytical value of serum protein of healthy person ... (A)
It is the process of obtaining.
　パーキンソン病患者は、以下の説明によればヒトであるが、それだけでなくイヌ、ネコなどのペット、あるいは、ウシ、ウマ、ブタ等の家畜であってもよい。したがって、本発明はヒトだけでなく、例えば、獣医学的にも適用することができる。また、パーキンソン病患者はパーキンソン病にかかっている者（患者）のみならず、パーキンソン病にかかっていることが疑われる者も包含される。 Parkinson's disease patients are humans according to the following explanation, but they may be pets such as dogs and cats, or livestock such as cows, horses and pigs. Therefore, the present invention can be applied not only to humans but also to veterinary medicine, for example. Parkinson's disease patients include not only those with Parkinson's disease (patients) but also those suspected of having Parkinson's disease.
　上記工程（ａ）において、パーキンソン病患者の組織に由来するたんぱく質および健常人の血清たんぱく質を解析する。本発明において、たんぱく質を解析するとは、パーキンソン病患者および健常人から得られたサンプル中のたんぱく質を同定し、それらの存在量を調べることである。存在量を相対的な比率で求めてもよい。 In the above step (a), proteins derived from the tissues of Parkinson's disease patients and serum proteins of healthy individuals are analyzed. In the present invention, analyzing a protein means identifying proteins in samples obtained from Parkinson's disease patients and healthy persons and examining their abundance. The abundance may be determined by a relative ratio.
　パーキンソン病患者の組織由来たんぱく質は、パーキンソン病患者の病変部位または病変の可能性のある部位に由来するたんぱく質であることが好ましい。このような部位に由来するたんぱく質を用いると、同定されるたんぱく質の種類が増え、診断の精度が向上する。しかしながら、後で説明するように、式（Ｃ）において、パーキンソン病患者の組織由来たんぱく質の解析値は相殺されるので、パーキンソン病患者の組織由来たんぱく質はいずれのパーキンソン病患者（健常人であってもよい）、いずれの部位から得られたたんぱく質であってもよい。パーキンソン病患者の組織として、例えば、市販のあるいは他の経路から入手可能な病変部位のサンプルを用いてもよい。したがって、技術的あるいは倫理的な問題等によりパーキンソン病患者の病変部位からたんぱく質サンプルの採取が困難あるいは不可能である場合であっても、上記同定手順を実施することができる。 It is preferable that the protein derived from the tissue of Parkinson's disease patient is a protein derived from a lesion site or a potential lesion site of Parkinson's disease patient. When a protein derived from such a site is used, the types of proteins to be identified increase and the accuracy of diagnosis improves. However, as will be described later, in the formula (C), the analysis value of the protein derived from the tissue of the Parkinson's disease patient is canceled out, and therefore the protein derived from the tissue of the Parkinson's disease patient is any Parkinson's disease patient (healthy person). Or a protein obtained from any site. As a tissue of a Parkinson's disease patient, for example, a sample of a lesion site that is commercially available or available from other routes may be used. Therefore, even if it is difficult or impossible to collect a protein sample from a lesion site of a Parkinson's disease patient due to technical or ethical problems, the above identification procedure can be performed.
　健常人の血清は、健常人のいずれの部位・組織から得たものであってもよく、例えば、健常人の末梢血から得てもよい。健常人の血清は、パーキンソン病患者の血清と同じ部位・組織から得たものであることが好ましい。例えば、健常人の血清が末梢血から得られたものである場合には、パーキンソン病患者の血清もその末梢血から得られたものであることが好ましい。 The serum of a healthy person may be obtained from any part / tissue of a healthy person, for example, from the peripheral blood of a healthy person. It is preferable that the serum of a healthy person is obtained from the same site / tissue as the serum of a Parkinson's disease patient. For example, when the serum of a healthy person is obtained from peripheral blood, it is preferable that the serum of Parkinson's disease patient is also obtained from the peripheral blood.
　上記同定手順におけるたんぱく質の解析手段・方法は当業者に公知のものを適宜選択しても用いることができる。好ましくは、たんぱく質の解析に同位体標識法を用いる。同位体標識法の例としてはｃＩＣＡＴ法やｉＴＲＡＱ法が挙げられる。以下において、ｃＩＣＡＴ法を用いて上記同定手順を実施する場合について説明するが、たんぱく質の解析手段・方法は上記のものに限定されない。 The protein analysis means / method in the above identification procedure can be used by appropriately selecting one known to those skilled in the art. Preferably, isotope labeling is used for protein analysis. Examples of the isotope labeling method include cICAT method and iTRAQ method. Although the case where the said identification procedure is implemented using cICAT below is demonstrated, the protein analysis means and method are not limited to the above.
　上記工程（ａ）を行う前に、たんぱく質を適当な同位体含有試薬（ｃＩＣＡＴ試薬）で標識する。たんぱく質を標識する前に、一般的な手法を用いて、各たんぱく質画分中に高濃度で存在する、アルブミン、ＩｇＧ、ＩｇＡ、ＩｇＭ、α２－マクログロブリン、α１－アンチトリプシン、トランスフェリン、ハプトグロビン、アポＡ－Ｉ、アポＡ－ＩＩ、Ｃ３およびトランスサイレチンなどのたんぱく質を除去してもよい。該工程に用いることができる一般的な除去の方法は、アジレント社の抗体カラム（Hu 14, Column 10x100mm）などの市販されている抗体カラムを用いて行ってもよいが、これだけに限らない。例えば、パーキンソン病患者の組織由来たんぱく質画分を重鎖（Ｈ鎖）試薬で、およびパーキンソン病患者の血清たんぱく質（ＮＨＳ）画分を軽鎖（Ｌ鎖）試薬で各々分別標識してもよい（あるいはその逆であってもよい）。ここで、パーキンソン病マーカーたんぱく質の同定手順で用いるパーキンソン病患者の組織由来たんぱく質画分は、複数のパーキンソン病患者の脳脊髄液（ＣＳＦ）をプールしたものに由来してもよい。また、健常人の血清たんぱく質画分は、複数の健常人の血清をプールしたものに由来するものであってもよい。 Before performing the above step (a), the protein is labeled with an appropriate isotope-containing reagent (cICAT reagent). Prior to labeling the protein, using general techniques, albumin, IgG, IgA, IgM, α2-macroglobulin, α1-antitrypsin, transferrin, haptoglobin, apo, present in high concentrations in each protein fraction. Proteins such as AI, Apo A-II, C3 and transthyretin may be removed. A general removal method that can be used in this step may be performed using a commercially available antibody column such as an antibody column (Hu 14, 10 Column 100 x 100 mm) manufactured by Agilent, but is not limited thereto. For example, a protein fraction derived from a tissue of Parkinson's disease patient may be separately labeled with a heavy chain (H chain) reagent, and a serum protein (NHS) fraction of a Parkinson's disease patient may be separately labeled with a light chain (L chain) reagent ( Or vice versa). Here, the tissue-derived protein fraction of Parkinson's disease patient used in the identification procedure of Parkinson's disease marker protein may be derived from a pool of cerebrospinal fluid (CSF) of a plurality of Parkinson's disease patients. Moreover, the serum protein fraction of a healthy person may be derived from a pool of a plurality of healthy person's sera.
　次いで、上記の手法を用いてたんぱく質を標識した後、当業者間で一般的な手法によってたんぱく質を精製処理してもよい。すなわち、標識したたんぱく質をトリプシンなどのペプチドを断片化する標準的なプロテアーゼで処理してもよい。その後、本発明では、標識したたんぱく質の精製工程において、ＳＣＸカラムクロマトグラフィー（4.6x100mm）などの一般的なカラムクロマトグラフィーを用いて複数画分にしてもよい。また、標準的なアビジンカラムを利用して標識したたんぱく質を回収してもよい。さらに、各たんぱく質画分をＴＦＡで処理してビオチン部分を切断してもよい。 Next, after labeling the protein using the above-described method, the protein may be purified by a general method among those skilled in the art. That is, the labeled protein may be treated with a standard protease that fragments a peptide such as trypsin. Thereafter, in the present invention, in the purification step of the labeled protein, a plurality of fractions may be obtained using general column chromatography such as SCX column chromatography (4.6 × 100 mm). Alternatively, the labeled protein may be recovered using a standard avidin column. Furthermore, each protein fraction may be treated with TFA to cleave the biotin moiety.
　続いて、分画を行った場合には、各たんぱく質画分中のたんぱく質を解析する。同定には、公知方法、例えば、一般的な質量分析法（ＭＳ）を組み合わせて用いてもよい。ＭＳの手段・方法は種々のものがあり、装置も多数市販されているので、適宜選択して使用することができる。また、分離能力と定性能力の向上を図るために、ガスクロマトグラフィー（ＧＣ）や液体クロマトグラフィー（ＬＣ）とＭＳとを組み合わせた分析方法（ＧＣ／ＭＳ、ＬＣ／ＭＳあるいはＬＣ／ＭＳ／ＭＳなど）も開発されており、そのための装置が多数市販されている。上記手順に使用できる一般的な質量分析装置として、例えば、nano-LC（LC-Packings）／QSTAR XL（AB、ESI-Q/TOF）質量分析装置等であってもよい。また、上記手順で同定されるたんぱく質を、当業者間で一般的なデータベースを用いて決定してもよい。例えば、NCBI（National Center for Biotechnology Information）が提供するThe Reference Sequence（RefSeq）などのデータベースを用いて、ｃＩＣＡＴペプチドレベルでランク１、スコア２０以上を示すたんぱく質であり、かつ、ペプチド精査の結果正しいと判断したたんぱく質を同定たんぱく質としてもよい。 Subsequently, when fractionation is performed, the protein in each protein fraction is analyzed. For identification, a known method, for example, general mass spectrometry (MS) may be used in combination. There are various MS means and methods, and many devices are commercially available, so that they can be appropriately selected and used. In addition, in order to improve separation ability and qualitative ability, an analysis method (GC / MS, LC / MS, LC / MS / MS, etc.) combining gas chromatography (GC), liquid chromatography (LC) and MS ) Has also been developed, and a large number of devices are commercially available. As a general mass spectrometer that can be used in the above procedure, for example, a nano-LC (LC-Packings) / QSTAR XL (AB, ESI-Q / TOF) mass spectrometer may be used. Moreover, you may determine the protein identified by the said procedure using a general database among those skilled in the art. For example, using a database such as NCBI (National Center for Biotechnology Information) The Reference Reference Sequence (RefSeq), etc., it is a protein that shows a rank of 1 at a cICAT peptide level and a score of 20 or more, and that the results of peptide scrutiny are correct. The determined protein may be used as the identified protein.
　次いで、上式（Ａ）の比較定量値を求める。式（Ａ）による各たんぱく質の比較定量値を、ＨｉＳｐｅｃ（日立製作所）などの一般に利用可能な統合データベースを用いて計算してもよい。 Next, the comparative quantitative value of the above formula (A) is obtained. The comparative quantitative value of each protein according to formula (A) may be calculated using a generally available integrated database such as HiSpec (Hitachi).
　上記同定手順の第２工程は、
　（ｂ）（ａ）と同一のパーキンソン病患者の組織由来たんぱく質およびパーキンソン病患者の血清たんぱく質を解析し、同定たんぱく質に関する比較定量値：
　　　パーキンソン病患者の組織由来たんぱく質の解析値／パーキンソン病患者の血清たんぱく質の解析値…（Ｂ）
を得る工程である。 The second step of the identification procedure is:
(B) Analyzing the same tissue-derived protein of Parkinson's disease patient as in (a) and serum protein of Parkinson's disease patient, and comparative quantitative value for the identified protein:
Analysis value of protein derived from tissue of Parkinson's disease patient / Analysis value of serum protein of Parkinson's disease patient ... (B)
It is the process of obtaining.
　この工程では、パーキンソン病患者の組織由来たんぱく質の解析値をパーキンソン病患者の血清たんぱく質の解析値で割ることによって比較定量値を求める。また、式（Ｂ）による各たんぱく質の比較定量値を、ＨｉＳｐｅｃ（日立製作所）などの一般に利用可能な統合データベースを用いて計算してもよい。 In this step, a comparative quantitative value is obtained by dividing the analysis value of the protein derived from the tissue of the Parkinson's disease patient by the analysis value of the serum protein of the Parkinson's disease patient. Moreover, you may calculate the comparative quantitative value of each protein by Formula (B) using generally available integrated databases, such as HiSpec (Hitachi).
　パーキンソン病患者の血清は、該患者のいずれの部位・組織から得たものであってもよい。例えば、パーキンソン病患者の末梢血から血清を得てもよい。 The serum of a Parkinson's disease patient may be obtained from any site / tissue of the patient. For example, serum may be obtained from the peripheral blood of Parkinson's disease patients.
　上記同定手順の第３工程は、
　（ｃ）（パーキンソン病患者の組織由来たんぱく質の解析値／健常人の血清たんぱく質の解析値）÷（パーキンソン病患者の組織由来たんぱく質の解析値／パーキンソン病患者の血清たんぱく質の解析値）…（Ｃ）
に従って、
　　　（パーキンソン病患者の血清たんぱく質の解析値／健常人の血清たんぱく質の解析値）
の値を得る工程である。 The third step of the identification procedure is:
(C) (analyzed value of protein derived from tissue of Parkinson's disease patient / analyzed value of serum protein of healthy person) / (analyzed value of protein derived from tissue of Parkinson's disease patient / analyzed value of serum protein of Parkinson's disease patient) (C )
According to
(Analyzed value of serum protein in patients with Parkinson's disease / Analyzed value of serum protein in healthy individuals)
Is a step of obtaining the value of.
　式（Ｃ）は、式（Ａ）より得られた各たんぱく質の比較定量値を式（Ｂ）より得られた各たんぱく質の比較定量値で割ることにより、（パーキンソン病患者の血清たんぱく質の解析値）／（健常人の血清たんぱく質の解析値）を計算するためのものである。この手順により、上述のごとく、健常人の血清とパーキンソン病患者の血清に関して、パーキンソン病患者の組織由来たんぱく質の解析値（あるいはいずれかのパーキンソン病患者のいずれかの組織由来のたんぱく質の解析値）を介する（式（Ｃ）において相殺される）ことで多種多様な低発現および低濃度たんぱく質の比較定量値を求めることができる。 The formula (C) is obtained by dividing the comparative quantitative value of each protein obtained from the formula (A) by the comparative quantitative value of each protein obtained from the formula (B) to obtain (analyzed value of serum protein of Parkinson's disease patient). ) / (Analyzed value of serum protein of healthy person). By this procedure, as described above, the analysis value of the protein derived from the tissue of the Parkinson's disease patient (or the analysis value of the protein derived from any tissue of the Parkinson's disease patient) regarding the serum of the healthy person and the serum of the Parkinson's disease patient. (Cancelled in the formula (C)), comparatively quantitative values of various low expression and low concentration proteins can be obtained.
　本発明は、上記同定手順によりパーキンソン病のマーカーたんぱく質と同定されたたんぱく質も提供する。パーキンソン病のマーカーたんぱく質と同定する基準は、（パーキンソン病患者の血清たんぱく質の解析値／健常人の血清たんぱく質の解析値）の比較定量値（上記工程（ｃ）中の式の値）が１より高くても、低くてもよい。好ましくは、比較定量値が１より高いたんぱく質を選択する場合、その値が２以上、好ましくは５以上、さらに好ましくは１０以上のものである。また、比較定量値が１より低いたんぱく質を選択する場合、その値が１／２以下、好ましくは１／５以下、さらに好ましくは１／１０以下のものである。パーキンソン病の病態の判定に用いる場合には、診断精度の観点から比較定量値が５以上または１／５以下のたんぱく質であることが最も好ましい。 The present invention also provides a protein identified as a marker protein for Parkinson's disease by the above identification procedure. The standard for identifying a marker protein of Parkinson's disease is that the comparative quantitative value (value of the expression in the above step (c)) of 1 (analyzed value of serum protein of Parkinson's disease patient / analyzed value of serum protein of healthy person) is 1. It can be high or low. Preferably, when a protein having a comparative quantitative value higher than 1 is selected, the value is 2 or more, preferably 5 or more, more preferably 10 or more. When a protein having a comparative quantitative value lower than 1 is selected, the value is 1/2 or less, preferably 1/5 or less, more preferably 1/10 or less. When used for determination of the pathological condition of Parkinson's disease, it is most preferable that the comparative quantitative value is a protein of 5 or more or 1/5 or less from the viewpoint of diagnostic accuracy.
　本発明の病態の判定方法における好ましいパーキンソン病のマーカーたんぱく質としては、上記同定手順にて同定されたたんぱく質のうち、（パーキンソン病患者の血清たんぱく質の解析値／健常人の血清たんぱく質の解析値）の比較定量値が５以上のたんぱく質が挙げられる。例えば、latrophilin 3（latrophilin homolog 3 (cow); lectomedin 3）（gi|14149677）およびcarnosinase 1（glutamate carboxypeptidase-like protein 2）（gi|21071039）である。これらのたんぱく質は、健常人と比較してパーキンソン病患者の体内、特に血清中において、その存在量が増加しうる。また、好ましいパーキンソン病のマーカーたんぱく質として前記比較解析値が１／５以下のたんぱく質が挙げられる。例えば、lactotransferrin（gi|54607120）、decorin isoform a preproprotein（small leucine-rich protein 1B）（gi|4503271）、superoxide dismutase 3, extracellular precursor（gi|118582275）およびamyloid beta (A4) precursor-like protein 2（gi|4502147）である。これらのたんぱく質は、健常人と比較してパーキンソン病患者の体内、特に血清中において、その存在量が減少しうる。特に、以前の報告より、上記たんぱく質のなかでもsuperoxide dismutase 3, extracellular precursor（gi|118582275）は、細胞障害の原因物質である活性酸素のスーパーオキサイド（Ｏ２－）を不活性化する酵素の一種であり、本酵素が不足すると細胞外スーパーオキサイド（Ｏ２－）による細胞障害が進行する可能性が考えられる（Li, Y. et al., Na. Genet. 11, 367-380(1995)およびMuller, F. et al., Free Radic. Biol. Med 40, 1993-2004(2006)）。一方、細胞内（細胞質）に存在するsuperoxide dismutase 1, soluble（gi|4507149）は、パーキンソン病患者と、健常人およびパーキンソン様症候群とで大きな差は認められない（表１）。したがって、パーキンソン病の大脳黒質中のドパミン産生神経細胞の変性に関して、superoxide dismutase 3, extracellular precursor（gi|118582275）の低下が細胞外スーパーオキサイド（Ｏ２－）の不活性化不全をもたらし、結果的にスーパーオキサイド（Ｏ２－）によるドパミン産生神経細胞の変性を引き起こすことが予想される。このことからも、上記基準により同定されたたんぱく質のパーキンソン病のマーカーとしての有効性を推定することができる。 As a preferable marker protein of Parkinson's disease in the method for determining a disease state of the present invention, among the proteins identified by the above identification procedure, (analyzed value of serum protein of Parkinson's disease patient / analyzed value of serum protein of healthy person) A protein having a comparative quantitative value of 5 or more may be mentioned. For example, latrophilin 3 (latrophilin homolog 3 (cow); lectomedin 3) (gi | 14149677) and carnosinase 1 (glutamate carboxypeptidase-like protein 2) (gi | 21071039). The amount of these proteins can be increased in the body of Parkinson's disease patients, particularly in serum, compared to healthy individuals. Moreover, as a preferable marker protein for Parkinson's disease, a protein having the comparative analysis value of 1/5 or less can be mentioned. For example, lactotransferrin (gi | 54607120), decorin isoform a preproprotein (small leucine-rich protein 1B) (gi | 4503271), superoxide dismutase 3, extracellular precursor (gi | 118582275) and amyloid beta (A4) precursor-like protein 2 ( gi | 4502147). The amount of these proteins can be reduced in the body of Parkinson's disease patients, particularly in serum, compared to healthy individuals. In particular, according to previous reports, among the above proteins, superoxide dismutase 3, extracellular precursor (gi | 118582275) is a kind of enzyme that inactivates superoxide of active oxygen (O 2− ), which is a causative agent of cell damage. When this enzyme is deficient, it is considered that cell damage caused by extracellular superoxide (O 2− ) may progress (Li, Y. et al., Na. Genet. 11, 367-380 (1995) and Muller, F. et al., Free Radic. Biol. Med 40, 1993-2004 (2006)). On the other hand, superoxide dismutase 1, soluble (gi | 4507149) present in the cell (cytoplasm) does not show a large difference between Parkinson's disease patients, healthy subjects and Parkinson's syndrome (Table 1). Therefore, regarding the degeneration of dopaminergic neurons in Parkinson's cerebral substantia nigra, the decrease in superoxide dismutase 3, extracellular precursor (gi | 118582275) leads to inactivation failure of extracellular superoxide (O 2− ). In particular, it is expected to cause degeneration of dopaminergic neurons by superoxide (O 2− ). Also from this, it is possible to estimate the effectiveness of the protein identified by the above criteria as a marker of Parkinson's disease.
　本発明のパーキンソン病の病態の判定方法は、患者から得た試料中のマーカーたんぱく質の量を調べることを含む。用いるマーカーたんぱく質は上記手順にて同定されたものである。本発明の病態の判定方法を適用できる患者は、イヌ、ネコ等のペット、あるいは、ウシ、ウマ、ブタ等の家畜を包含するが、好ましくは霊長類、特に好ましくはヒトである。したがって、本発明の病態の判定方法はヒトだけでなく、例えば、獣医学的にも適用することができる。また、患者は、パーキンソン病にかかっている患者のみならずパーキンソン病であることが疑われる者であってもよい。 The method of determining the pathological condition of Parkinson's disease of the present invention includes examining the amount of marker protein in a sample obtained from a patient. The marker protein to be used has been identified by the above procedure. The patient to which the method for determining a disease state of the present invention can be applied includes pets such as dogs and cats, and domestic animals such as cows, horses and pigs, preferably primates, particularly preferably humans. Therefore, the disease state determination method of the present invention can be applied not only to humans but also to veterinary medicine, for example. The patient may be a person suspected of having Parkinson's disease as well as a patient suffering from Parkinson's disease.
　上述の如く、本発明の病態の判定方法では、患者からの試料の種類は特に限定されない。試料は体液であってもよく、体液試料としては全血、血清、尿、汗、唾液、腹水などが例示されるが、好ましいのは血清である。試料の採取方法は当該分野において様々なものが知られ、用いられており、適宜選択して用いることができる。例えば、血清試料を得る場合には、腕の静脈から採血し、遠心分離等の通常の分離方法により血清を得ることができる。好ましくは、患者および／または健常者において、同じ採取方法にて試料を得て、同じ測定方法にて上記マーカーたんぱく質の量を測定することが、データの精度向上の点から好ましい。 As described above, in the disease state determination method of the present invention, the type of sample from the patient is not particularly limited. The sample may be a bodily fluid, and examples of the bodily fluid sample include whole blood, serum, urine, sweat, saliva, ascites, and the like is preferable. Various sample collection methods are known and used in the field, and can be appropriately selected and used. For example, when obtaining a serum sample, blood can be collected from an arm vein, and serum can be obtained by a normal separation method such as centrifugation. Preferably, from the viewpoint of improving the accuracy of data, it is preferable that a patient and / or a healthy person obtain a sample by the same collection method and measure the amount of the marker protein by the same measurement method.
　本発明の病態の判定方法において、たんぱく質の検出および定量方法は、特に限定されるものではなく、当該分野で知られた方法を使用することができる。その例としては、アフィニティークロマトグラフ法、二次元電気泳動法、あるいは上述のようなｃＩＣＡＴ法もしくはｉＴＲＡＱ法などの同位体標識法、マーカーたんぱく質に特異的な抗体を用いる免疫学的方法等が挙げられる。二次元電気泳動法や同位体標識法等のプロテオーム解析に用いられる方法を適用してもよい。免疫学的方法を用いてマーカーたんぱく質を検出および定量してもよい。マーカーたんぱく質に対する抗体を公知の方法により調製して、ＥＬＩＳＡ法や免疫染色法などの公知の手法により分析を行うことができる。マイクロチップやマイクロタイターディッシュ、あるいはビーズ等の当該分野で公知の器具・装置、ならびに公知の固定化手段等を用いてハイスループット分析を行って大量の検体を分析することもできる。公知の種々の標識（例えば、同位体標識、蛍光標識、発光標識、呈色標識、金コロイド標識、ビオチンなどの特異的結合物質での標識等）を用いて、マーカーたんぱく質の検出および定量を容易化してもよい。 In the pathological condition determination method of the present invention, the protein detection and quantification method is not particularly limited, and methods known in the art can be used. Examples thereof include affinity chromatography, two-dimensional electrophoresis, isotope labeling such as the above-described cICAT or iTRAQ, and immunological methods using an antibody specific to the marker protein. . Methods used for proteomic analysis such as two-dimensional electrophoresis and isotope labeling may be applied. Immunological methods may be used to detect and quantify the marker protein. An antibody against the marker protein can be prepared by a known method and analyzed by a known method such as an ELISA method or an immunostaining method. It is also possible to analyze a large amount of samples by performing high-throughput analysis using a known device / device such as a microchip, a microtiter dish, or a bead, and a known immobilization means. Easy detection and quantification of marker proteins using various known labels (eg, isotope labels, fluorescent labels, luminescent labels, color labels, colloidal gold labels, labels with specific binding substances such as biotin) May be used.
　特に、同位体標識法は定性のみならず定量性にも優れた方法である。同位体標識法のなかでもｃＩＣＡＴ法（Hansen, K.C. et al, Mol. Cell Proteomics, 2, 299-314(2003)）がさらに好ましい。ｃＩＣＡＴ法は、患者と健常人あるいは治療前と治療後のたんぱく質の網羅的発現差解析を行うことにより、疾患関連たんぱく質を分析する有効な手段として用いられており、本発明にも有効に用いられる。同位体標識法には質量分析法（ＭＳ）を組み合わせるのが一般的である。ＭＳの手段・方法は種々のものがあり、装置も多数市販されているので、適宜選択して使用することができる。また、分離能力と定性能力の向上を図るために、ガスクロマトグラフィー（ＧＣ）や液体クロマトグラフィー（ＬＣ）とＭＳとを組み合わせた分析方法（ＧＣ／ＭＳ、ＬＣ／ＭＳあるいはＬＣ／ＭＳ／ＭＳなど）も開発されており、そのための装置の多数市販されている。 In particular, the isotope labeling method is not only qualitative but also excellent in quantitativeness. Among the isotope labeling methods, the cICAT method (Hansen, K.C. et al, Mol. Cell Proteomics, 2, 299-314 (2003)) is more preferable. The cICAT method is used as an effective means for analyzing a disease-related protein by conducting a comprehensive expression difference analysis between a patient and a healthy person or a protein before and after treatment, and is also used effectively in the present invention. . In general, isotope labeling is combined with mass spectrometry (MS). There are various MS means and methods, and many devices are commercially available, so that they can be appropriately selected and used. In addition, in order to improve separation ability and qualitative ability, an analysis method (GC / MS, LC / MS, LC / MS / MS, etc.) combining gas chromatography (GC), liquid chromatography (LC) and MS ) Has also been developed, and a large number of devices are commercially available.
　さらに、本発明の病態の判定方法およびキットでは、上記たんぱく質をコードするヌクレオチド配列から転写されるｍＲＮＡを検出および／または定量してもよい。ｍＲＮＡの検出および定量は一般的な方法を用いて行われてもよく、例えば、本発明のたんぱく質をコードするヌクレオチド配列から転写されるｍＲＮＡの全長または一部を増幅する定量リアルタイムＲＴ－ＰＣＲ法などにより行ってもよいが、ｍＲＮＡを検出および定量できればこれだけに限定しない。 Furthermore, in the method and kit for determining a disease state of the present invention, mRNA transcribed from the nucleotide sequence encoding the protein may be detected and / or quantified. The detection and quantification of mRNA may be performed using a general method, for example, a quantitative real-time RT-PCR method for amplifying the full length or a part of mRNA transcribed from the nucleotide sequence encoding the protein of the present invention. However, the present invention is not limited to this as long as mRNA can be detected and quantified.
　上で説明した本発明の病態の判定方法において、治療中の患者から得た試料中の上記マーカーたんぱく質の量と、治療前および／または治療後の同じ患者、および／または健常人から同様に得て同様に調べた試料中の上記マーカーたんぱく質とを比較してもよい。本明細書において、治療中とは、Ｌ－ＤＯＰＡ投与や化学療法などの処置を患者に施している期間をいう。治療前、治療中および／または治療後の各々において少なくとも１回患者から試料を得る。好ましくは、治療前、治療中そして治療後の各々において数回、適当な時間間隔をおいて患者から試料をサンプリングする。そうすることによってパーキンソン病患者の試料中の上記マーカーたんぱく質の量の経時変化をより詳細に調べることができ、パーキンソン病の病態をさらに詳細に解析することができる。例えば、治療に対する患者の応答の有無、程度、あるいは患者がいずれの治療段階にあるのか等の状況をさらに詳細に調べることができ、今後の病態の変化をさらに高い精度で予測することができる。 In the method for determining a disease state of the present invention described above, the amount of the marker protein in a sample obtained from a patient under treatment and the same patient before and / or after treatment and / or from a healthy person are obtained in the same manner. The marker protein in the sample examined in the same manner may be compared. In this specification, the term “during treatment” refers to a period during which a patient is treated with L-DOPA or chemotherapy. A sample is obtained from the patient at least once before, during and / or after treatment. Preferably, the sample is sampled from the patient at appropriate time intervals before, during and after treatment. By doing so, the time-dependent change of the amount of the marker protein in the sample of Parkinson's disease patient can be examined in more detail, and the pathological condition of Parkinson's disease can be analyzed in more detail. For example, the presence / absence and extent of the patient's response to the treatment, or the state of treatment of the patient can be examined in more detail, and future changes in the disease state can be predicted with higher accuracy.
　したがって、本発明のパーキンソン病の病態の判定方法は、パーキンソン病の治療モニタリングおよび予後にも用いることができる。 Therefore, the method for determining the pathological condition of Parkinson's disease of the present invention can also be used for treatment monitoring and prognosis of Parkinson's disease.
　本発明は、別の態様において、パーキンソン病のマーカーと同定されたたんぱく質の量を測定するための手段を必須として含む、パーキンソン病の病態の判定用キットを提供する。該キットは、上記の本発明の方法において試料中のマーカーたんぱく質の量を測定するための手段を必須として含む。試料中のマーカーたんぱく質の量を測定する方法は上述のごとく種々のものが公知であり、適宜選択して用いることができる。例えばマーカーたんぱく質の測定がＥＬＩＳＡ法を用いて行われる場合には、本発明のキットは、マーカーたんぱく質特異的モノクローナル抗体、ＥＬＩＳＡ用マイクロタイタープレート、適当な発色試薬等を含む。本発明のキットの好ましい具体例の１つは、上記たんぱく質の測定が同位体標識法、特にｃＩＣＡＴ法を用いて行われるものである。そのようなキットは同位体での標識に必要な試薬、器具を含んでいてもよい。本発明のキットはパーキンソン病の病態の解析に好ましく用いられる。 In another aspect, the present invention provides a kit for determining the pathological condition of Parkinson's disease, which essentially comprises means for measuring the amount of the protein identified as a marker of Parkinson's disease. The kit essentially includes means for measuring the amount of the marker protein in the sample in the above-described method of the present invention. As described above, various methods for measuring the amount of marker protein in a sample are known, and can be appropriately selected and used. For example, when the measurement of the marker protein is carried out using the ELISA method, the kit of the present invention contains a marker protein-specific monoclonal antibody, an ELISA microtiter plate, an appropriate coloring reagent, and the like. One of the preferable specific examples of the kit of the present invention is that the above-mentioned protein is measured using an isotope labeling method, particularly a cICAT method. Such a kit may contain reagents and instruments necessary for isotope labeling. The kit of the present invention is preferably used for analysis of the pathology of Parkinson's disease.
　本発明は、さらなる別の態様において、パーキンソン病の治療薬のスクリーニング方法を提供する。該方法は、パーキンソン病にかかっている動物に候補薬剤を投与すること、次いで該候補薬剤の投与前および投与後に該動物から採取した試料中の上記マーカーたんぱく質の量を調べることを含む。パーキンソン病が治療され改善されるにつれてマーカーたんぱく質の量が減少または増加し、悪化するにつれマーカーたんぱく質の量が増加または減少する。すなわち、マーカーたんぱく質はパーキンソン病の発症および進行に関与していると考えられる。したがって、該候補薬剤の投与により試料中のマーカーたんぱく質の量がどれほど増加または減少したかを調べることによって、パーキンソン病の治療効果の良否を判断することができる。例えば、上記マーカーたんぱく質の阻害剤、アンタゴニスト、あるいは抗体などはパーキンソン病の治療薬または予防薬としての候補である。かかる方法によりスクリーニングされ、得られたパーキンソン病の治療薬もまた本発明に包含される。 In still another aspect, the present invention provides a screening method for a therapeutic agent for Parkinson's disease. The method includes administering a candidate agent to an animal suffering from Parkinson's disease, and then examining the amount of the marker protein in a sample taken from the animal before and after administration of the candidate agent. As Parkinson's disease is treated and improved, the amount of marker protein decreases or increases, and as it worsens, the amount of marker protein increases or decreases. That is, it is considered that the marker protein is involved in the onset and progression of Parkinson's disease. Therefore, whether the therapeutic effect of Parkinson's disease is good or not can be determined by examining how much the amount of the marker protein in the sample is increased or decreased by administration of the candidate drug. For example, the above-described marker protein inhibitors, antagonists, antibodies and the like are candidates as therapeutic or preventive agents for Parkinson's disease. The therapeutic agent for Parkinson's disease obtained by screening by such a method is also encompassed in the present invention.
　また、本発明では、上記方法およびキットにパーキンソン病の一般的なマーカーたんぱく質を併用してもよい。該マーカーとして、Ｐａｒｋｉｎ、ＤＪ－１、α－シヌクレイン、ＰＩＮＫ１、ＵＣＨＬ－１、およびＬＲＲＫ２／ｄａｒｄａｒｉｎ等のたんぱく質を含むが、これらだけに限らない。これらのたんぱく質によるデータを併用することにより、さらに精度の高いパーキンソン病の病態の判定を行うことができる。 In the present invention, a general marker protein for Parkinson's disease may be used in combination with the above method and kit. Such markers include, but are not limited to, proteins such as Parkin, DJ-1, α-synuclein, PINK1, UCHL-1, and LRRK2 / dardarin. By using these protein data together, it is possible to determine the pathological condition of Parkinson's disease with higher accuracy.
　本発明は、さらに別の態様において、パーキンソン病の病態の判定に使用されるパーキンソン病のマーカーの同定方法を提供する。該方法は下記工程（ａ）～（ｃ）を含む：
　（ａ）パーキンソン病患者の組織由来たんぱく質および健常人の血清たんぱく質を解析し、同定たんぱく質に関する比較定量値：
　　パーキンソン病患者の組織由来たんぱく質の解析値／健常人の血清たんぱく質の解析値…（Ａ）
を得て；
　（ｂ）（ａ）と同一のパーキンソン病患者の組織由来たんぱく質およびパーキンソン病患者の血清たんぱく質を解析し、同定たんぱく質に関する比較定量値：
　　（ａ）と同一のパーキンソン病患者の組織由来たんぱく質の解析値／パーキンソン病患者の血清たんぱく質の解析値…（Ｂ）
を得て；次いで
　（ｃ）（パーキンソン病患者の組織由来たんぱく質の解析値／健常人の血清たんぱく質の解析値）÷（パーキンソン病患者の組織由来たんぱく質の解析値／パーキンソン病患者の血清たんぱく質の解析値）…（Ｃ）
に従って、（パーキンソン病患者の血清たんぱく質の解析値／健常人の血清たんぱく質の解析値）の値を得ること。
　各工程の説明は、すでに上で行ったとおりである。 In still another aspect, the present invention provides a method for identifying a marker for Parkinson's disease used for determination of the pathological condition of Parkinson's disease. The method includes the following steps (a) to (c):
(A) Analyzing tissue-derived protein of Parkinson's disease patient and serum protein of healthy person, and comparative quantitative value for the identified protein:
Analytical value of protein derived from tissue of Parkinson's disease patient / Analytical value of serum protein of healthy person ... (A)
To obtain;
(B) Analyzing the same tissue-derived protein of Parkinson's disease patient as in (a) and serum protein of Parkinson's disease patient, and comparative quantitative value for the identified protein:
Analysis value of protein derived from tissue of Parkinson's disease patient same as (a) / Analysis value of serum protein of Parkinson's disease patient ... (B)
(C) (analyzed value of protein derived from tissue of Parkinson's disease patient / analyzed value of serum protein of healthy subject) ÷ (analyzed value of protein derived from tissue of Parkinson's disease patient / analysis of serum protein of Parkinson's disease patient) Value) ... (C)
To obtain the value of (analyzed value of serum protein of Parkinson's disease patient / analyzed value of serum protein of healthy subject).
The description of each step has already been made above.
　上記のパーキンソン病の病態の判定に使用されるパーキンソン病のマーカーの同定方法において、５以上または１／５以下の（パーキンソン病患者の血清たんぱく質の解析値／健常人の血清たんぱく質の解析値）の値を有するたんぱく質を、パーキンソン病の病態の判定に使用されるパーキンソン病のマーカーと同定することが好ましい。 In the method for identifying a marker of Parkinson's disease used for the determination of the above-mentioned pathological condition of Parkinson's disease, 5 or more or 1/5 or less (analyzed value of serum protein of Parkinson's disease patient / analyzed value of serum protein of healthy person) It is preferable to identify a protein having a value as a marker for Parkinson's disease to be used for the determination of the pathology of Parkinson's disease.
　本発明は、もう１つの態様において、パーキンソン病の病態の判定のためにパーキンソン病のマーカーとして使用される、latrophilin 3（gi|14149677）、carnosinase 1（gi|21071039）、lactotransferrin（gi|54607120）、decorin isoform a preproprotein（gi|4503271）、superoxide dismutase 3, extracellular precursor（gi|118582275）およびamyloid beta (A4) precursor-like protein 2（gi|4502147）からなる群より選択される少なくとも１種のたんぱく質を提供する。これらのたんぱく質は、５以上または１／５以下の（パーキンソン病患者の血清たんぱく質の解析値／健常人の血清たんぱく質の解析値）の値を有するもので、該マーカーとして好ましいものである。 The present invention, in another embodiment, is used as a marker for Parkinson's disease for the determination of the pathology of Parkinson's disease, latrophilin 3 (gi | 14149677), carnosinase 1 (gi | 21071039), lactotransferrin (gi | 54607120) , Decorin isoform a preproprotein (gi | 4503271), superoxide dismutase 3, extracellular precursor (gi | 118582275) and amyloid beta (A4) precursor-like protein 2 (gi | 4502147) I will provide a. These proteins have a value of 5 or more or 1/5 or less (analyzed value of serum protein of Parkinson's disease patient / analyzed value of serum protein of healthy person), and is preferable as the marker.
　これらのうち、superoxide dismutase 3, extracellular precursor（gi|118582275）およびamyloid beta (A4) precursor-like protein 2（gi|4502147）は、パーキンソン病の病態の判定のためにパーキンソン病のマーカーとして特に好ましく使用される。 Of these, superoxide dismutase 3, extracellular precursor (gi | 118582275) and amyloid beta (A4) precursor-like protein 2 (gi | 4502147) are particularly preferably used as markers of Parkinson's disease for the determination of Parkinson's disease Is done.
　以下に参考例および実施例を示して本発明をさらに詳細かつ具体的に説明するが、実施例は本発明を限定するものではない。 Hereinafter, the present invention will be described in more detail and specifically with reference to Examples and Examples, but the Examples are not intended to limit the present invention.
　具体的には、パーキンソン病患者血清（ＰＤＳ：１０名分をプールしたもの）と日本人健常者標準血清（ＪＮＳ：１０名分をプールしたもの）及びパーキンソン病患者脳脊髄液（ＰＤ－ＣＳＦ：パーキンソン病患者１０名のＣＳＦをプールしたもの（病変組織由来たんぱく質として））のたんぱく質を、ｃＩＣＡＴ法により、同定・比較定量解析を行った。すなわち、アジレント社製抗体カラム（Ｈｕ１４、カラム　１０ｘ１００ｍｍ）で、アルブミン、ＩｇＧ、ＩｇＡ、ＩｇＭ、α２－マクログロブリン、α１－アンチトリプシン、トランスフェリン、ハプトグロブリン、Ａｐｏ　Ａ－Ｉ、Ａｐｏ　Ａ－ＩＩ、Ｃ３、トランスサイレチン、α１－酸性糖たんぱく質、α２－酸性たんぱく質を除去したＰＤ－ＣＳＦ（１００μｇ　たんぱく質）及びＪＮＳ（１００μｇ）を、それぞれｃＩＣＡＴ－Ｈ鎖試薬およびｃＩＣＡＴ－Ｌ鎖試薬で標識し、常法により得られたｃＩＣＡＴペプチドを、ｎａｎｏ－ＬＣ／Ｑ－Ｓｔａｒ　ＸＬで解析を行い、日立統合データベースシステム（ＨｉＳｐｅｃ）を用いて、脳脊髄液（ＰＤ－ＣＳＦ、Ｈ鎖標識）と健常者血清（ＪＮＳ、Ｌ鎖標識）との各たんぱく質の比較定量値（Ｈ／Ｌ）を計算した。また、ＲｅｆＳｅｑのＤＢを用いて、ｃＩＣＡＴペプチドレベルでランク１、スコア２０以上を示すたんぱく質及びペプチド精査の結果正しいと判断されたたんぱく質を同定たんぱく質とした。その結果、３５６種類のたんぱく質の同定が可能であり、そのうち、ピーク混雑で比較定量不能のものは２種類、ＰＤ－ＣＳＦにのみ存在する（Ｈ／Ｌ＝∞）は９９種類、血清のみに存在するもの（Ｈ／Ｌ＝０）は６種類であり、残りの２４９種類は０から無限大までの間の比較定量値が計算可能であった（図１）。この結果は、同様に処理した血清同士（ＰＤＳとＪＮＳ）の比較解析で得られるたんぱく質数（約１２０種類）よりもはるかに多くのたんぱく質の解析が可能であることを示す。また、上記同定したたんぱく質の約８０％はＰＤ－ＣＳＦをｃＩＣＡＴ法で同定したものと同一のたんぱく質であった。このことから、上記の結果は、血清では同定限界以下の病変組織由来の低発現たんぱく質がＰＤ－ＣＳＦ中の存在するたんぱく質により同定され、比較定量が可能になったためと考えられる。さらに、この原理を利用して、ｃＩＣＡＴ法で同様にＰＤ－ＣＳＦをＨ試薬で、パーキンソン病患者血清（ＰＤＳ）をＬ試薬で分別標識し、たんぱく質の同定と比較定量解析を行った。その結果、３０６種類のたんぱく質の同定が可能であり、ピーク混雑で比較定量不可のものは８種類、ＰＤ－ＣＳＦにのみ存在する（Ｈ／Ｌ＝∞）は２９種類、血清のみに存在するもの（Ｈ／Ｌ＝０）は４種類であり、残りの２６５種類は０から無限大までの間の比較定量値（ＰＤ－ＣＳＦ／ＰＤＳ）が計算可能であった（図２）。 Specifically, Parkinson's disease patient serum (PDS: pooled for 10 patients), Japanese healthy standard blood serum (JNS: pooled for 10 patients) and Parkinson's disease patient cerebrospinal fluid (PD-CSF: Proteins obtained by pooling CSF of 10 Parkinson's disease patients (as lesion tissue-derived proteins) were identified and subjected to comparative quantitative analysis by the cICAT method. That is, with an antibody column (Hu14, column 10 × 100 mm) manufactured by Agilent, albumin, IgG, IgA, IgM, α2-macroglobulin, α1-antitrypsin, transferrin, haptoglobulin, Apo AI, Apo A-II, C3, PD-CSF (100 μg protein) and JNS (100 μg) from which transthyretin, α1-acidic glycoprotein, α2-acidic protein had been removed were labeled with cICAT-H chain reagent and cICAT-L chain reagent, respectively. The obtained cICAT peptide was analyzed with nano-LC / Q-Star XL, and using Hitachi integrated database system (HiSpec), cerebrospinal fluid (PD-CSF, H chain labeled) and healthy subject serum (JNS, Comparison of each protein with L chain label) The amount value (H / L) was calculated. In addition, using the RefSeq DB, a protein showing rank 1 at the cICAT peptide level and a score of 20 or more and a protein judged to be correct as a result of peptide scrutiny were identified proteins. As a result, it is possible to identify 356 types of proteins, of which 2 are peak-congested and cannot be quantified comparatively, 2 are present only in PD-CSF (H / L = ∞), 99 are present only in serum. There were six types (H / L = 0), and the remaining 249 types were able to calculate comparative quantitative values between 0 and infinity (FIG. 1). This result shows that much more proteins can be analyzed than the number of proteins (about 120 kinds) obtained by comparative analysis of sera treated in the same manner (PDS and JNS). In addition, about 80% of the identified protein was the same protein as that obtained by identifying PD-CSF by the cICAT method. From this, it is considered that the above result is that a low-expressing protein derived from a lesion tissue below the identification limit in serum was identified by a protein present in PD-CSF, and comparative quantification was possible. Furthermore, using this principle, PD-CSF was similarly labeled with H reagent and Parkinson's disease patient serum (PDS) was separately labeled with L reagent by cICAT method, and protein identification and comparative quantitative analysis were performed. As a result, it is possible to identify 306 types of proteins, 8 types that are peak-congested and cannot be quantified comparatively, only 29 types are present in PD-CSF (H / L = ∞), and only exist in serum. (H / L = 0) was 4 types, and the remaining 265 types were able to calculate comparative quantitative values (PD-CSF / PDS) between 0 and infinity (FIG. 2).
　そこで、上記の２つの比較定量値すなわち、ＰＤ－ＣＳＦ／ＪＮＳおよびＰＤ－ＣＳＦ／ＰＤＳが得られた各たんぱく質に関して、（ＰＤ－ＣＳＦ／ＪＮＳ）値を（ＰＤ－ＣＳＦ／ＰＤＳ）値で割る（ＰＤ－ＣＳＦ／ＪＮＳ÷ＰＤ－ＣＳＦ／ＰＤＳ＝ＰＤＳ／ＪＮＳ）ことにより、ＰＤＳ／ＪＮＳを計算し、なお、分母および分子が∞の時および０の時は、比較定量計算不能とした。その結果、ＰＤＳ／ＪＮＳに関しては、比較定量不能なものは１０４種類であり、比較定量可能なものは２０２種類であった。図３に比較定量可能なたんぱく質（２０２種類）の比較定量値をＧｉ＃順に並び替えたものを示す。 Therefore, the (PD-CSF / JNS) value is divided by the (PD-CSF / PDS) value for each protein from which the above two comparative quantitative values, ie, PD-CSF / JNS and PD-CSF / PDS were obtained ( PD-CSF / JNS ÷ PD-CSF / PDS = PDS / JNS), and PDS / JNS was calculated. When the denominator and numerator were ∞ and 0, comparative quantitative calculation was impossible. As a result, regarding PDS / JNS, there were 104 types that could not be comparatively quantified, and 202 types that could be comparatively quantified. FIG. 3 shows the comparative quantitative values of proteins (202 types) that can be comparatively quantified, rearranged in the order of Gi #.
　また、健常者血清との比較定量値（ＰＤＳ／ＪＮＳ）が５以上、もしくは１／５以下を示す特異性の高いたんぱく質はパーキンソン病患者血清では合計１０種類以上存在し、そのうち代表的な６種類のたんぱく質を表１に示した。なお、参照として、関連たんぱく質で殆ど変動していないたんぱく質２種類の比較定量値（ＰＤＳ／ＪＮＳ）も示す。また、パーキンソン病患者血清の代わりに、パーキンソン様症候群患者血清（ＰＳＳ：１０名分をプールしたもの）を用い、日本人健常者標準血清（ＪＮＳ：１０名分をプールしたもの）及びパーキンソン病患者脳脊髄液（ＰＤ－ＣＳＦ：パーキンソン病患者１０名のＣＳＦをプールしたもの（病変組織由来たんぱく質として）のたんぱく質を、同様にｃＩＣＡＴ法により、同定・比較定量解析を行い、得られた上述たんぱく質の比較定量値（ＰＳＳ／ＪＮＳ）も参考として表１に示す。
Figure JPOXMLDOC01-appb-T000001
 ＊表中のＮＡは、ピーク混雑のため、定量不能であることを示す。 In addition, there are a total of 10 or more proteins with high specificity showing a comparative quantitative value (PDS / JNS) of 5 or more or 1/5 or less with that of healthy subjects, and 6 of them are representative of them. The proteins are shown in Table 1. For reference, the comparative quantitative values (PDS / JNS) of two types of proteins that hardly change in related proteins are also shown. Also, instead of Parkinson's disease patient serum, Parkinson's syndrome patient serum (PSS: pooled for 10 patients), Japanese healthy standard serum (JNS: pooled for 10 persons) and Parkinson's disease patients Proteins of cerebrospinal fluid (PD-CSF: pooled CSF of 10 Parkinson's disease patients (as lesion tissue-derived proteins) were similarly subjected to identification and comparative quantification analysis by cICAT method. Comparative quantitative values (PSS / JNS) are also shown in Table 1 for reference.
Figure JPOXMLDOC01-appb-T000001
 * NA in the table indicates that quantification is impossible due to peak congestion.
　好ましくは、これらのたんぱく質を測定することにより、パーキンソン病の病態の判定を正確に行うことが可能となり、さらに健常者とパーキンソン病患者を区別して診断することが可能になる。 Preferably, by measuring these proteins, it becomes possible to accurately determine the pathological condition of Parkinson's disease, and further, it is possible to make a diagnosis by distinguishing healthy subjects from Parkinson's disease patients.
　本発明は、パーキンソン病の研究分野ならびにそれらの病態判定のためのキットの製造などの分野、さらにはパーキンソン病の新規治療薬の創薬やスクリーニングの分野において利用可能である。 The present invention can be used in the field of research on Parkinson's disease, the manufacture of kits for determining their pathology, and the field of drug discovery and screening for new therapeutic drugs for Parkinson's disease.

Claims (15)

  1. 　パーキンソン病の病態の判定方法であって、下記工程（ａ）～（ｃ）を含む方法によりパーキンソン病のマーカーと同定されたたんぱく質の量を調べることを含む方法：
    　（ａ）パーキンソン病患者の組織由来たんぱく質および健常人の血清たんぱく質を解析し、同定たんぱく質に関する比較定量値：
    　　パーキンソン病患者の組織由来たんぱく質の解析値／健常人の血清たんぱく質の解析値…（Ａ）
    を得て；
    　（ｂ）（ａ）と同一のパーキンソン病患者の組織由来たんぱく質およびパーキンソン病患者の血清たんぱく質を解析し、同定たんぱく質に関する比較定量値：
    　　（ａ）と同一のパーキンソン病患者の組織由来たんぱく質の解析値／パーキンソン病患者の血清たんぱく質の解析値…（Ｂ）
    を得て；次いで
    　（ｃ）（パーキンソン病患者の組織由来たんぱく質の解析値／健常人の血清たんぱく質の解析値）÷（パーキンソン病患者の組織由来たんぱく質の解析値／パーキンソン病患者の血清たんぱく質の解析値）…（Ｃ）
    に従って、（パーキンソン病患者の血清たんぱく質の解析値／健常人の血清たんぱく質の解析値）の値を得ること。     A method for determining a pathological condition of Parkinson's disease, which comprises examining the amount of a protein identified as a marker for Parkinson's disease by a method comprising the following steps (a) to (c):
    (A) Analyzing tissue-derived protein of Parkinson's disease patient and serum protein of healthy person, and comparative quantitative value for the identified protein:
    Analytical value of protein derived from tissue of Parkinson's disease patient / Analytical value of serum protein of healthy person ... (A)
    To obtain;
    (B) Analyzing the same tissue-derived protein of Parkinson's disease patient as in (a) and serum protein of Parkinson's disease patient, and comparative quantitative value for the identified protein:
    Analysis value of protein derived from tissue of Parkinson's disease patient same as (a) / Analysis value of serum protein of Parkinson's disease patient ... (B)
    (C) (analyzed value of protein derived from tissue of Parkinson's disease patient / analyzed value of serum protein of healthy subject) ÷ (analyzed value of protein derived from tissue of Parkinson's disease patient / analysis of serum protein of Parkinson's disease patient) Value) ... (C)
    To obtain the value of (analyzed value of serum protein of Parkinson's disease patient / analyzed value of serum protein of healthy subject).
  2. 　該マーカーと同定されたたんぱく質が、５以上または１／５以下の（パーキンソン病患者の血清たんぱく質の解析値／健常人の血清たんぱく質の解析値）の値のたんぱく質である、請求項１に記載の方法。 The protein identified as the marker is a protein having a value of 5 or more or 1/5 or less (analyzed value of serum protein of Parkinson's disease patient / analyzed value of serum protein of healthy person). Method.
  3. 　該マーカーと同定されたたんぱく質が、latrophilin 3（gi|14149677）、carnosinase 1（gi|21071039）、lactotransferrin（gi|54607120）、decorin isoform a preproprotein（gi|4503271）、superoxide dismutase 3, extracellular precursor（gi|118582275）およびamyloid beta (A4) precursor-like protein 2（gi|4502147）からなる群より選択される少なくとも１種のたんぱく質である、請求項１または２に記載の方法。 Proteins identified as the marker are latrophilin 3 (gi | 14149677), carnosinase 1 (gi | 21071039), lactotransferrin (gi | 54607120), decorin isoform a preproprotein (gi | 4503271), superoxide dismutase 3, extracellular precursor (gi | 118582275) and amyloid beta (A4) precursor-like protein 2 (gi | 4502147). The method according to claim 1 or 2, wherein the protein is at least one protein selected from the group consisting of
  4. 　該マーカーと同定されたたんぱく質が、superoxide dismutase 3, extracellular precursor（gi|118582275）である、請求項１または２に記載の方法。 The method according to claim 1, wherein the protein identified as the marker is superoxide dismutase 3, extracellular precursor (gi | 118582275).
  5. 　該マーカーと同定されたたんぱく質の量が同位体標識法を用いて調べられる、請求項１～４のいずれか１項に記載の方法。 The method according to any one of claims 1 to 4, wherein the amount of the protein identified as the marker is examined using an isotope labeling method.
  6. 　同位体標識法がｃＩＣＡＴ法である、請求項５に記載の方法。 The method according to claim 5, wherein the isotope labeling method is a cICAT method.
  7. 　病態の判定がパーキンソン病患者の治療モニタリングである、請求項１～６のいずれか１項に記載の方法。 The method according to any one of claims 1 to 6, wherein the determination of the disease state is treatment monitoring of a patient with Parkinson's disease.
  8. 　病態の判定がパーキンソン病患者の予後である、請求項１～７のいずれか１項に記載の方法。 The method according to any one of claims 1 to 7, wherein the determination of the pathological condition is a prognosis of a Parkinson's disease patient.
  9. 　治療中のパーキンソン病患者から得た試料中の請求項１～４のいずれかに記載のマーカーと同定されたたんぱく質の量と、治療前および／または治療後の同じ患者、ならびに／あるいは健常人から得た試料中の該たんぱく質の量とを比較することをさらに含む、請求項１～８のいずれか１項に記載の方法。 The amount of the protein identified as the marker according to any of claims 1 to 4 in a sample obtained from a patient with Parkinson's disease being treated, and the same patient before and / or after treatment and / or from a healthy person The method according to any one of claims 1 to 8, further comprising comparing the amount of the protein in the obtained sample.
  10. 　請求項１～８のいずれか１項に記載の方法において、請求項１～４のいずれかに記載のマーカーと同定されたたんぱく質の量を測定するための手段を必須として含む、パーキンソン病の病態の判定用キット。 The method according to any one of claims 1 to 8, wherein the pathological condition of Parkinson's disease essentially comprises means for measuring the amount of the protein identified as the marker according to any one of claims 1 to 4. Evaluation kit.
  11. 　パーキンソン病の治療薬のスクリーニング方法であって、
    　（ａ）パーキンソン病患者に候補薬剤を投与すること；次いで
    　（ｂ）該候補薬剤の投与前および投与後に該患者から採取した試料中の請求項１～４のいずれかに記載のマーカーと同定されたたんぱく質の量を調べることを含む方法。     A screening method for a therapeutic drug for Parkinson's disease,
    (A) administering a candidate drug to a patient with Parkinson's disease; then (b) identified as a marker according to any one of claims 1 to 4 in a sample collected from the patient before and after administration of the candidate drug A method comprising examining the amount of protein.
  12. 　下記工程（ａ）～（ｃ）を含む、パーキンソン病の病態の判定に使用されるパーキンソン病のマーカーの同定方法：
    　（ａ）パーキンソン病患者の組織由来たんぱく質および健常人の血清たんぱく質を解析し、同定たんぱく質に関する比較定量値：
    　　パーキンソン病患者の組織由来たんぱく質の解析値／健常人の血清たんぱく質の解析値…（Ａ）
    を得て；
    　（ｂ）（ａ）と同一のパーキンソン病患者の組織由来たんぱく質およびパーキンソン病患者の血清たんぱく質を解析し、同定たんぱく質に関する比較定量値：
    　　（ａ）と同一のパーキンソン病患者の組織由来たんぱく質の解析値／パーキンソン病患者の血清たんぱく質の解析値…（Ｂ）
    を得て；次いで
    　（ｃ）（パーキンソン病患者の組織由来たんぱく質の解析値／健常人の血清たんぱく質の解析値）÷（パーキンソン病患者の組織由来たんぱく質の解析値／パーキンソン病患者の血清たんぱく質の解析値）…（Ｃ）
    に従って、（パーキンソン病患者の血清たんぱく質の解析値／健常人の血清たんぱく質の解析値）の値を得ること。     A method for identifying a marker for Parkinson's disease used for determination of the pathological condition of Parkinson's disease, comprising the following steps (a) to (c):
    (A) Analyzing tissue-derived protein of Parkinson's disease patient and serum protein of healthy person, and comparative quantitative value for the identified protein:
    Analytical value of protein derived from tissue of Parkinson's disease patient / Analytical value of serum protein of healthy person ... (A)
    To obtain;
    (B) Analyzing the same tissue-derived protein of Parkinson's disease patient as in (a) and serum protein of Parkinson's disease patient, and comparative quantitative value for the identified protein:
    Analysis value of protein derived from tissue of Parkinson's disease patient same as (a) / Analysis value of serum protein of Parkinson's disease patient ... (B)
    (C) (analyzed value of protein derived from tissue of Parkinson's disease patient / analyzed value of serum protein of healthy subject) ÷ (analyzed value of protein derived from tissue of Parkinson's disease patient / analysis of serum protein of Parkinson's disease patient) Value) ... (C)
    To obtain the value of (analyzed value of serum protein of Parkinson's disease patient / analyzed value of serum protein of healthy subject).
  13. 　５以上または１／５以下の（パーキンソン病患者の血清たんぱく質の解析値／健常人の血清たんぱく質の解析値）の値を有するたんぱく質を、パーキンソン病の病態の判定に使用されるパーキンソン病のマーカーと同定する、請求項１２記載の方法。 A protein having a value of 5 or more or 1/5 or less (analyzed value of serum protein of Parkinson's disease patient / analyzed value of serum protein of healthy subject) is used as a marker of Parkinson's disease used for the determination of the pathology of Parkinson's disease 13. The method of claim 12, wherein the method is identified.
  14. 　パーキンソン病の病態の判定のためにパーキンソン病のマーカーとして使用される、latrophilin 3（gi|14149677）、carnosinase 1（gi|21071039）、lactotransferrin（gi|54607120）、decorin isoform a preproprotein（gi|4503271）、superoxide dismutase 3, extracellular precursor（gi|118582275）およびamyloid beta (A4) precursor-like protein 2（gi|4502147）からなる群より選択される少なくとも１種のたんぱく質。 Used as a marker of Parkinson's disease for the determination of Parkinson's disease, latrophilin 3 (gi | 14149677), carnosinase 1 (gi | 21071039), lactotransferrin (gi | 54607120), decorin isoform a preproprotein (gi | 4503271) , Superoxide た dismutase 3, extracellular precursor (gi | 118582275) and amyloid beta (A4) precursor-like protein 2 (gi | 4502147).
  15. 　パーキンソン病の病態の判定のためにパーキンソン病のマーカーとして使用されるsuperoxide dismutase 3, extracellular precursor（gi|118582275）。 Superoxide dismutase 3, extracellular precursor (gi | 118582275) used as a marker for Parkinson's disease to determine the pathology of Parkinson's disease.
PCT/JP2009/062595 2008-07-11 2009-07-10 Disease-related protein for parkinson’s disease, and use thereof WO2010005077A1 (en)

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