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WO2019129029A1 - 作为帕金森症诊治标志物的基因及其用途 - Google Patents

作为帕金森症诊治标志物的基因及其用途 Download PDF

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Publication number
WO2019129029A1
WO2019129029A1 PCT/CN2018/123794 CN2018123794W WO2019129029A1 WO 2019129029 A1 WO2019129029 A1 WO 2019129029A1 CN 2018123794 W CN2018123794 W CN 2018123794W WO 2019129029 A1 WO2019129029 A1 WO 2019129029A1
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Prior art keywords
gene
protein
detecting
product
disease
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English (en)
French (fr)
Inventor
肖枫
汪冰怡
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Beijing Medintell Bioinformatic Technology Co Ltd
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Beijing Medintell Bioinformatic Technology Co Ltd
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Priority claimed from CN201711483559.4A external-priority patent/CN108048554B/zh
Priority claimed from CN201711477253.8A external-priority patent/CN107904304B/zh
Priority claimed from CN201711481574.5A external-priority patent/CN108103183B/zh
Priority claimed from CN201711481575.XA external-priority patent/CN108220419B/zh
Priority claimed from CN201810292179.0A external-priority patent/CN108611413B/zh
Application filed by Beijing Medintell Bioinformatic Technology Co Ltd filed Critical Beijing Medintell Bioinformatic Technology Co Ltd
Publication of WO2019129029A1 publication Critical patent/WO2019129029A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • 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

Definitions

  • the invention belongs to the field of molecular diagnosis, and relates to a gene as a marker for diagnosis and treatment of Parkinson's disease and a use thereof.
  • PD is the second most common, slow-onset, age-related neurodegenerative disease following Alzheimer's disease, also known as tremor.
  • Pathologically the selective loss of dopaminergic neurons (DNs) in the substantia nigra, the formation of Lewy body (LB) in the remaining neurons, and the reduction of striatum dopamine content, resulting in resting tremor, Four major clinical symptoms of muscle rigidity, bradykinesia, and abnormal posture gait (Braak H, Tedici K D, et al. Staging of brain pathology related to sporadic Parkinson, s disease. Neurobiology of Aging, 2003, 24(2) :197-211).
  • PD patients account for 3% of the total population, accounting for 1% of the population over 60 years old, and 8-18 PD patients per 100,000 people worldwide every year (de Lau L M, Breteler MM. Epidemiology of parkinson's Disease. The Lancet Neurology, 2006, 5(6): 525-535). Stephen (K S. Incidence of parkinson's Disease: Variation by age, Gender, and Race/Ethnicity, 2003: 157, 1015-1022) studied the relationship between PD incidence and age, gender, and ethnicity, and found incidence rates above 60 years of age. It will increase rapidly, and only 4% of cases under 50 years old; 1.9 times of male patients are female; the incidence of PD in different races is changing. In general, the incidence of white PD is higher than that of Asians, and blacks are black. It is the lowest.
  • the Unified Pakinson's Disease Rating Scale (UPDRS) and the modified Hoehn and Yahr Parkinson's Disease Staging Scale (HY staging) are clinical diagnostics for PD. And common methods of treatment evaluation.
  • exclusion of brain trauma, cerebrovascular disease, metal poisoning, etc. can cause the cause of secondary Parkinson's disease, and there are no signs of extraocular muscle paralysis, cerebellar sign, cone system damage and muscle atrophy, according to the British brain.
  • the library's diagnostic criteria for Parkinson's disease, patients with less movement, plus one of three symptoms of static tremor, myotonia, and abnormal posture gait can be diagnosed as PD.
  • decreased levels of dopamine and its metabolites in urine and cerebrospinal fluid, and a good response to the drug levodopa also help distinguish between PD and other neurodegenerative diseases.
  • Neurodegenerative diseases including Parkinson's disease, Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis, which are characterized by the storage and specialization of abnormal proteins in different parts of the central nervous system.
  • Loss of sex neurons Ross C A, Poirier M A. Protein aggregation and neurodegenerative disease. Nat Med, 2004, 10 Suppl: S10-S17
  • Neurodegenerative diseases occur in the brain because of pathological changes, and brain tissue is obtained after necropsy after death, so the pathological mechanism is progressing slowly. In general, neurodegenerative diseases are the result of a combination of genes and the environment.
  • the gene expression regulatory network system has made remarkable progress in the field of cancer research.
  • tumor tissues such as lung cancer, breast cancer, gastric cancer, colorectal cancer, etc.
  • the expression levels of certain genes are stable and distinct from the normal tissues of the source.
  • Tumors of different tissue origins even tumors with different differentiation status of the same tissue source, have different gene expression profiles, indicating that gene expression has obvious tissue specificity and developmental stage specificity.
  • the pathological direct feature of neurodegenerative diseases is the overexpression of disease-specific proteins, it is speculated that abnormalities in specific genes alter the expression levels of functional proteins, thereby causing diseases.
  • a molecular marker that can be used for early diagnosis of Parkinson's disease.
  • the use of genetic markers to diagnose Parkinson's disease is timely, specific and sensitive, so that patients can know the disease risk in the early stage of the disease, and take appropriate measures for the risk level. Preventive and therapeutic measures.
  • the invention provides the use of a gene in the preparation of a tool for the diagnosis of Parkinson's disease, the gene being selected from the group consisting of THBD, KCTD20, DNASE2, NACC2, FGFBP2.
  • the present invention provides the use of a product for detecting a gene as described above for the preparation of a tool for the diagnosis of Parkinson's disease.
  • the product for detecting the gene described above includes a product for detecting expression of the gene described above.
  • the product for detecting expression of the gene described above includes a product for detecting the mRNA expression level of the aforementioned gene.
  • the product for detecting the expression level of the mRNA of the aforementioned gene comprises a product for detecting the mRNA expression level of the aforementioned gene by reverse transcription PCR, real-time quantitative PCR, in situ hybridization, gene chip or high-throughput sequencing platform.
  • the product for detecting the expression level of the mRNA of the aforementioned gene by the reverse transcription PCR comprises at least one pair of primers for specifically amplifying the gene described above; and the product for detecting the expression level of the mRNA of the aforementioned gene by the real-time quantitative PCR includes at least a pair of primers for specifically amplifying the aforementioned gene; the product for detecting the expression level of the mRNA of the aforementioned gene by the in situ hybridization comprises: a probe which hybridizes with the nucleic acid sequence of the gene described above; the gene chip detects the aforementioned Products of gene mRNA expression levels include probes that hybridize to the nucleic acid sequences of the genes described above.
  • the real-time quantitative PCR detects at least one pair of primers for specifically amplifying the aforementioned gene, and the primer is:
  • the primer sequence for specifically amplifying the THBD gene is shown in SEQ ID NOS. 1 to 2;
  • the primer sequences for specifically amplifying the KCTD20 gene are shown in SEQ ID NOS. 3 to 4;
  • the primer sequences for specifically amplifying the DNASE2 gene are shown in SEQ ID NOS. 5 to 6;
  • the primer sequences for specifically amplifying the NACC2 gene are shown in SEQ ID NOS. 7 to 8;
  • the primer sequence for specifically amplifying the FGFBP2 gene is shown in SEQ ID NOS. 9 to 10.
  • the product for detecting expression of the aforementioned gene comprises a product for detecting the expression level of a protein encoded by the aforementioned gene.
  • the product for detecting the expression level of the gene-encoded protein described above includes a product for detecting the expression level of the protein encoded by the aforementioned gene by ELISA, immunoblotting, or protein chip.
  • the ELISA detects a product of a protein-coding protein expression level of the aforementioned gene, comprising: an antibody or a ligand that specifically binds to a protein encoded by the aforementioned gene; and the immunoblot detects a product of a protein expression level of the aforementioned gene, including An antibody or a ligand which specifically binds to a protein encoded by a gene as described above; the protein chip detects a product of a protein expression level of the aforementioned gene, and comprises an antibody or a ligand which specifically binds to a protein encoded by the gene described above.
  • the present invention provides a tool for diagnosing Parkinson's disease that is capable of diagnosing Parkinson's disease by detecting a gene described above in a sample.
  • the tool is capable of diagnosing Parkinson's disease by detecting gene expression as described above in the sample.
  • the tool is capable of diagnosing Parkinson's disease by detecting mRNA expression of a gene as described above in the sample, and/or by encoding the protein encoded by the aforementioned gene.
  • the tool comprises a chip, a kit, a test strip or a high throughput sequencing platform.
  • the high-throughput sequencing platform is a special diagnostic tool, and the product detecting the gene expression described above can be applied to the platform to realize the detection of the expression of the aforementioned gene.
  • the chip comprises a gene chip, a protein chip;
  • the gene chip comprises a solid phase carrier and an oligonucleotide probe immobilized on a solid phase carrier, wherein the oligonucleotide probe comprises a gene for detecting the aforementioned gene Transcriptional level of an oligonucleotide probe directed against a gene as described above;
  • the protein chip comprising a solid phase carrier and a specific antibody or ligand immobilized on the solid phase vector of the gene encoding the protein;
  • the gene chip is available The expression level of a plurality of genes including the aforementioned genes (for example, a plurality of genes associated with Parkinson's disease) is detected.
  • the protein chip can be used to detect expression levels of a plurality of proteins (e.g., a plurality of proteins associated with Parkinson's disease) including the protein encoded by the aforementioned gene. By simultaneously detecting multiple markers of Parkinson's disease, the accuracy of Parkinson's disease diagnosis can be greatly improved.
  • a plurality of proteins e.g., a plurality of proteins associated with Parkinson's disease
  • the kit comprises a gene detection kit and a protein immunoassay kit;
  • the gene detection kit comprises an reagent for detecting a transcription level of the gene described above;
  • the protein immunoassay kit comprises the gene coding described above Protein specific antibody.
  • the reagent includes a reagent required for detecting a gene expression level of the aforementioned gene using RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or a microarray method.
  • the reagent comprises a primer and/or a probe for the gene described above. Primers and probes which can be used to detect the expression levels of the aforementioned genes are easily designed based on the nucleotide sequence information of the aforementioned genes.
  • the probe that hybridizes to the nucleic acid sequence of the aforementioned gene may be DNA, RNA, DNA-RNA chimera, PNA or other derivative.
  • the length of the probe is not limited, and any length can be used as long as the specific hybridization is completed and specifically bound to the nucleotide sequence of interest.
  • the length of the probe can be as short as 25, 20, 15, 13 or 10 bases in length. Also, the length of the probe can be as long as 60, 80, 100, 150, 300 base pairs or longer, or even the entire gene.
  • the length of the probe is usually at least 14 base pairs, and the longest is generally no more than 30 base pairs, and the nucleotide sequence of interest Complementary lengths are best at 15-25 base pairs.
  • the probe self-complementary sequence is less than 4 base pairs so as not to affect the efficiency of hybridization.
  • the high throughput sequencing platform includes reagents for detecting the level of transcription of a gene as described above.
  • the test paper includes a test paper carrier and an oligonucleotide immobilized on a test paper carrier capable of detecting the transcription level of the aforementioned gene.
  • the specific antibody encoding the protein encoded by the aforementioned gene includes a monoclonal antibody and a polyclonal antibody.
  • Specific antibodies to the previously encoded genes include intact antibody molecules, any fragments or modifications of antibodies (eg, chimeric antibodies, scFv, Fab, F(ab')2, Fv, etc. as long as the fragments are capable of retaining The ability to bind to the gene-encoded protein described above is sufficient.
  • the preparation of antibodies for protein levels is well known to those skilled in the art, and the present invention can be used to prepare the antibodies using any method.
  • the primer sequence for the gene described above is:
  • the primer sequences for the THBD gene are shown in SEQ ID NOS. 1 to 2;
  • the primer sequences for the KCTD20 gene are shown in SEQ ID NOS. 3 to 4.
  • Primer sequences for the DNASE2 gene are set forth in SEQ ID NOS. 5-6;
  • Primer sequences for the NACC2 gene are set forth in SEQ ID NOS. 7-8;
  • Primer sequences directed against the FGFBP2 gene are set forth in SEQ ID NOS. 9-10.
  • Sources of the aforementioned genes and their expression products for diagnosing Parkinson's disease include, but are not limited to, blood, tissue fluid, urine, saliva, spinal fluid, and the like which can obtain genomic DNA.
  • the source of the aforementioned gene and its expression product for diagnosing Parkinson's disease is blood.
  • the present invention provides the use of the aforementioned gene or an expression product thereof for screening a drug for treating Parkinson's disease.
  • the test compound was added to the culture system, and the expression level of the above-described gene was determined; in the control group, the test compound was not added to the same culture system, and the aforementioned gene was determined.
  • the expression level wherein, if the expression level of the aforementioned gene in the experimental group is smaller than that of the control group, it indicates that the substance to be screened is a drug that inhibits the gene described above.
  • the method further comprises: further testing the drug obtained in the above step to inhibit the effect of Parkinson, and if the test compound has a significant inhibitory effect on Parkinson, the compound is for preventing or treating Parkinson's. drug.
  • the culture system includes, but is not limited to, a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system (such as an animal model, preferably an animal model of a non-human mammal such as a mouse, a rabbit, a sheep, or a monkey. and many more.
  • a compound isolated by the screening method of the present invention is administered as a medicament to a human or other mammal, including but not limited to a mouse, a rat, a guinea pig, a rabbit, a cat, a dog, a sheep, a pig, a cow, a monkey, a donkey,
  • the isolated compounds may be administered directly or may be formulated into various dosage forms using known pharmaceutical preparation methods.
  • the drug may be administered orally as a sugar-coated tablet, a capsule, an elixir, and a microcapsule, as needed; or a sterile solution or suspension with water or any other pharmaceutically acceptable liquid, orally in the form of an injection. Apply.
  • the compound may be mixed in a unit dosage form, such as, but not limited to, sterile water, physiological saline, in a unit dosage required for the generally accepted pharmaceutical administration.
  • a unit dosage form such as, but not limited to, sterile water, physiological saline, in a unit dosage required for the generally accepted pharmaceutical administration.
  • Vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, and the like According to the content of the active ingredient in these preparations, a suitable administration amount within a specified range can be obtained.
  • the present invention provides the use of the aforementioned gene and/or its expression product for the preparation of a medicament for treating Parkinson's disease.
  • the medicament includes an inhibitor of the aforementioned gene and/or an expression product thereof.
  • the inhibitor includes a substance which inhibits expression of the gene described above, a substance which affects the stability of the gene expression product described above, and/or a substance which inhibits the activity of the gene expression product described above.
  • the inhibitor comprises: a double-stranded ribonucleic acid which inhibits expression of a gene described above by interfering with RNA, or a tumor vaccine based on a protein encoded by the aforementioned gene, or for inhibiting a protein encoded by the aforementioned gene Active protein.
  • the inhibitor is an siRNA directed against the aforementioned gene.
  • RNA interference refers to endogenous or exogenous double-stranded RNA (dsRNA)-mediated degradation of intracellular mRNA, resulting in a target gene. Silence of expression results in a corresponding loss of functional phenotype.
  • RNAi technology is a typical negative regulation mechanism that can specifically knock out or turn off the expression of specific genes. This technology has been widely used to explore gene function, gene therapy and new drug development.
  • Cell-based RNAi screening has many advantages in functional genetics studies, mainly in the use of RNAi methods in most cell types, and relatively easy to down-regulate or silence expression of the gene of interest.
  • siRNA-specific fragments were designed based on the mRNA sequences of the genes described above.
  • the design of siRNA is based on published general design principles (Elbashir et. al 2001, Schwarz et. al 2003, Khvorova et. al 2003, Reynolds et. al 2004, Hsieh et. al 2004, Ui-Tei et.
  • the present invention provides a pharmaceutical composition for treating Parkinson, which comprises siRNA against the aforementioned gene and a pharmaceutically acceptable carrier.
  • sequence of the siRNA is shown in SEQ ID NO. 15 to 32.
  • the pharmaceutically acceptable carrier in the present invention includes, but is not limited to, a diluent, an excipient such as water, a filler such as starch, sucrose, etc.; a binder such as a cellulose derivative, an alginate, gelatin, and polyethylene. Pyrrolidone; wetting agent such as glycerin; disintegrating agent such as agar, calcium carbonate and sodium hydrogencarbonate; absorption enhancer quaternary ammonium compound; surfactant such as cetyl alcohol; adsorption carrier such as kaolin and soap clay; lubricant such as talc, Calcium stearate and magnesium, polyethylene glycol and the like.
  • a diluent an excipient such as water, a filler such as starch, sucrose, etc.
  • a binder such as a cellulose derivative, an alginate, gelatin, and polyethylene.
  • Pyrrolidone such as glycerin
  • disintegrating agent such as agar,
  • the medicament of the present invention can also be combined with other drugs for treating Parkinson's, and the combination of a plurality of drugs can greatly mention the success rate of the treatment.
  • a "DNASE2 gene” includes a polynucleotide of the DNASE2 gene and any functional equivalent of the DNASE2 gene.
  • the DNASE2 gene (Chromosome 19, NC_000019.10 (12875211..12881521, complement)) sequence can be found in the international public nucleic acid sequence database GeneBank.
  • THBD gene includes a BRAD gene as well as a polynucleotide of any functional equivalent of the THBD gene.
  • the THBD gene (Chromosome 20, NC_000020.11 (23045633.. 23049664, complement)) sequence can be found in the international public nucleic acid sequence database GeneBank.
  • the "KCTD20 gene” includes a polynucleotide of the KCTD20 gene and any functional equivalent of the KCTD20 gene.
  • the KCTD20 gene (Chromosome 6, NC_000006.12 (36442767.. 36491143)) sequence can be found in the international public nucleic acid sequence database GeneBank.
  • a "NACC2 gene” includes a polynucleotide of the NACC2 gene and any functional equivalent of the NACC2 gene.
  • the NACC2 gene (Chromosome 9, NC_000009.12 (136006537..136095285, complement)) sequence can be found in the international public nucleic acid sequence database GeneBank.
  • the "FGFBP2 gene” includes the human FGFBP2 gene and a polynucleotide equivalent to any function of the human FGFBP2 gene.
  • the sequence of a representative FGFBP2 gene has the same DNA sequence as the FGFBP2 gene (NM_031950.3) in the current international public nucleic acid sequence database GeneBank.
  • gene expression products include proteins as well as partial peptides of proteins.
  • a partial peptide of the protein contains a functional domain associated with Parkinson's disease.
  • DNASE2 protein includes the DNASE2 protein as well as any functional equivalent of the DNASE2 protein.
  • the functional equivalents include a DNASE2 protein conserved variant protein, or an active fragment thereof, or a reactive derivative thereof, an allelic variant, a natural mutant, an inducible mutant, DNA capable of binding to DNASE2 under high or low stringency conditions A protein encoded by a hybridized DNA.
  • THBD protein includes the THTHB protein as well as any functional equivalent of the THBD protein.
  • the functional equivalents include a conservative variant protein of the THBD protein, or an active fragment thereof, or a reactive derivative thereof, an allelic variant, a natural mutant, an inducible mutant, a DNA capable of interacting with THBD under high or low stringency conditions A protein encoded by a hybridized DNA.
  • KCTD20 protein includes the KCTD20 protein as well as any functional equivalent of the KCTD20 protein.
  • the functional equivalents include a KCTD20 protein conserved variant protein, or an active fragment thereof, or a reactive derivative thereof, an allelic variant, a natural mutant, an inducible mutant, and a DNA capable of interacting with KCTD20 under high or low stringency conditions.
  • a protein encoded by a hybridized DNA includes a KCTD20 protein conserved variant protein, or an active fragment thereof, or a reactive derivative thereof.
  • NACC2 protein includes the NACC2 protein as well as any functional equivalent of the NACC2 protein.
  • the functional equivalents include a conservative variant protein of the NACC2 protein, or an active fragment thereof, or a reactive derivative thereof, an allelic variant, a natural mutant, an inducible mutant, and a DNA capable of interacting with NACC2 under high or low stringency conditions.
  • a protein encoded by a hybridized DNA includes a conservative variant protein of the NACC2 protein, or an active fragment thereof, or a reactive derivative thereof, an allelic variant, a natural mutant, an inducible mutant, and a DNA capable of interacting with NACC2 under high or low stringency conditions.
  • FGFBP2 protein includes the FGFBP2 protein as well as any functional equivalent of the FGFBP2 protein.
  • the functional equivalents include a FGFBP2 protein conserved variant protein, or an active fragment thereof, or a reactive derivative thereof, an allelic variant, a natural mutant, an inducible mutant, and a DNA capable of interacting with FGFBP2 under high or low stringency conditions.
  • a protein encoded by a hybridized DNA is a protein that encoded by a hybridized DNA.
  • modification of one or more amino acids in a protein does not affect the function of the protein.
  • One skilled in the art will recognize that altering a single amino acid or a small percentage of amino acids or individual additions, deletions, insertions, substitutions to an amino acid sequence is a conservative modification in which a protein change produces a protein with similar function.
  • Conservative substitution tables that provide functionally similar amino acids are well known in the art.
  • a protein modified by the addition of one amino acid or a plurality of amino acid residues is a fusion protein of a protein encoded by the aforementioned gene.
  • a fusion protein of a protein encoded by the aforementioned gene There is no limitation on the peptide or protein fused to the gene-encoded protein described above, as long as the resulting fusion protein retains the biological activity of the protein.
  • diagnosing Parkinson's disease includes both determining whether a subject already has Parkinson's disease and also determining whether the subject is at risk of having Parkinson's disease.
  • the present invention finds for the first time that the expression of THBD, KCTD20, DNASE2, NACC2, and FGFBP2 is associated with Parkinson's disease.
  • detecting the expression of the former gene in the subject it is possible to determine whether the subject has Parkinson's disease or to judge the subject. Whether there is a risk of Parkinson's disease, instructing the clinician to provide a preventive or treatment plan for the subject.
  • the invention discovers a new molecular marker, which is more timely, more specific and more sensitive than the traditional detection means, and can realize the early diagnosis of Parkinson's disease, thereby reducing the mortality of Parkinson's disease.
  • Figure 1 shows the difference in expression of DNASE2 gene in Parkinson's disease patients and normal persons using a gene chip
  • Figure 2 shows the difference in expression of DNASE2 gene in Parkinson's disease patients and normal persons by QPCR
  • Figure 3 shows the difference in expression of the THBD gene in Parkinson's disease patients and normal persons using a gene chip
  • Figure 4 shows the difference in expression of the THBD gene in Parkinson's disease patients and normal persons by QPCR
  • Figure 5 shows the difference in expression of KCTD20 gene in Parkinson's disease patients and normal persons using a gene chip
  • Figure 6 shows the difference in expression of KCTD20 gene in Parkinson's disease patients and normal persons by QPCR
  • Figure 7 shows the difference in expression of the NACC2 gene in Parkinson's disease patients and normal persons using a gene chip
  • Figure 8 shows the difference in expression of the NACC2 gene in Parkinson's disease patients and normal persons by QPCR
  • FIG. 9 shows the expression of FGFBP2 gene in the blood of patients with Parkinson's disease by QPCR
  • Figure 10 shows the effect of detecting siRNA on gene expression by using QPCR
  • Figure 11 shows the effect of KCTD20 on the content of ⁇ -syn by ELISA
  • Figure 12 shows the effect of siRNA on the expression of THBD gene by QPCR
  • Figure 13 shows the effect of THBD on the content of ⁇ -syn by ELISA
  • Figure 14 shows the effect of siRNA on the expression of FGFBP2 gene by QPCR
  • Figure 15 shows the effect of detecting FGFBP2 gene expression on the growth of Parkinson's nerve cells using MTT.
  • PD inclusion criteria Diagnostic criteria are in line with PD clinical diagnostic criteria (refer to "Jiang Yuping, Wang Jian, Ding Zhengtong, et al., Diagnostic criteria for primary Parkinson's disease, 2005, Chinese Journal of Clinical Neuroscience, 2006, 14:40" ). Exclusion criteria: (1) idiopathic tremor; (2) secondary Parkinson's syndrome; (3) severe dementia, dysarthria; (4) patients with other mental disorders. The study was approved by the hospital ethics committee and all patients signed informed consent.
  • Normal group 10 healthy volunteers aged 32-80 years old, 5 males and 5 females.
  • Fresh blood (peripheral blood) was taken directly, and 3 volumes of red blood cell lysate was added, mixed, left at room temperature for 10 minutes, and centrifuged at 10,000 rpm for 1 minute. Thoroughly discard the supernatant and collect leukocyte pellets. The leukocyte pellet collected per 100-200 ⁇ l of blood was added to 1 ml of TRIzol.
  • RNA is mainly in the aqueous phase, and the aqueous phase (usually 550 ⁇ l can be absorbed) is transferred to a new tube.
  • RNA-free water 50-100 ⁇ l was added to the pellet, and the tube wall was lightly shaken to fully dissolve the RNA and stored at -70 °C.
  • RNA quality expressed by RNA integrity, integrity can be detected by ordinary agarose gel electrophoresis (electrophoresis conditions: 1.2% gel; 0.5 x TBE running buffer; 150v, 15 minutes).
  • RNA purity The OD260/OD280 ratio is an indicator of the extent of protein contamination in an RNA sample. High quality RNA samples with an OD260/OD280 value (10 mM Tris, pH 7.5) around 2.0.
  • Agilent DNA Microarray Scanner scanner was used to perform hybridization at 65 ° C for 17 h in a chip hybridization oven using a human whole gene expression profiling chip (4x 44K gene) from Agilent, USA.
  • the hybridized chip After the hybridized chip reads the data point by the chip scanner, the data is introduced into the analysis software, and the gene with the natural logarithm absolute value of the two ratios greater than 2.0 or less than 0.5 is used as the differentially expressed gene.
  • RNA in total RNA was removed using the Ribo-Zero kit.
  • the cDNA library was constructed using the Illumina TruseqTM RNA sample Prep Kit, and the specific procedures were carried out according to the instructions.
  • the cDNA library was sequenced using the Illumina X-Ten sequencing platform, and the specific procedures were performed according to the instructions.
  • RNA-seq read position was mapped by TopHat v1.3.1, the number of RNA-seq fragments was normalized by Cufflinks v1.0.3 to calculate the relative abundance of transcripts, and the differential expression was detected by cuffdiff.
  • the p value is ⁇ 0.05, the gene is considered to be significantly differentially expressed.
  • RNA-seq results showed that the expression level of FGFBP2 gene in the blood of patients with Parkinson's disease was significantly higher than that in normal human blood.
  • the screening criteria were the same as in Example 1, 50 patients with Parkinson's disease and 50 normal subjects.
  • the cDNA was reverse transcribed into 1 ⁇ g of total RNA using reverse transcription buffer.
  • 1 ⁇ g of total RNA was used as template RNA for each sample, and the following components were added to the PCR tube: DEPC water, 5 ⁇ reverse transcription buffer, 10 mmol/L dNTP, 0.1 mmol/l DTT, 30 ⁇ mmol/l Oligo dT, 200 U/ ⁇ l M-MLV, template RNA.
  • Incubate at 42 ° C for 1 h, 72 ° C for 10 min, and briefly centrifuge.
  • the QPCR amplification primers were designed according to the coding sequences of DNASE2 gene and GAPDH gene in Genbank, and synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd.
  • the specific primer sequences are as follows:
  • the forward primer was 5'-CAGCATCACACTCACATC-3' (SEQ ID NO. 5);
  • the reverse primer is 5'-AGTACAGGTCATCTCCAA-3' (SEQ ID NO. 6),
  • the forward primer was 5'-CTCAATGCCAGTCAGATC-3' (SEQ ID NO. 1);
  • the reverse primer is 5'-GTTCAGTAGCAAGGAAATG-3' (SEQ ID NO. 2)
  • the forward primer was 5'-GCTGCTTAGTGGATGATAC-3' (SEQ ID NO. 3);
  • the reverse primer was 5'-AGATTATGAGGACCAGATGA-3' (SEQ ID NO. 4),
  • the forward primer was 5'-ATGGTGGAGGAGCAGTAC-3' (SEQ ID NO. 7);
  • the reverse primer was 5'-TGGCTCAGGCTTCTCTTG-3' (SEQ ID NO. 8),
  • the forward primer was 5'-TTTAACTCTGGTAAAGTGGATAT-3' (SEQ ID NO. 11);
  • the reverse primer was 5'-GGTGGAATCATATTGGAACA-3' (SEQ ID NO. 12).
  • the SYBR Green polymerase chain reaction system was purchased from Invitrogen.
  • Reagent volume Forward primer 1 ⁇ l Reverse primer 1 ⁇ l SYBR Green polymerase chain reaction system 12.5 ⁇ l template 2 ⁇ l Deionized water Make up 25 ⁇ l
  • PCR reaction conditions 95 ° C for 10 min, (95 ° C 10 s, 60 ° C 40 s) * 45 cycles.
  • the SYBR Green was used as a fluorescent marker, and the PCR reaction was carried out on a Light Cycler quantitative PCR machine.
  • the target band was determined by melting curve analysis and electrophoresis, and the ⁇ CT method was used for relative quantification.
  • the screening criteria were the same as in Example 1, 90 patients with Parkinson's disease and normal subjects.
  • RNA extraction step is the same as in Example 1.
  • a 25 ⁇ l reaction system was constructed, which included 5 ⁇ l of 5 ⁇ reverse transcription buffer, 5 ⁇ l of dNTP (2.5 mM), 40 U/ ⁇ l of RNasin, and 200 U/ ⁇ l of M-MLV, and the nuclease-free water was added to the expected volume.
  • the QPCR amplification primers were designed according to the coding sequences of FGFBP2 gene and GAPDH gene in Genbank, and synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd.
  • the specific primer sequences are as follows:
  • the forward primer was 5'-ATGAGGAAGCAAAGAAGA-3' (SEQ ID NO. 9);
  • the reverse primer was 5'-GGAAGAAGCTGATGAGAA-3' (SEQ ID NO. 10).
  • the forward primer was 5'-AACTCTGGTAAAGTGGATATTG-3' (SEQ ID NO. 13);
  • the reverse primer was 5'-GGTGGAATCATATTGGAACA-3' (SEQ ID NO. 14).
  • the SYBR Green polymerase chain reaction system was purchased from Invitrogen.
  • PCR reaction conditions 95 ° C for 10 min, (95 ° C 15 s, 60 ° C 60 s) * 45 cycles.
  • the SYBR Green was used as a fluorescent marker, and the PCR reaction was carried out on a Light Cycler quantitative PCR machine.
  • the target band was determined by melting curve analysis and electrophoresis, and the ⁇ CT method was used for relative quantification.
  • the experiment used 3 repeated experiments, the results were expressed in the mean ⁇ standard deviation, using SPSS13.0 statistical software for statistical analysis, the difference between the two using t test, that when P ⁇ 0.05 has statistical significane.
  • Example 3 Western blotting experiments confirmed the expression products of differentially expressed genes in Parkinson's disease patients and normal subjects
  • HBSS Na 2 HPO 4 0.132g, KH 2 PO 4 0.06g, KCl 0.4g, phenol red 1ml, NaHCO 3 0.35g, D-glucose 1.0g, dissolved in 1000ml
  • Double distilled water Double distilled water
  • the cell suspension obtained in the above experiment (concentration: 1 ⁇ 10 6 /ml) was centrifuged at room temperature 1 000 r/min for 10 min, the supernatant was discarded, 100 ⁇ l of lysis buffer was added, and the mixture was shaken at 4 ° C for 1 h, and the cells were disrupted by a sonicator for 10 s each time. 10 times, centrifuged at 12000r/min for 1h at 4°C; the supernatant was used to quantify the protein by Brandford method, dispensed into 2.5 ⁇ g/ ⁇ l, and stored in a -80°C refrigerator for later use.
  • Total cellular protein was quantified by Brandford method, mixed with sample buffer for 5 min, cooled for 5 min; 30 pg of protein was applied to the prepared 15% polyacrylamide gel, electrophoresed, and set to 80V constant pressure, see Marker After the increase to 120V; the gel after electrophoresis was removed and transferred to 100V for 50 min using Bio.Rad semi-dry transfer system; after the membrane was finished, it was washed once with 1 ⁇ PBS, immersed in blocking solution, and overnight at 40 C; the blocking solution was drained and added to Western.
  • washing solution for 5-10min add to the anti-shaker bed for 2h at room temperature; dilute in the appropriate buffer with Western secondary antibody dilution solution in blocking buffer for 60min; wash the membrane solution 3 times for 10min each time; use ECL reagent to develop Fixation detects protein expression.
  • the gray value of the protein band was analyzed using Image J software, and the gray value of the target white band was normalized by using ⁇ -actin as an internal parameter. The results were expressed as mean ⁇ standard deviation. Statistical analysis was performed using SPSS 13.0 statistical software. The difference between the two was analyzed by t test, which was considered statistically significant when P ⁇ 0.05.
  • the diluted siRNA and the transfection reagent were mixed (the ratio of siRNA to transfection reagent was 1:2), allowed to stand at room temperature for 10-15 min, added to the cell culture medium, and gently mixed. After the cells were further cultured for 72 hours, they were centrifuged at 12,000 rpm for 10 min at 4 ° C, and the supernatant was collected for subsequent ELISA detection. RNA was extracted from the lysed cells, and QPCR was used to detect the effect of siRNA silencing KCTD20.
  • siRNA sequence of KCTD20 was designed according to the sequence of rat KCTD20 gene (Chromosome 20, NC_005119.4 (6205897..6222818)) as follows:
  • KCTD20-SIRNA1 (referred to as KCTD-1)
  • the sense strand is 5'-GGUCUGAAGUUAUCUACAATT-3' (SEQ ID NO. 15),
  • the antisense strand is 5'-UUGUAGAUAACUUCAGACCTT-3' (SEQ ID NO. 16);
  • KCTD20-SIRNA2 (referred to as KCTD-2)
  • the sense strand is 5'-CCAGGAGAUAAUGAUGCAUTT-3' (SEQ ID NO. 17),
  • the antisense strand is 5'-AUGCAUCAUUAUCUCCUGGTT-3' (SEQ ID NO. 18);
  • KCTD20-SIRNA3 (referred to as KCTD-3)
  • the sense strand is 5'-GCGACUAUCUCUGCAUUAATT-3' (SEQ ID NO. 19),
  • the antisense strand is 5'-UUAAUGCAGAGAUAGUCGCTT-3' (SEQ ID NO. 20).
  • RNA extraction Add 400 ⁇ L of Trizol to each well of a 12-well plate, gently pipette a few times to fully lyse the cells, then transfer the cell lysate to a 1.5 mL centrifuge tube, add 80 ⁇ L of chloroform to each tube, and vortex well. It was allowed to stand at room temperature for 3 min, centrifuged at 12,000 rpm for 15 min at 4 °C. Take the uppermost layer into a new centrifuge tube, add 200 ⁇ L of isopropanol to each tube, gently mix upside down, let stand for 10 min at room temperature, and centrifuge at 10 ° C for 10 min at 12000 rpm.
  • the ⁇ -syn-mAb (Abeam, ab138501) was coated into a 96-well plate (0.5 ⁇ g/well) at 4 ° C overnight. After washing three times with 0.1% PBST, 3% BSA was blocked. The PC12- ⁇ -syn cell supernatant was concentrated with a 3k ultrafiltration tube, added to a 96-well plate, 100 ⁇ L per well, and reacted at 37 ° C for 1 hour, and washed with 0.1% PBST six times.
  • Syn-1 (BD, 610787) was added as a detection antibody, and after reacting for 1 hour at 37 ° C, HRP-labeled goat anti-mouse secondary antibody was added, and TMB was developed, and the SpectraMax M5 microplate reader (Molecular Devices, LLC, Sunnyvale) was used. , CA, USA) Measurement of absorbance (450 nm).
  • the amount of ⁇ -syn in the cell supernatant was increased by 2.12 times after the siRNA inhibited the expression of KCTD20 as compared with the control group.
  • siRNA sequence of THBD was designed according to the sequence of rat THBD gene (Chromosome 3, NC_005102.4 (142748673..142752325, complement)) as follows:
  • THBD-siRNA1 (abbreviated as THBD-1)
  • the sense strand is 5'-GCUUCGAAUGCCGCUGCUATT-3' (SEQ ID NO. 21),
  • the antisense strand is 5'-UAGCAGCGGCAUUCGAAGCTT-3' (SEQ ID NO. 22);
  • THBD-siRNA2 (abbreviated as THBD-2)
  • the sense strand is 5'-GGUGCGAAAUGUUCUGCAATT-3' (SEQ ID NO. 23),
  • the antisense strand is 5'-UUGCAGAACAUUUCGCACCTT-3' (SEQ ID NO. 24);
  • THBD-siRNA3 (abbreviated as THBD-3)
  • the sense strand is 5'-GCGCAGAGCUGGAGUACAATT-3' (SEQ ID NO. 25),
  • the antisense strand is 5'-UUGUACUCCAGCUCUGCGCTT-3' (SEQ ID NO. 26).
  • RNA extraction Add 400 ⁇ L of Trizol to each well of a 12-well plate, gently pipette a few times to fully lyse the cells, then transfer the cell lysate to a 1.5 mL centrifuge tube, add 80 ⁇ L of chloroform to each tube, and vortex well. It was allowed to stand at room temperature for 3 min, centrifuged at 12,000 rpm for 15 min at 4 °C. Take the uppermost layer into a new centrifuge tube, add 200 ⁇ L of isopropanol to each tube, gently mix upside down, let stand for 10 min at room temperature, and centrifuge at 10 ° C for 10 min at 12000 rpm.
  • the ⁇ -syn-mAb (Abeam, ab138501) was coated into a 96-well plate (0.5 ⁇ g/well) at 4 ° C overnight. After washing three times with 0.1% PBST, 3% BSA was blocked. The PC12- ⁇ -syn cell supernatant was concentrated with a 3k ultrafiltration tube, added to a 96-well plate, 100 ⁇ L per well, and reacted at 37 ° C for 1 hour, and washed with 0.1% PBST six times.
  • Syn-1 (BD, 610787) was added as a detection antibody, and after reacting for 1 hour at 37 ° C, HRP-labeled goat anti-mouse secondary antibody was added, and TMB was developed, and the SpectraMax M5 microplate reader (Molecular Devices, LLC, Sunnyvale) was used. , CA, USA) Measurement of absorbance (450 nm).
  • the amount of ⁇ -syn in the cell supernatant was increased by 4.88 times after the siRNA inhibited the expression of THBD as compared with the control group.
  • Dopamine neuron cell SH-SY5Y in DMEM containing 10% fetal bovine serum, 1% penicillin/streptomycin (pH 7.2-7.4), at 37 ° C, 5% CO 2 , relative humidity of 90% Cultivate in an incubator. Change the liquid every 2 days, pass the cells until the cells grow to 90% contact, wash with PBS, add 0.25%-EDTA trypsin to separate the cells from the bottle wall, and terminate with DMEM medium containing fetal bovine serum.
  • the sense chain is 5'-UUAGAAACUCUCUUCUUCCAG-3' (SEQ ID NO. 27),
  • the antisense strand is 5'-GGAAGAAGAGAGUUUCUAAUC-3' (SEQ ID NO. 28);
  • the sense strand is 5'-AAGUAGUUGUGUAUGCUUGUC-3' (SEQ ID NO. 29),
  • the antisense strand is 5'-CAAGCAUACACAACUACUUAU-3' (SEQ ID NO. 30);
  • the sense strand is 5'-UACAGAUAAUAAGUAGUUGUG-3' (SEQ ID NO. 31),
  • the antisense strand is 5'-CAACUACUUAUUAUCUGUAGA-3' (SEQ ID NO. 32).
  • adenovirus-mediated siRNA the cells were divided into five groups, SH-SY5Y group: SH-SY5Y cells not transfected with any viral vector, as a blank control; Ad group: infected empty adenovirus plasmid cell group, siRNA1 Group: Adenovirus-mediated interference sequence 1 infected cell group: siRNA2 group: adenovirus-mediated interference sequence 2 infected cell group; siRNA3 group: adenovirus-mediated interference sequence 3 infected cell group.
  • the cells were inoculated into a 6-well cell culture plate at 1 ⁇ 10 5 /well, 2 ml per well, and cultured in a 37 ° C, 5% CO 2 incubator for 24 h, at which time the cell fusion density was about 50%-60%; The supernatant was aspirated, and washed twice with 1 ml of serum-free medium.
  • the adenoviruses of 50% MOI diluted in 1 ml of serum-free medium were shaken once every 20 minutes to increase the infection effect. After 48 hours of infection, Further, the concentration of 1000 ⁇ mol/L MPP + complete medium was added and incubated for 24 hours. Collecting cells for extracting RNA;
  • the specific method is:
  • the culture solution was aspirated, washed once with PBS, and an appropriate amount of TRIzol reagent was added. 1 mL was added to each well of a six-well plate, and the cells were lysed for 5 min at room temperature, uniformly blown, and dispensed into a 1.5 mL Eppendorf tube at 1 mL/tube. Chloroform was added to 400 ⁇ l of chloroform/ml Trizol, and the mixture was shaken up and down 30-50 times by hand and left at room temperature for 5 minutes.
  • RNA precipitate was transferred to a clean Eppendorf tube, 0.4 mL of isopropanol was added, and the mixture was gently mixed, left at room temperature for 10 min, and centrifuged at 7500 g for 10 min at 4 ° C. Discard the supernatant, wash the RNA precipitate with 75% ethanol, centrifuge at 7500g for 5min, dry the RNA precipitate at room temperature, and dissolve it in an appropriate amount of DEPC water after 5-10min. The integrity of the RNA samples was determined by agarose gel electrophoresis with a mass fraction of 1.0%, and the extracted RNA was quantitatively determined using a Bio-Photometer.
  • the 3.3QPCR amplification step is the same as in Example 2.
  • FGFBP2 gene The effect of FGFBP2 gene on cell proliferation of SH-SY5Y Parkinson's disease cell model was examined by MTT assay.
  • the SH-SY5Y cell density was adjusted to 5 ⁇ 10 4 /mL, and 100 ⁇ l of cells per well were seeded in a 96-well culture plate, and the cells were treated as in Example 3, and MTT assay was applied every 12 hours after the treatment until 72 h. MTT reduction assay was used to detect cell viability: the solution in the well was discarded, 100 ⁇ l of medium was added, and 10 ⁇ l of 5 mg/mL MTT solution was added to each well. After incubating at 37 ° C for 4 h, the medium was aspirated and 100 ⁇ l of DMSO was added to each well.
  • the experiment used 3 replicate experiments and statistical analysis using SPSS18.0 statistical software. The difference between the two was analyzed by t test, which was considered statistically significant when P ⁇ 0.05.

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Abstract

本发明公开了THBD基因、KCTD20基因、DNASE2基因、NACC2基因、FGFBP2基因及其表达产物在制备诊断帕金森症的产品中的应用。通过检测THBD基因、KCTD20基因、DNASE2基因、NACC2基因、FGFBP2基因及其表达产物的水平可以判断受试者将来患有帕金森症的风险或者确诊帕金森症的状态。本发明还公开了以上基因在制备治疗帕金森症的药物中的应用，为临床开发治疗帕金森症药物提供候选靶标。

Description

作为帕金森症诊治标志物的基因及其用途 技术领域
本发明属于分子诊断领域，涉及作为帕金森症诊治标志物的基因及其用途。
背景技术
PD是继阿尔兹海默病后的第二大常见的、缓慢发生的、与年龄相关的神经退行性疾病，又名震颤麻搏。病理上中脑黑质多巴胺能神经元(dopaminergic neurons,DNs)选择性丧失、残存神经元中路易小体化(Lewy body,LB)形成和纹状体多巴胺含量明显减少，导致了静止性震颤、肌强直、运动迟缓、姿势步态异常这4个主要的临床症状(Braak H,Tredici K D，et al.Staging of brain pathology related to sporadic Parkinson,s disease.Neurobiology of Aging,2003,24(2)：197-211)。在工业化国家，PD患者占总人口的化3％，占60岁以上人口的1％，全世界每年每100000人中新増8-18例PD患者(de Lau L M,Breteler MM.Epidemiology of parkinson’s disease.The Lancet Neurology,2006,5(6):525-535)。Stephen(K S.Incidence of parkinson’s Disease:Variation by age,Gender,and Race/Ethnicity,2003:157,1015-1022)研究了PD发生率与年龄、性别和种族的关系，发现在60岁以上发病率会快速増加，而50岁以下的病例只占4％；男性患者是女性患者的1.9倍；不同种族PD的发生率则是变化的，总的来说白人PD发生率比亚洲人高，黑人则是最低。
目前除了SPECT或者PET影像方法具有部分辅助诊断作用外，帕金森病统一评分量表(Unified Pakinson’s Disease Rating Scale,UPDRS)和改良的Hoehn和Yahr帕金森病分期量表(H-Y分期)是PD临床诊断和治疗评估的常用方法。一般来说，排除如脑外伤、脑血管疾病、金属中毒等可引起继发性帕金森病的病因，也没有眼外肌麻痹、小脑征、锥体系损害以及 肌萎缩等体征下，根据英国脑库的帕金森病诊断标准，患者有少动，再加上静止性震颤、肌强直、姿势步态异常三种症状中的一种，即可诊断为PD。此外，尿和脑脊液中多巴胺及其代谢产物降低，对药物左旋多巴的反应良好也有助于区分PD和其他神经退行性疾病。
事实上多巴胺能神经元变性缺失50％-70％以上方出现临床症状，这往往使患者错失了早期诊断、早期治疗的机会，所以极需方便有效的生物标志物能帮助PD早期诊断并提高诊断的正确性。
神经退行性疾病，包括帕金森病、阿尔茨海默病、亨廷顿病、和肌萎缩性脊髓侧索硬化症等，它们共同的特征是在中枢神经系统的不同部位有异常蛋白的存储和特发性神经元丢失(Ross C A，Poirier M A.Protein aggregation and neurodegenerative disease.Nat Med，2004,10Suppl：S10-S17)从而导致了运动和认知等功能障碍。神经退行性疾病因为病理改变发生在脑部，脑组织取得都是在死后剖尸所得，所以病理机制研究进展缓慢。总的来说，神经退行性疾病是各种基因和环境共同影响的结果。
基因表达调控网络系统在癌症研究领域取得引人注目的进展，在许多肿瘤组织中如肺癌、乳腺癌、胃癌、结直肠癌等，某些基因的表达水平与其来源正常组织存在稳定而明显的差异。不同组织来源的肿瘤，甚至同一组织来源不同分化状态的加瘤，基因表达谱都不尽相同，表明基因表达具有明显的组织特异性和发育阶段特异性。因为神经退行性疾病病理上的直接特征是疾病特异性蛋白质的过量表达，所以推测特异性基因的异常改变了功能蛋白的表达水平，从而引起了疾病。现已越来越多的研究发现特异基因能够成为诊断帕金森的生物标志物，如以下申请号的专利公开的：201510713527.3、201510464905.9、201510463593.X、201510463617.1、201510809837.5。
大部分疾病都是多基因调控的疾病，为了提高疾病的诊断准确率，将大量基因联合诊断成为必然，因此需要发掘更多与帕金森病相关的分子比标志物以便临床应用。
发明内容
为了弥补现有技术的不足，本发明的目的在于提供一种可用于帕金森症早期诊断的分子标志物。相比传统的帕金森症的诊断方法，使用基因标志物来诊断帕金森症的具有及时性、特异性和灵敏性，从而使患者在疾病早期就能知晓疾病风险，针对风险高低，采取相应的预防和治疗措施。
为了实现上述目的，本发明采用如下技术方案：
本发明提供了基因在制备诊断帕金森症的工具中的应用，所述基因选自以下组：THBD、KCTD20、DNASE2、NACC2、FGFBP2。
本发明提供了检测前面所述基因的产品在制备诊断帕金森症的工具中的应用。
进一步，所述检测前面所述基因的产品包括检测前面所述基因表达的产品。
进一步，所述检测前面所述基因表达的产品包括检测前面所述基因mRNA表达水平的产品。
更进一步，所述检测前面所述基因mRNA表达水平的产品包括通过反转录PCR、实时定量PCR、原位杂交、基因芯片或高通量测序平台检测前面所述基因mRNA表达水平的产品。
更进一步，所述反转录PCR检测前面所述基因mRNA表达水平的产品至少包括一对特异扩增前面所述基因的引物；所述实时定量PCR检测前面所述基因mRNA表达水平的产品至少包括一对特异扩增前面所述基因的引物；所述原位杂交检测前面所述基因mRNA表达水平的产品包括：与前面所述基因的核酸序列杂交的探针；所述基因芯片检测前面所述基因mRNA表达水平的产品包括：与前面所述基因的核酸序列杂交的探针。
优选地，所述实时定量PCR检测前面所述基因mRNA表达水平的产品至少包括的一对特异扩增前面所述基因的引物，所述引物为：
特异扩增THBD基因的引物序列如SEQ ID NO.1～2所示；
特异扩增KCTD20基因的引物序列如SEQ ID NO.3～4所示；
特异扩增DNASE2基因的引物序列如SEQ ID NO.5～6所示；
特异扩增NACC2基因的引物序列如SEQ ID NO.7～8所示；
特异扩增FGFBP2基因的引物序列如SEQ ID NO.9～10所示。
进一步，所述检测前面所述基因表达的产品包括检测前面所述基因编码蛋白表达水平的产品。
进一步，所述检测前面所述基因编码蛋白表达水平的产品包括通过ELISA、免疫印迹、蛋白芯片检测前面所述基因编码蛋白表达水平的产品。
进一步，所述ELISA检测前面所述基因编码蛋白表达水平的产品包括：与前面所述基因编码蛋白特异性结合的抗体或配体；所述免疫印迹检测前面所述基因编码蛋白表达水平的产品包括：与前面所述基因编码蛋白特异性结合的抗体或配体；所述蛋白芯片检测前面所述基因编码蛋白表达水平的产品包括：与前面所述基因编码蛋白特异性结合的抗体或配体。
本发明提供了一种用于诊断帕金森症的工具，所述工具能够通过检测样本中前面所述基因来诊断帕金森症。
进一步，所述工具能够通过检测样本中前面所述基因表达来诊断帕金森症。
进一步，所述工具能够通过检测样本中前面所述基因mRNA表达，和/或，前面所述基因编码蛋白表达来诊断帕金森症。
优选地，所述工具包括芯片、试剂盒、试纸或高通量测序平台。其中，高通量测序平台是一种特殊的诊断工具，检测前面所述基因表达的产品可以应用于该平台实现对前面所述基因的表达情况的检测。随着高通量测序技术的发展，对一个人的基因表达谱的构建将成为十分便捷的工作。通过对比疾病患者和正常人群的基因表达谱，容易分析出哪个基因的异常与疾病相关。因此，在高通量测序中获知前面所述基因的异常与帕金森症相关也属于前面所述基因的用途，同样在本发明的保护范围之内。
其中，所述芯片包括基因芯片、蛋白质芯片；所述基因芯片包括固相载体以及固定在固相载体的寡核苷酸探针，所述寡核苷酸探针包括用于检测前面所述基因转录水平的针对前面所述基因的寡核苷酸探针；所述蛋白质芯片包括固相载体以及固定在固相载体的前面所述基因编码蛋白的特异性抗体或配体；所述基因芯片可用于检测包括前面所述基因在内的多个基因(例如，与帕金森症相关的多个基因)的表达水平。所述蛋白质芯片可用于检测包括前面所述基因编码蛋白在内 的多个蛋白质(例如与帕金森症相关的多个蛋白质)的表达水平。通过将多个与帕金森症的标志物同时检测，可大大提高帕金森症诊断的准确率。
其中，所述试剂盒包括基因检测试剂盒和蛋白免疫检测试剂盒；所述基因检测试剂盒包括用于检测前面所述基因转录水平的试剂；所述蛋白免疫检测试剂盒包括前面所述基因编码蛋白的特异性抗体。进一步，所述试剂包括使用RT-PCR、实时定量PCR、免疫检测、原位杂交或芯片方法检测前面所述基因表达水平过程中所需的试剂。优选度，所述试剂包括针对前面所述基因的引物和/或探针。根据前面所述基因的核苷酸序列信息容易设计出可以用于检测前面所述基因表达水平的引物和探针。
与前面所述基因的核酸序列杂交的探针可以是DNA、RNA、DNA-RNA嵌合体、PNA或其它衍生物。所述探针的长度没有限制，只要完成特异性杂交、与目的核苷酸序列特异性结合，任何长度都可以。所述探针的长度可短至25、20、15、13或10个碱基长度。同样，所述探针的长度可长至60、80、100、150、300个碱基对或更长，甚至整个基因。由于不同的探针长度对杂交效率、信号特异性有不同的影响，所述探针的长度通常至少是14个碱基对，最长一般不超过30个碱基对，与目的核苷酸序列互补的长度以15-25个碱基对最佳。所述探针自身互补序列最好少于4个碱基对，以免影响杂交效率。
所述高通量测序平台包括检测前面所述基因转录水平的试剂。
所述试纸包括试纸载体和固定在试纸载体上的寡核苷酸，所述寡核苷酸能够检测前面所述基因的转录水平。
进一步，所述前面所述基因编码蛋白的特异性抗体包括单克隆抗体、多克隆抗体。前面所述基因编码蛋白的特异性抗体包括完整的抗体分子、抗体的任何片段或修饰(例如，，嵌合抗体、scFv、Fab、F(ab’)2、Fv等。只要所述片段能够保留与前面所述基因编码蛋白的结合能力即可。用于蛋白质水平的抗体的制备时本领域技术人员公知的，并且本发明可以使用任何方法来制备所述抗体。
在本发明的具体实施方案中，所述针对前面所述基因的引物序列为：
针对THBD基因的引物序列如SEQ ID NO.1～2所示；
针对KCTD20基因的引物序列如SEQ ID NO.3～4所示；
针对DNASE2基因的引物序列如SEQ ID NO.5～6所示；
针对NACC2基因的引物序列如SEQ ID NO.7～8所示；
针对FGFBP2基因的引物序列如SEQ ID NO.9～10所示。
用于诊断帕金森症的前面所述基因及其表达产物的来源包括但不限于血液、组织液、尿液、唾液、脊髓液等可以获得基因组DNA的体液。在本发明的具体实施方案中，用于诊断帕金森症的前面所述基因及其表达产物的来源是血液。
本发明提供了前面所述的基因或其表达产物在筛选治疗帕金森的药物中的应用。
筛选治疗帕金森的药物的步骤如下：
在实验组中，向培养体系中加入待测化合物，并测定前面所述的基因的表达水平；在对照组中，向同样的培养体系中不加入待测化合物，并测定前面所述基因的的表达水平；其中，如果实验组中前面所述基因的表达水平小于对照组，则说明该待筛选的物质为抑制前面所述基因的药物。
在本发明中，所述的方法还包括：对上面步骤获得的药物进一步测试其抑制帕金森的效果，若测试化合物对帕金森有显著的抑制效果，则说明该化合物为预防或治疗帕金森的药物。
所述培养体系包括(但不限于)细胞体系、亚细胞体系、溶液体系、组织体系、器官体系或动物体系(如动物模型，优选非人哺乳动物的动物模型，如鼠、兔、羊、猴等)等。
当将通过本发明的筛选方法分离的化合物作为药物施用于人或其它哺乳动物，其包括但不限于小鼠、大鼠、豚鼠、兔、猫、犬、羊、猪、牛、猴子、狒狒、黑猩猩时，分离的化合物可以直接施用，或者可以利用已知的药物制备方法配制成各种剂型。例如，根据需要，所述药物可以作为糖衣片剂、胶囊剂、酏剂和微胶囊口服施用；或者用水或任何其它药物可接受的液体配制成无菌溶液或悬浮液，以注射剂的形式非口服施用。例如，可以将化合物以一般接受的药物施用方式所需的单位剂型(unit dose)、与药学可接受的载体或介质混合在一起，所述载体或介质包括但不限于无菌水、生理盐水、植物油、乳化剂、悬浮剂、表面活性剂、稳定剂、调味剂、赋形剂(excipient)、媒介物(vehicle)、防腐剂、粘合剂等。 根据这些制剂中有效成分的含量，可以获得在指定范围内的合适的给药量。
本发明提供了前面所述基因和/或其表达产物在制备治疗帕金森病药物中的应用。
进一步，所述药物包括前面所述基因和/或其表达产物的抑制剂。
更进一步，所述抑制剂包括抑制前面所述基因表达的物质、影响前面所述基因表达产物稳定性的物质、和/或抑制前面所述基因表达产物活性的物质。
作为可选择的实施方式，所述抑制剂包括：通过干扰RNA抑制前面所述基因表达的双链核糖核酸，或基于前面所述基因编码蛋白的肿瘤疫苗、或用于抑制前面所述基因编码蛋白活性的蛋白质。
作为本发明的一种优选的实施方式，所述抑制剂是针对前面所述基因的siRNA。
在本发明中，所述RNA干扰(RNA interference，RNAi)是指内源性或外源性双链RNA(double-stranded RNA，dsRNA)介导的细胞内mRNA发生特异性降解，从而导致靶基因的表达沉默，产生相应的功能表型缺失的现象。RNAi技术是一种典型的负调控机制，使用该技术可以特异性剔除或关闭特定基因的表达，该技术已被广泛用于探索基因功能、基因治疗及新药开发领域。以细胞为基础的RNAi筛选在功能基因学研究方面具有许多优势，主要表现在大多数细胞类型都能使用RNAi方法，并且相对较容易下调或沉默目的基因的表达。
为了确保前面所述基因能够被高效剔除或沉默，根据前面所述基因的mRNA序列设计了siRNA特异性片段。siRNA的设计根据已发表的通用设计原则(Elbashir et.al 2001，Schwarz et.al 2003，Khvorova et.al 2003，Reynolds et.al 2004，Hsieh et.al 2004，Ui-Tei et.al 2004)，通过在线工具完成设计，该在线工具为：siRNASelectionProgram of Whitehead Institute(BingbingYuan et.al 2004，http://jura.wi.mit.edu/bi℃/siRNAext/)和BL℃K-iTTM RNAi Designer ofINVITROGEN(winner of the 2004Frost&Sullivan Excellence in Research Award，https://rnaidesigner.invitrogen.com/sirna/)。为了进一步提高siRNA片断的有效性，综合两个在线设计工具的优点来设计用于筛选的多对siRNA片断，通过同源性比对(NCBI BLAST)来过滤siRNA序列，以提高siRNA片断的特异性并减少 RNAi干扰的脱靶效应，最后，通过实验筛选干扰效果最佳的siRNA应用于本发明中。
本发明提供了一种治疗帕金森的药物组合物，所述药物组合物包括针对前面所述基因的siRNA以及药学上可接受的载体。
进一步，siRNA的序列如SEQ ID NO.15～32所示。
在本发明中药学上可接受的载体包括但并不限于：稀释剂、赋形剂如水等、填充剂如淀粉、蔗糖等；粘合剂如纤维素衍生物、藻酸盐、明胶和聚乙烯吡咯烷酮；湿润剂如甘油；崩解剂如琼脂、碳酸钙和碳酸氢钠；吸收促进剂季铵化合物；表面活性剂如十六烷醇；吸附载体如高岭土和皂粘土；润滑剂如滑石粉、硬脂酸钙和镁、聚乙二醇等。
本发明的药物还可与其他治疗帕金森的药物联用，多种药物联合使用可以大大提到治疗的成功率。
在本发明的上下文中，“DNASE2基因”包括DNASE2基因以及DNASE2基因的任何功能等同物的多核苷酸。DNASE2基因(Chromosome 19,NC_000019.10(12875211..12881521,complement))序列可在国际公共核酸序列数据库GeneBank中查询到。
在本发明的上下文中，“THBD基因”包括THBD基因以及THBD基因的任何功能等同物的多核苷酸。THBD基因(Chromosome 20,NC_000020.11(23045633..23049664,complement))序列可在国际公共核酸序列数据库GeneBank中查询到。
在本发明的上下文中，“KCTD20基因”包括KCTD20基因以及KCTD20基因的任何功能等同物的多核苷酸。KCTD20基因(Chromosome 6,NC_000006.12(36442767..36491143))序列可在国际公共核酸序列数据库GeneBank中查询到。
在本发明的上下文中，“NACC2基因”包括NACC2基因以及NACC2基因的任何功能等同物的多核苷酸。NACC2基因(Chromosome 9,NC_000009.12(136006537..136095285,complement))序列可在国际公共核酸序列数据库GeneBank中查询到。
在本发明的上下文中，“FGFBP2基因”包括人FGFBP2基因以及与人FGFBP2 基因的任何功能等同的多核苷酸。在本发明的具体实施方案中一种代表性的FGFBP2的基因的序列具有与目前国际公共核酸序列数据库GeneBank中FGFBP2基因(NM_031950.3)相同的DNA序列。
在本发明的上下文中，基因表达产物包括蛋白以及蛋白的部分肽。所述蛋白的部分肽含有与帕金森症相关的功能域。
“DNASE2蛋白”包括DNASE2蛋白以及DNASE2蛋白的任何功能等同物。所述功能等同物包括DNASE2蛋白保守性变异蛋白质、或其活性片段，或其活性衍生物，等位变异体、天然突变体、诱导突变体、在高或低的严紧条件下能与DNASE2的DNA杂交的DNA所编码的蛋白质。
“THBD蛋白”包括THBD蛋白以及THBD蛋白的任何功能等同物。所述功能等同物包括THBD蛋白保守性变异蛋白质、或其活性片段，或其活性衍生物，等位变异体、天然突变体、诱导突变体、在高或低的严紧条件下能与THBD的DNA杂交的DNA所编码的蛋白质。
“KCTD20蛋白”包括KCTD20蛋白以及KCTD20蛋白的任何功能等同物。所述功能等同物包括KCTD20蛋白保守性变异蛋白质、或其活性片段，或其活性衍生物，等位变异体、天然突变体、诱导突变体、在高或低的严紧条件下能与KCTD20的DNA杂交的DNA所编码的蛋白质。
“NACC2蛋白”包括NACC2蛋白以及NACC2蛋白的任何功能等同物。所述功能等同物包括NACC2蛋白保守性变异蛋白质、或其活性片段，或其活性衍生物，等位变异体、天然突变体、诱导突变体、在高或低的严紧条件下能与NACC2的DNA杂交的DNA所编码的蛋白质。
“FGFBP2蛋白”包括FGFBP2蛋白以及FGFBP2蛋白的任何功能等同物。所述功能等同物包括FGFBP2蛋白保守性变异蛋白质、或其活性片段，或其活性衍生物，等位变异体、天然突变体、诱导突变体、在高或低的严紧条件下能与FGFBP2的DNA杂交的DNA所编码的蛋白质。
通常，已知的是，一个蛋白质中一个或多个氨基酸的修饰不会影响蛋白质的功能。本领域技术人员会认可改变单个氨基酸或小百分比的氨基酸或对氨基酸序 列的个别添加、缺失、插入、替换是保守修饰，其中蛋白质的改变产生具有相似功能的蛋白质。提供功能相似的氨基酸的保守替换表是本领域公知的。
通过添加一个氨基酸或多个氨基酸残基修饰的蛋白质的例子是前面所述基因编码蛋白的融合蛋白。对于与前面所述基因编码蛋白融合的肽或者蛋白质没有限制，只要所得的融合蛋白保留蛋白的生物学活性即可。
在本发明的上下文中，“诊断帕金森症”既包括判断受试者是否已经患有帕金森症、也包括判断受试者是否存在患有帕金森症的风险。
本发明的优点和有益效果：
本发明首次发现了THBD、KCTD20、DNASE2、NACC2、FGFBP2基因表达与帕金森症相关，通过检测受试者中前面基因的表达，可以判断受试者是否患有帕金森症、或者判断受试者是否存在患有帕金森症的风险，从而指导临床医师给受试者提供预防方案或者治疗方案。
本发明发现了一种新的分子标记物，相比传统的检测手段，基因诊断更及时、更特异、更灵敏，能够实现帕金森症的早期诊断，从而降低帕金森症的死亡率。
附图说明
图1显示利用基因芯片检测DNASE2基因在帕金森症患者和正常人中的表达差异；
图2显示利用QPCR检测DNASE2基因在帕金森症患者和正常人中的表达差异；
图3显示利用基因芯片检测THBD基因在帕金森症患者和正常人中的表达差异；
图4显示利用QPCR检测THBD基因在帕金森症患者和正常人中的表达差异；
图5显示利用基因芯片检测KCTD20基因在帕金森症患者和正常人中的表达差异；
图6显示利用QPCR检测KCTD20基因在帕金森症患者和正常人中的表达差异；
图7显示利用基因芯片检测NACC2基因在帕金森症患者和正常人中的表达差异；
图8显示利用QPCR检测NACC2基因在帕金森症患者和正常人中的表达差异；
图9显示利用QPCR检测FGFBP2基因在帕金森病患者血液中的表达情况；
图10显示利用利用QPCR检测siRNA对基因表达的影响；
图11显示利用ELISA检测KCTD20对α-syn含量的影响；
图12利用QPCR检测siRNA对THBD基因表达的影响；
图13利用利用ELISA检测THBD对α-syn含量的影响；
图14显示利用QPCR检测siRNA对FGFBP2基因表达的影响；
图15显示利用MTT检测FGFBP2基因表达对帕金森神经细胞生长的影响。
具体的实施方式
下面结合附图和实施例对本发明作进一步详细的说明。以下实施例仅用于说明本发明而不用于限制本发明的范围。实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring HarborLaboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。
实施例1 筛选帕金森症患者和正常人中差异表达的基因
1、研究对象：
收集原发性PD患者10例，男5例，女5例，年龄32-85岁，病程5个月-20年。PD入组标准：诊断标准均符合PD临床诊断标准(参考“蒋雨平，王坚，丁正同，等，原发性帕金森病的诊断标准，2005，中国临床神经科学，2006，14:40”)。排除标准：(1)特发性震颤；(2)继发性帕金森综合征；(3)严重痴呆、构音障碍者；(4)患有其他精神疾患者。此项研究己通过医院伦理委员会批准且所有患者签署知情同意书。
正常组：选取年龄32-80岁的健康志愿者10例，男女各5例。
两组之间年龄、性别差异无统计学意义(P>0.10)，具有可比性。
2、血液中总RNA的提取
(1)匀浆处理(Homogenization)
直接取新鲜的血液(外周血)，加入3倍体积红细胞裂解液，混匀后室温放置10分钟，10,000rpm离心1分钟。彻底吸弃上清，收集白细胞沉淀。每100-200μl血液收集的白细胞沉淀加入1ml TRIzol。
(2)分层(Phase Separation)
a.样品加入TRIzol后，室温放置5min，使样品充分裂解。
b.每1ml TRIzol加入200μl氯仿，剧烈振荡混匀后室温放置3-5min使其自然分相。
(3)RNA沉淀(RNA Precipitation)
a.4℃12,000rpm离心10-15min。样品会分成三层：黄色的有机相，中间层和无色的水相，RNA主要在水相中，把水相(通常可吸取550μl)转移到新管中。
b.在上清中加入等体积冰冷的异丙醇，室温放置10-20min。4℃12,000rpm离心10min，弃上清，RNA沉淀于管底。
(4)RNA漂洗(RNA Wash)
a.RNA沉淀中加入1ml 75％乙醇(用RNase-free水配制)，温和振荡离心管，悬浮沉淀。每1ml TRIzol加入1ml 75％乙醇。
b.4℃5,000-8,000rpm离心1-2min，弃上清；短暂快速离心，用移液器小心吸弃上清，室温放置1-2分钟晾干沉淀。
(5)溶解RNA(Redissolving the RNA)
沉淀中加入50-100μl RNase-free水，轻弹管壁，以充分溶解RNA，-70℃保存。
3、RNA质量和纯度检测
RNA质量：通过RNA完整性来表示，可用普通琼脂糖凝胶电泳(电泳条件：1.2％胶；0.5×TBE电泳缓冲液；150v，15分钟)检测完整性。
RNA纯度：OD260/OD280比值是衡量RNA样品中蛋白质污染程度的指标。高质量的RNA样品，OD260/OD280值(10mM Tris,pH7.5)在2.0左右。
4、基因芯片检测
4.1基因芯片杂交及扫描
总RNA经线性化扩增后，cy3-UTP标记，荧光标记后的cRNAs采用RNEASY Mini Kit纯化，用Amhion的RNA Fragmentation Reagents对标记好的cRNAs进行片段化处理。采用美国Agilent公司的人全基因表达谱芯片(4x 44K基因)，在 芯片杂交炉中65℃杂交17h，然后洗脱、染色，最后用Agilent DNA MicroarrayScanner扫描仪扫描。
4.2芯片数据处理与分析
杂交后的芯片经芯片扫描仪读取数据点后，将数据导入分析软件，对于两组比值的自然对数绝对值大于2.0或小于0.5的基因作为差异表达基因。
4.3统计学处理
采用SPSS 13.0统计软件进行数据分析，组间差异比较采用单因素方差分析法，P<0.05差异有显著性意义。
4.4结果
结果显示(如图1所示)，与正常人相比，帕金森症患者血液中DNASE2基因的mRNA水平显著升高，差异具有统计学意义(P<0.05)。
结果显示(如图3所示)，与正常人相比，帕金森症患者血液中THBD基因的mRNA水平显著升高，差异具有统计学意义(P<0.05)。
结果显示(如图5所示)，与正常人相比，帕金森症患者血液中KCTD20基因的mRNA水平显著升高，差异具有统计学意义(P<0.05)。
结果显示(如图7所示)，与正常人相比，帕金森症患者血液中NACC2基因的mRNA水平显著升高，差异具有统计学意义(P<0.05)。
5、RNA-seq检测
5.1去除rRNA
使用Ribo-Zero试剂盒除去总RNA中的核糖体RNA。
5.2构建cDNA文库
利用Illumina TruseqTM RNA sample Prep Kit进行cDNA文库的构建，具体操作按说明书进行。
5.3上机测序
使用Illumina X-Ten测序平台对cDNA文库进行测序，具体操作按说明书进行。
5.4高通量转录组测序数据分析
对测序结果进行生物信息学分析，利用TopHat v1.3.1进行RNA-seq读段定位，通过Cufflinks v1.0.3将RNA-seq片段数目进行标准化计算转录本的相对丰度，利用cuffdiff检测差异表达，当p值<0.05时，认为基因显著差异表达。
5.5结果
RNA-seq结果显示，FGFBP2基因在帕金森病患者血液中的表达量显著高于正常人血液中的水平。
实施例2 QPCR实验验证帕金森症患者和正常人中差异表达的基因
一、THBD基因、KCTD20基因、DNASE2基因、NACC2基因大样本验证
1、研究对象：
筛选标准同实施例1，帕金森症患者和正常人各50例。
2、血液中总RNA的提取
步骤同实施例1。
3、逆转录
用逆转录缓冲液对lμg总RNA进行逆转录合成cDNA。采用25μl反应体系，每个样品取1μg总RNA作为模板RNA，在PCR管中分别加入以下组分：DEPC水，5×逆转录缓冲液，10mmol/L dNTP，0.1mmol/l DTT，30μmmol/l Oligo dT,200U/μl M-MLV，模板RNA。42℃孵育1h，72℃10min，短暂离心。
4、QPCR
(1)引物设计
根据Genbank中DNASE2基因和GAPDH基因的编码序列设计QPCR扩增引物，由上海生工生物工程技术服务有限公司合成。具体引物序列如下：
DNASE2基因：
正向引物为5’-CAGCATCACACTCACATC-3’(SEQ ID NO.5)；
反向引物为5’-AGTACAGGTCATCTCCAA-3’(SEQ ID NO.6)，
THBD基因：
正向引物为5’-CTCAATGCCAGTCAGATC-3’(SEQ ID NO.1)；
反向引物为5’-GTTCAGTAGCAAGGAAATG-3’(SEQ ID NO.2)，
KCTD20基因：
正向引物为5’-GCTGCTTAGTGGATGATAC-3’(SEQ ID NO.3)；
反向引物为5’-AGATTATGAGGACCAGATGA-3’(SEQ ID NO.4)，
NACC2基因：
正向引物为5’-ATGGTGGAGGAGCAGTAC-3’(SEQ ID NO.7)；
反向引物为5’-TGGCTCAGGCTTCTCTTG-3’(SEQ ID NO.8)，
GAPDH基因：
正向引物为5’-TTTAACTCTGGTAAAGTGGATAT-3’(SEQ ID NO.11)；
反向引物为5’-GGTGGAATCATATTGGAACA-3’(SEQ ID NO.12)。
(2)按照表1配制PCR反应体系：
其中，SYBR Green聚合酶链式反应体系购自Invitrogen公司。
表1 PCR反应体系
试剂 体积
正向引物 1μl
反向引物 1μl
SYBR Green聚合酶链式反应体系 12.5μl
模板 2μl
去离子水 补足25μl
(3)PCR反应条件：95℃10min，(95℃10s，60℃40s)*45个循环。以SYBR Green作为荧光标记物，在Light Cycler荧光定量PCR仪上进行PCR反应，通过融解曲线分析和电泳确定目的条带，ΔΔCT法进行相对定量。
5、统计学方法
结果数据都是以平均值±标准差的方式来表示，采用SPSS13.0统计软件来进行统计分析的，两者之间的差异采用t检验，认为当P<0.05时具有统计学意义。
6、结果
结果如图2所示，与正常人相比，帕金森症患者血液中DNASE2基因的mRNA水平显著增加，差异具有统计学意义(P<0.05)，结果同基因芯片实验。
结果如图4所示，与正常人相比，帕金森症患者血液中THBD基因的mRNA水平显著增加，差异具有统计学意义(P<0.05)，结果同基因芯片实验。
结果如图6所示，与正常人相比，帕金森症患者血液中KCTD20基因的mRNA水平显著增加，差异具有统计学意义(P<0.05)，结果同基因芯片实验。
结果如图8所示，与正常人相比，帕金森症患者血液中NACC2基因的mRNA水平显著增加，差异具有统计学意义(P<0.05)，结果同基因芯片实验。
二、FGFBP2大样本验证
1、筛选标准同实施例1，帕金森症患者和正常人各90例。
2、RNA提取步骤同实施例1。
3、逆转录：
(1)取总RNA 2μg进行逆转录，加入Oligo(dT)2μl，充分混匀。70℃水浴5分钟后立即冰浴2-3min。
(2)构建25μl反应体系，其中包括5×逆转录缓冲液5μl，dNTP(2.5mM)5μl，RNasin 40U/μl，M-MLV 200U/μl，补无核酶水至预期体积。
(3)42℃水浴60分钟后，95℃水浴5分钟以灭活M-MLV。
(4)-20℃储存备用。
4、QPCR扩增
(1)引物设计
根据Genbank中FGFBP2基因和GAPDH基因的编码序列设计QPCR扩增 引物，由上海生工生物工程技术服务有限公司合成。具体引物序列如下：
FGFBP2基因：
正向引物为5’-ATGAGGAAGCAAAGAAGA-3’(SEQ ID NO.9)；
反向引物为5’-GGAAGAAGCTGATGAGAA-3’(SEQ ID NO.10)。
GAPDH基因：
正向引物为5’-AACTCTGGTAAAGTGGATATTG-3’(SEQ ID NO.13)；
反向引物为5’-GGTGGAATCATATTGGAACA-3’(SEQ ID NO.14)。
(2)按照表2配制PCR反应体系：
其中，SYBR Green聚合酶链式反应体系购自Invitrogen公司。
表2 PCR反应体系
试剂 体积
正向引物 1μl
反向引物 1μl
SYBR Green聚合酶链式反应 12.5μl
模板 2μl
去离子水 补足25μl
(3)PCR反应条件：95℃10min，(95℃15s，60℃60s)*45个循环。以SYBR Green作为荧光标记物，在Light Cycler荧光定量PCR仪上进行PCR反应，通过融解曲线分析和电泳确定目的条带，ΔΔCT法进行相对定量。
5、统计学方法
实验采用3次重复实验，结果数据都是以平均值±标准差的方式来表示，使用SPSS13.0统计软件来进行统计分析的，两者之间的差异采用t检验，认为当P<0.05时具有统计学意义。
6、结果
结果如图9所示，与正常人血液相比，FGFBP2基因在帕金森病患者血液中 的表达显著上调，差异具有统计学意义(P<0.05)，同RNA-sep结果一致。
实施例3 免疫印迹实验验证帕金森症患者和正常人中差异表达基因的表达产物
1、临床对象：同实施例2。
2、单核细胞分离
帕金森症患者和正常人取静脉血10ml，注入盛肝素的无菌小瓶中，加盖后立即轻轻摇匀。用无菌吸管加入等体积的HBSS(NaCl 8.0g，Na 2HPO 4 0.132g，KH 2PO 4 0.06g，KCl 0.4g，酚红1ml，NaHCO 3 0.35g，D-葡萄糖1.0g，溶于1000ml双蒸水)，以降低红细胞的凝聚。吸取8ml淋巴细胞分层液置50ml离心管中，将稀释血液沿管壁缓慢加入，保持界面清楚，勿使两者相混，在20℃2000r/min离心30min，小心吸取分层液与血浆交接部位混浊的灰白色层，即淋巴细胞层，加入另一支离心管中，用5倍体积的HBSS洗涤2次，依次以2000r/min、1500r/min在室温下离心10min，以便去除大部分混杂的血小板，用10ml双蒸水与细胞团块混合1min，使残余红细胞裂解，然后迅速加入等量1.8％NaCl溶液，2000r/min离心，去上清，经细胞计数后用HBSS溶液调整细胞至1×10 6个/ml备用。
3、单核细胞总蛋白质提取
将上述实验所得细胞悬液(浓度为1×10 6个/ml)室温1 000r/min离心10min，弃上清后加入100μl裂解缓冲液，4℃震荡1h，用超声波仪破碎细胞，每次10s，共10次，于4℃12000r/min离心1h；取上清用Brandford法定量蛋白，分装成2.5μg/μl，-80℃冰箱保存备用。
4、Western blot检测
细胞总蛋白用Brandford法定量，取适量与样品缓冲液混合煮沸5min，冷却5min；取30pg蛋白上样到制备好的15％聚丙烯酰胺凝胶，进行电泳，开始设为80V恒压，看见Marker后增加至120V；将电泳后的胶取出，使用Bio.Rad半干转印系统于100V转移50min；转膜完毕后，用1xPBS洗一次，浸入封闭液，40C过夜；倒掉封闭液，加入Western洗涤液洗涤5-10min，加入一抗摇床室温 杂交2h；按照适当比例用Western二抗稀释液稀释于封闭缓冲液中，孵育60min；洗膜液洗3次，每次10min；使用ECL试剂显影、定影检测蛋白表达。
5、统计学处理
将蛋白条带的灰度值使用Image J软件进行分析，以β-actin为内参，将目的白条带的灰度值进行归一化处理。结果数据都是以平均值±标准差的方式来表示，采用SPSS13.0统计软件来进行统计分析的，两者之间的差异采用t检验，认为当P<0.05时具有统计学意义。
6、结果
结果显示，以正常人组血液中的蛋白相对表达水平为1，帕金森症组血液中DNASE2蛋白水平为5.13±1.57，显著升高，差异具有统计学意义(P<0.05)。
结果显示，以正常人组血液中的蛋白相对表达水平为1，帕金森症组血液中THBD蛋白水平为4.14±1.12，显著升高，差异具有统计学意义(P<0.05)。
结果显示，以正常人组血液中的蛋白相对表达水平为1，帕金森症组血液中KCTD20蛋白水平为5.87±1.45，显著升高，差异具有统计学意义(P<0.05)。
结果显示，以正常人组血液中的蛋白相对表达水平为1，帕金森症组血液中NACC2蛋白水平为8.29±2.01，显著升高，差异具有统计学意义(P<0.05)。
实施例4 抑制KCTD20表达对α-syn的影响
1、siRNA转染
取对数期的PC12-α-syn细胞，12孔细胞培养板每孔接种3×10 5细胞/1mL，37℃培养12h后吸去培养基，加入800μL新鲜培养基。用100μL新鲜无血清的Opti-MEM培养基稀释KCTD20及对照(NC)siRNA(由上海吉玛制药技术有限公司提供)，siRNA终浓度为40pmol，并用100μL新鲜无血清的Opti-MEM培养基稀释转染试剂
Figure PCTCN2018123794-appb-000001
3000Reagent。将稀释的siRNA和转染试剂混匀(siRNA与转染试剂体积比为1：2)，室温静置10-15min后加入到细胞培养基中，轻晃混匀。细胞继续培养72h后，4℃，12000rpm离心10min，收集上清用于后续ELISA检测。裂解细胞提取RNA，进行QPCR检测siRNA沉默 KCTD20效果。
KCTD20的siRNA序列根据大鼠KCTD20基因(Chromosome 20,NC_005119.4(6205897..6222818))序列设计，如下所示：
KCTD20-SIRNA1(简称为KCTD-1)
正义链为5’-GGUCUGAAGUUAUCUACAATT-3’(SEQ ID NO.15)，
反义链为5’-UUGUAGAUAACUUCAGACCTT-3’(SEQ ID NO.16)；
KCTD20-SIRNA2(简称为KCTD-2)
正义链为5’-CCAGGAGAUAAUGAUGCAUTT-3’(SEQ ID NO.17)，
反义链为5’-AUGCAUCAUUAUCUCCUGGTT-3’(SEQ ID NO.18)；
KCTD20-SIRNA3(简称为KCTD-3)
正义链为5’-GCGACUAUCUCUGCAUUAATT-3’(SEQ ID NO.19)，
反义链为5’-UUAAUGCAGAGAUAGUCGCTT-3’(SEQ ID NO.20)。
2、QPCR检测siRNA抑制KCTD20表达情况
RNA提取：向12孔板细胞中每孔加入400μL Trizol，轻轻吹打几下使细胞充分裂解，然后将细胞裂解液转移到1.5mL离心管中，每管加入80μL氯仿，充分涡旋混匀，室温静置3min，4℃，12000rpm离心15min。取最上层到新离心管中，每管加入200μL异丙醇，轻轻上下颠倒混匀，室温静置10min，4℃，12000rpm离心10min。弃上清，每管加入预冷的75％乙醇，吸打混匀，4℃，12000rpm离心5min。弃上清，沉淀干燥后加入适量ddH 2O溶解RNA，60℃促溶10min，nano-job测定RNA质量及浓度。并通过反转录试剂盒(promega,USA)将RNA反转录成cDNA。
QPCR:在96孔板中每孔加入10μL mix，4μL引物，4.5μL ddH 2O，1.5μL cDNA，混匀，用实时定量基因扩增仪(Applied Biosystems，USA)检测siRNA沉默KCTD20基因的情况。
QPCR结果如图10所示，三条siRNA分别抑制78.4％，75.8％，60.2％的KCTD20表达，因此KCTD-1能最有效抑制KCTD20的表达，使用转染KCTD-1的细胞进行ELISA实验。
3、ELISA检测抑制KCTD20表达对α-syn的影响
包被α-syn-mAb(Abcam，ab138501)至96孔板中(0.5μg/孔)，4℃过夜。用0.1％PBST洗涤三遍后，3％BSA封闭。将PC12-α-syn细胞上清用3k超滤管浓缩后加入96孔板中，每孔加100μL，并在37℃下反应1小时，0.1％PBST洗涤六遍。加入syn-1(BD,610787)作为检测抗体，在37℃下反应1小时后再加入HRP标记的山羊抗小鼠二抗，TMB显色，于SpectraMax M5酶标仪(Molecular Devices，LLC，Sunnyvale，CA，USA)测量吸光度(450nm)。
结果如图11所示，与对照组相比，siRNA抑制KCTD20表达后，细胞上清中α-syn的量升高2.12倍。
实施例5 抑制THBD表达对α-syn的影响
1、siRNA转染
取对数期的PC12-α-syn细胞，12孔细胞培养板每孔接种3×10 5细胞/1mL，37℃培养12h后吸去培养基，加入800μL新鲜培养基。用100μL新鲜无血清的Opti-MEM培养基稀释THBD及对照(NC)siRNA(由上海吉玛制药技术有限公司提供)，siRNA终浓度为40pmol，并用100μL新鲜无血清的Opti-MEM培养基稀释转染试剂
Figure PCTCN2018123794-appb-000002
3000Reagent。将稀释的siRNA和转染试剂混匀(其体积比为1：2)，室温静置10-15min后加入到细胞培养基中，轻晃混匀。细胞继续培养72h后，4℃，12000rpm离心10min，收集上清用于后续ELISA检测。裂解细胞提取RNA，进行QPCR检测siRNA沉默效果。
THBD的siRNA序列根据大鼠THBD基因(Chromosome 3,NC_005102.4(142748673..142752325,complement))序列设计，如下所示：
THBD-siRNA1(简称为THBD-1)
正义链为5’-GCUUCGAAUGCCGCUGCUATT-3’(SEQ ID NO.21)，
反义链为5’-UAGCAGCGGCAUUCGAAGCTT-3’(SEQ ID NO.22)；
THBD-siRNA2(简称为THBD-2)
正义链为5’-GGUGCGAAAUGUUCUGCAATT-3’(SEQ ID NO.23)，
反义链为5’-UUGCAGAACAUUUCGCACCTT-3’(SEQ ID NO.24)；
THBD-siRNA3(简称为THBD-3)
正义链为5’-GCGCAGAGCUGGAGUACAATT-3’(SEQ ID NO.25)，
反义链为5’-UUGUACUCCAGCUCUGCGCTT-3’(SEQ ID NO.26)。
2、QPCR检测siRNA抑制THBD表达情况
RNA提取：向12孔板细胞中每孔加入400μL Trizol，轻轻吹打几下使细胞充分裂解，然后将细胞裂解液转移到1.5mL离心管中，每管加入80μL氯仿，充分涡旋混匀，室温静置3min，4℃，12000rpm离心15min。取最上层到新离心管中，每管加入200μL异丙醇，轻轻上下颠倒混匀，室温静置10min，4℃，12000rpm离心10min。弃上清，每管加入预冷的75％乙醇，吸打混匀，4℃，12000rpm离心5min。弃上清，沉淀干燥后加入适量ddH 2O溶解RNA，60℃促溶10min，nano-job测定RNA质量及浓度。并通过反转录试剂盒(promega,USA)将RNA反转录成cDNA。
QPCR:在96孔板中每孔加入10μL mix，4μL引物，4.5μL ddH 2O，1.5μL cDNA，混匀，用实时定量基因扩增仪(Applied Biosystems，USA)检测siRNA沉默THBD基因的情况。
QPCR结果如图12，三条siRNA分别抑制99.3％，99.9％，99.9％的THBD表达，因此THBD-1,THBD-2，THBD-3均能够很有效的抑制THBD的表达，选择转染THBD-3的细胞进行ELISA实验。
3、ELISA检测抑制THBD表达对α-syn的影响
包被α-syn-mAb(Abcam，ab138501)至96孔板中(0.5μg/孔)，4℃过夜。用0.1％PBST洗涤三遍后，3％BSA封闭。将PC12-α-syn细胞上清用3k超滤管浓缩后加入96孔板中，每孔加100μL，并在37℃下反应1小时，0.1％PBST洗涤六遍。加入syn-1(BD,610787)作为检测抗体，在37℃下反应1小时后再加入HRP标记的山羊抗小鼠二抗，TMB显色，于SpectraMax M5酶标仪(Molecular Devices，LLC，Sunnyvale，CA，USA)测量吸光度(450nm)。
结果如图13所示，与对照组相比，siRNA抑制THBD表达后，细胞上清中 α-syn的量升高4.88倍。
实施例6 FGFBP2基因的沉默
1、细胞培养
多巴胺神经元细胞SH-SY5Y，以含10％胎牛血清、1％青霉素/链霉素的DMEM培养液中(pH7.2～7.4)，在37℃、5％CO 2、相对湿度为90％的培养箱中培养。每隔2天换液一次，待细胞生长至90％接触时进行传代，用PBS清洗后加入0.25％-EDTA胰蛋白酶消化使细胞从瓶壁上分离下来，用含胎牛血清的DMEM培养液终止胰酶消化反应，1000g离心2min，弃上清，用新配置的培养液重悬，以1:3～1:4比例传代，24小时后细胞进入对数生长期更换培养液，并根据实验要求给予不同的干预。
2、siRNA设计
针对FGFBP2的siRNA序列：
siRNA1：
正义链为5’-UUAGAAACUCUCUUCUUCCAG-3’(SEQ ID NO.27)，
反义链为5’-GGAAGAAGAGAGUUUCUAAUC-3’(SEQ ID NO.28)；
siRNA2：
正义链为5’-AAGUAGUUGUGUAUGCUUGUC-3’(SEQ ID NO.29)，
反义链为5’-CAAGCAUACACAACUACUUAU-3’(SEQ ID NO.30)；
siRNA3：
正义链为5’-UACAGAUAAUAAGUAGUUGUG-3’(SEQ ID NO.31)，
反义链为5’-CAACUACUUAUUAUCUGUAGA-3’(SEQ ID NO.32)。
3、重组腺病毒
根据腺病毒介导的siRNA的不同，将细胞分为五组，SH-SY5Y组：不转染任何病毒载体的SH-SY5Y细胞，作为空白对照；Ad组：感染空腺病毒质粒细胞组，siRNA1组：腺病毒介导干扰序列1感染细胞组：siRNA2组：腺病毒介导干扰序列2感染细胞组；siRNA3组：腺病毒介导干扰序列3感染细胞组。
将细胞按1×10 5/孔接种到6孔细胞培养板中，每孔2ml，在37℃、5％CO 2培养箱中细胞培养24h，此时细胞融合密度约为50％-60％；吸出上清弃掉，用无血清培养基1ml洗两遍，用1ml无血清培养基稀释的MOI为50的各组腺病毒，每间隔20min摇晃培养板一次，以增加感染效果，感染48h后，再加入浓度为1000μmol/L MPP +完全培养基，孵育24h后。收集细胞用于提取RNA；
3、QPCR检测FGFBP2基因的转录水平
3.1细胞总RNA的提取
采用TRIzol Reagent(Invitrogen Cat.No.15596-018)总RNA提取试剂，按说明书提供方法提取SH-SY5Y细胞的总RNA。具体方法为：
吸去培养液，用PBS清洗一遍，加入适量TRIzol试剂，六孔板中每孔加入1mL，室温放置5min裂解细胞，吹打均匀后以1mL/管分装至1.5mL Eppendorf管中。按400μl氯仿/ml Trizol加入氯仿，用手上下摇动30-50次，室温放置5min。4℃、12000g离心15min，将上层水相移至干净Eppendorf管中，加入0.4mL异丙醇，轻轻混匀，室温放置10min，4℃、7500g离心10min。弃上清，75％乙醇洗涤RNA沉淀，7500g离心5min，室温干燥RNA沉淀，5-10min后溶于适量DEPC水。质量分数为1.0％的琼脂糖凝胶电泳检测RNA样本的完整性，应用Bio-Photometer对提取的RNA进行定量测定。
3.2逆转录步骤同实施例2。
3.3QPCR扩增步骤同实施例2。
4、统计学方法
实验采用3次重复实验，结果数据都是以平均值±标准差的方式来表示，使用SPSS13.0统计软件来进行统计分析的，干扰FGFBP2基因表达组与对照组之间的差异采用t检验，认为当P<0.05时具有统计学意义。
5、结果
结果如图14显示，相比对照组，实验组能够不同程度的抑制FGFBP2基因的表达，而siRNA1组抑制效果最为明显，因此选择siRNA1进行后续的实验。
实施例7 FGFBP2基因对神经细胞的影响
采用MTT实验检测FGFBP2基因对SH-SY5Y帕金森病细胞模型细胞增殖能力的影响。
1、细胞培养与腺病毒感染步骤同实施例6。
2、MTT检测
调整SH-SY5Y细胞密度至5×10 4/mL，以每孔100μl细胞接种于96孔培养板中，按照实施例3处理细胞，在处理后每隔12h应用MTT检测，直至72h。MTT还原分析法检测细胞活性：将孔内溶液弃掉，加入100μl培养基，再每孔加入5mg/mL的MTT液10μl，37℃培养4h后，吸去培养基，每孔加入DMSO 100μl，室震荡10min，使紫蓝色沉淀充分溶解，用酶标仪在490nm波长测吸光度值(OD值)。将OD值作为反映SH-SY5Y细胞活性的参数。本实验重复3次，每次实验中相同条件设5个平行孔。
3、统计学方法
实验采用3次重复实验，使用SPSS18.0统计软件来进行统计分析，两者之间的差异采用t检验，认为当P<0.05时具有统计学意义。
4、结果
图15所示的结果显示：siRNA1组的细胞生长速度高于对照组组的细胞生长速度，差异具有统计学意义(P<0.05)。上述结果表明FGFBP2过表达不利于SH-SY5Y细胞的生长，通过抑制FGFBP2基因的表达可以促进神经细胞的生长。
上述实施例的说明只是用于理解本发明的方法及其核心思想。应当指出，对于本领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也将落入本发明权利要求的保护范围内。

Claims (27)

  1. 基因在制备诊断帕金森症的工具中的应用，所述基因选自以下组：THBD、KCTD20、DNASE2、NACC2、FGFBP2。
  2. 检测权利要求1所述的基因的产品在制备诊断帕金森症的工具中的应用。
  3. 根据权利要求2所述的应用，其特征在于，所述产品包括检测所述基因表达的产品。
  4. 根据权利要求3所述的应用，其特征在于，所述检测所述基因表达的产品包括检测所述基因的mRNA表达水平的产品。
  5. 根据权利要求4所述的应用，其特征在于，所述检测所述基因的mRNA表达水平的产品包括通过反转录PCR、实时定量PCR、原位杂交、基因芯片或高通量测序平台检测所述基因的mRNA表达水平的产品。
  6. 根据权利要求5所述的应用，其特征在于，所述反转录PCR检测所述基因的mRNA表达水平的产品至少包括一对特异扩增所述基因的引物；所述实时定量PCR检测所述基因的mRNA表达水平的产品至少包括一对特异扩增所述基因的引物；所述原位杂交检测所述基因的mRNA表达水平的产品包括：与所述基因的核酸序列杂交的探针；所述基因芯片检测所述基因的mRNA表达水平的产品包括：与所述基因的核酸序列杂交的探针。
  7. 根据权利要求6所述的应用，其特征在于，所述实时定量PCR检测所述基因的mRNA表达水平的产品至少包括一对特异扩增所述基因的引物，所述引物序列为：
    特异扩增THBD基因的引物序列如SEQ ID NO.1～2所示；
    特异扩增KCTD20基因的引物序列如SEQ ID NO.3～4所示；
    特异扩增DNASE2基因的引物序列如SEQ ID NO.5～6所示；
    特异扩增NACC2基因的引物序列如SEQ ID NO.7～8所示；
    特异扩增FGFBP2基因的引物序列如SEQ ID NO.9～10所示。
  8. 根据权利要求3所述的应用，其特征在于，所述检测所述基因表达的产品包括检测所述基因编码蛋白表达水平的产品。
  9. 根据权利要求8所述的应用，其特征在于，检测所述基因编码蛋白表达水平的产品包括通过ELISA、免疫印迹、蛋白芯片检测蛋白表达水平的产品。
  10. 根据权利要求9所述的应用，其特征在于，所述ELISA检测所述基因编码蛋白表达水平的产品包括：与所述基因编码蛋白特异性结合的抗体或配体；所述免疫印迹检测所述基因编码蛋白表达水平的产品包括：与所述基因编码蛋白特异性结合的抗体或配体；所述蛋白芯片检测所述基因编码蛋白表达水平的产品包括：与所述基因编码蛋白特异性结合的抗体或配体。
  11. 一种用于诊断帕金森症的工具，其特征在于，所述工具能够通过检测样本中权利要求1所述的基因来诊断帕金森症。
  12. 根据权利要求11所述的工具，其特征在于，所述工具能够通过检测样本中所述基因表达来诊断帕金森症。
  13. 根据权利要求12所述的工具，其特征在于，所述工具能够通过检测样本中所述基因mRNA表达来诊断帕金森症。
  14. 根据权利要求12所述的工具，其特征在于，所述工具能够通过检测样本中所述基因编码蛋白的表达来诊断帕金森症。
  15. 根据权利要求11-14中任一项所述的工具，其特征在于，所述工具包括芯片、试剂盒、试纸或高通量测序平台。
  16. 根据权利要求15所述的工具，其特征在于，所述芯片包括基因芯片、蛋白质芯片；所述基因芯片包括固相载体以及固定在固相载体的寡核苷酸探针，所述寡核苷酸探针包括用于检测所述基因转录水平的针对所述基因的寡核苷酸探针；所述蛋白质芯片包括固相载体以及固定在固相载体的所述基因编码蛋白的特异性抗体或配体。
  17. 根据权利要求15所述的工具，其特征在于，所述试剂盒包括基因检测试剂盒和蛋白免疫检测试剂盒；所述基因检测试剂盒包括用于检测所述基因转录水平的试剂；所述蛋白免疫检测试剂盒包括所述基因编码蛋白的特异性抗体或配体。
  18. 根据权利要求15所述的工具，其特征在于，所述试纸包括用于检测所述基因转录水平的试剂。
  19. 根据权利要求15所述的工具，其特征在于，所述高通量测序平台包括用于检测所述基因转录水平的试剂。
  20. 根据权利要求17-19中任一项所述的工具，其特征在于，所述检测所述基因转录水平的试剂包括针对所述基因的引物和/或探针。
  21. 根据权利要求20所述的工具，其特征在于，所述针对所述基因的引物序列如下：
    针对THBD基因的引物序列如SEQ ID NO.1～2所示；
    针对KCTD20基因的引物序列如SEQ ID NO.3～4所示；
    针对DNASE2基因的引物序列如SEQ ID NO.5～6所示；
    针对NACC2基因的引物序列如SEQ ID NO.7～8所示；
    针对FGFBP2基因的引物序列如SEQ ID NO.9～10所示。
  22. 根据权利要求11-21中任一项所述的工具，其特征在于，所述样本是血液。
  23. 权利要求1所述的基因或其表达产物在筛选治疗帕金森的药物中的应用。
  24. 权利要求1所述的基因和/或其表达产物在制备治疗帕金森病药物中的应用。
  25. 根据权利要求24所述的应用，其特征在于，所述药物包括针对所述基因的siRNA。
  26. 一种治疗帕金森的药物组合物，其特征在于，所述药物组合物包括针对权利要求1所述的基因的siRNA以及药学上可接受的载体。
  27. 根据权利要求26所述的药物组合物，其特征在于，siRNA的序列为：SEQ ID NO.15～32所示。
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WO2012074933A1 (en) * 2010-11-29 2012-06-07 Genentech, Inc. Methods for detecting neurodegenerative diseases or disorders
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