CN108048554A - The molecular marker that THBD genes are diagnosed as parkinsonism - Google Patents
The molecular marker that THBD genes are diagnosed as parkinsonism Download PDFInfo
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- 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
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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
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- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/50—Determining the risk of developing a disease
Abstract
The invention discloses purposes of the THBD genes in parkinsonism is diagnosed.THBD gene contents before normal person with that, there are significant difference, Parkinson's disease patients and non-Parkinson's disease patients can be distinguished according to difference in the blood of Parkinson's disease patients.The invention also discloses the diagnostic products of parkinsonism, which can realize the diagnostic purpose of parkinsonism by detecting in blood THBD genes.
Description
Technical field
The invention belongs to molecular diagnosis fields, are related to a kind of molecular marker for parkinsonism diagnosis, and in particular to
Application of the molecular marker-THBD genes in the product for preparing diagnosis parkinsonism in blood.
Background technology
Parkinson's disease (Parkinson ' s disease；PD) it is the Common neurologic that occurs mainly in mid-aged population
Degenerative disease.With the aggravation of global aging, the diseased colonies of Parkinson's disease constantly expand, it not only seriously affect patient and
The quality of life of household also brings increasingly heavier financial burden for society.
As most commonly seen neurodegenerative movement disorder disease, Parkinson's disease (PD) is characterized by several movements
In symptom, for example static tremor, myotonia, position are unstable, while including some non-motor symptoms, such as autonomic disorder,
Insanity, sensory disturbance, cognitive disorder and dementia (Volta M, Milnerwood A J, Farrer M J.Insights
from late-onset familial parkinsonism on the pathogenesis of idiopathic
Parkinson's disease[J].The Lancet Neurology,2015,14(10):1054-1064).PD is as a kind of
Complicated neurological disease and the nerve degenerative diseases the most main for being only second to alzheimer disease, affect about 1% it is super
Spend 60 years old elderly population (Samii A, Nutt J G, Ransom B R.Parkinson's disease [J] .Lancet,
2004,363(9423):1783-1793), and in the world, cause every year more than 100,000 people death (Mortality G
B D,Causes of Death C.Global,regional,and national age-sex specific all-cause
and cause-specific mortality for 240 causes of death,1990-2013:a systematic
analysis for the Global Burden of Disease Study 2013[J].Lancet,2015,385
(9963):117-171).PD not only reduces the quality of life of patient and its care-giver, also causes very heavy warp to society
Ji burden.According to conservative estimation, only in the U.S., up to 23,000,000,000 dollars every year of the financial burden triggered by PD.PD is as a kind of
Chronic, progressive disease, non-athletic property symptom, which typically precedes dyskinesia, to be occurred for many years.Although the pathological characteristics of PD are
Lewy body cynapse core egg in the generation of the nigrostriatal dopamine serotonergic neuron of degeneration and activity deficiency and neuronal cell
White and other protein accumulations, other neurotransmitters in addition to dopamine and other brain areas also assist in PD occurrence and development
In the process.Parkinson's disease is being considered mainly being caused and being influenced by environmental factor before, but it has recently been demonstrated that the disease
Come from complicated interaction (Kalia L V, the Lang A E.Parkinson's disease of inherent cause and environmental factor
[J].Lancet,2015,386(9996):896-912).It is past during the decade, several researchs are from animal model, gene table
It reaches, the angle of whole-genome association (GWAS) and systems biology, is directed to understanding causing a disease and molecule mechanism and taking for PD
Obtain some achievements.The discovery of several potential core proteins, as leucine-rich repeat kinase 2 (LRRK2),
PTEN-induced putative kinase 1(PINK1)、parkin(PARK2)、PARK7、ubiquitin carboxyl-
Terminal esterase L1 (UCHL1), glucocerebrosidase (GBA) and alpha-synuclein (SNCA) are
Understand that its molecule mechanism has established important basis.Nevertheless, the pathogenesis of Parkinson's disease is still unilateral, local,
Distance thoroughly understands that its pathogenesis also has longer stretch.
The complexity of PD is also embodied in challenge clinically.Up to the present, there is not yet and determined for PD early stages
The diagnostic test of diagnosis.Gene expression regulation network system obtains noticeable progress in cancer research field, many swollen
Such as lung cancer, breast cancer, stomach cancer, colorectal cancer in tumor tissue, the expression of some genes exist with its source normal structure
Stable and apparent difference.The tumour of different tissue sources or even same tissue-derived different differentiation states plus knurl, gene table
It is all not quite similar up to spectrum, shows that gene expression has apparent tissue specificity and stage of development specificity.Because neurological
Property disease pathology on be directly characterized in the overexpression of disease specific protease matter, so speculating that the exception of specific gene changes
The expression of functional protein is become, so as to cause disease.More and more researchs find that specific gene can become
The biomarker of Parkinson is diagnosed, as disclosed in the patent of following application number：201510713527.3、
201510464905.9、201510463593.X、201510463617.1、201510809837.5。
Most of disease is all the disease of polygenes regulation and control, and in order to improve the accuracy rate of diagnosis of disease, lots of genes is joined
Closing diagnosis becomes inevitable, it is therefore desirable to excavate more with the relevant molecular proportion marker of Parkinson's disease so as to clinical practice.
The content of the invention
In order to make up for the deficiencies of the prior art, it is an object of the invention to provide one kind can be used for parkinsonism to early diagnose
Molecular marker.Compared to the diagnostic method of traditional parkinsonism, having for parkinsonism is diagnosed using gene marker
Promptness, specificity and sensitivity so that patient just can know disease risks in disease early stage, for risk height, are taken
Corresponding prevention and treatment measure.
To achieve these goals, the present invention adopts the following technical scheme that：
Application in the instrument for diagnosing parkinsonism in preparation the present invention provides the product of detection THBD gene expressions.
Further, the product of detection THBD gene expressions mentioned above includes：By RT-PCR, real-time quantitative PCR,
Immune detection, in situ hybridization, chip or high-flux sequence detection of platform THBD gene expression doses are to diagnose the production of parkinsonism
Product.
Further, the product with RT-PCR diagnosis parkinsonisms includes at least drawing for a pair of of specific amplified THBD genes
Object；The product with real-time quantitative PCR diagnosis parkinsonism includes at least the primer of a pair of of specific amplified THBD genes；It is described
Included with the product of immune detection diagnosis parkinsonism：The antibody combined with THBD protein-specifics；It is described to be examined in situ hybridization
The product of disconnected parkinsonism includes：With the probe of the nucleic acid array hybridizing of THBD genes；It is described to diagnose parkinsonism with chip
Product includes：Protein chip and genetic chip；Wherein, protein chip includes the antibody combined with THBD protein-specifics, gene
Chip includes the probe with the nucleic acid array hybridizing of THBD genes.
In specific embodiments of the present invention, the product with real-time quantitative PCR diagnosis parkinsonism includes at least
The sequence of the primer of a pair of of specific amplified THBD genes is as shown in SEQ ID NO.1 and SEQ ID NO.2.
Preferably, the diagnostic tool includes chip, kit, test paper or high-flux sequence platform.Wherein, high pass measures
Sequence platform is a kind of special diagnostic tool, and the product of detection THBD gene expressions can be applied to the platform and realize to THBD bases
The detection of the expression of cause.With the development of high throughput sequencing technologies, the structure of the gene expression profile of a people will be become
Very easily work.By comparing the gene expression profile of Disease and normal population, the different of which gene is easily analyzed
It is often related to disease.Therefore, know that the exception of THBD genes is related to parkinsonism in high-flux sequence and fall within THBD bases
The purposes of cause, equally within protection scope of the present invention.
The present invention also provides a kind of instrument for diagnosing parkinsonism, the diagnostic tool includes chip, kit, examination
Paper or high-flux sequence platform.
Wherein, the chip includes genetic chip, protein-chip；The genetic chip includes solid phase carrier and fixation
In the oligonucleotide probe of solid phase carrier, the oligonucleotide probe includes detecting being directed to for THBD gene transcription levels
The oligonucleotide probe of THBD genes；The protein-chip includes solid phase carrier and is fixed on the THBD albumen of solid phase carrier
Specific antibody；The genetic chip can be used for multiple genes of the detection including THBD genes (for example, and parkinsonism
Relevant multiple genes) expression.The protein-chip can be used for multiple albumen of the detection including THBD albumen
The expression of matter (such as with the relevant multiple protein of parkinsonism).By by multiple markers with parkinsonism simultaneously
Detection is greatly improved the accuracy rate of parkinsonism diagnosis.
Wherein, the kit includes gene detecting kit and protein immunization detection kit；The genetic test examination
Agent box includes the reagent for detecting THBD gene transcription levels；The protein immunization detection kit includes the spy of THBD albumen
Heterogenetic antibody.Further, the reagent includes the use of RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or chip side
Reagent needed for method detection THBD gene expression dose processes.Preference, the reagent include the primer for THBD genes
And/or probe.It is easily designed according to the nucleotide sequence information of THBD genes and can be used for detecting THBD gene expression doses
Primer and probe.
Probe with the nucleic acid array hybridizing of THBD genes can be that DNA, RNA, DNA-RNA chimera, PNA or other spread out
Biology.There is no limit as long as completing specific hybrid, being specifically bound with purpose nucleotide sequence, appoint the length of the probe
What length can.The length of the probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the length of the probe
Degree can be grown to 60,80,100,150,300 base-pairs or longer or even whole genes.Since different probe lengths is to hybridization
Efficiency, signal specificity have different influences, and the length of the probe is typically at least 14 base-pairs, most long to be usually no more than
30 base-pairs, it is optimal with 15-25 base-pair with the length of purpose nucleotide sequence complementation.The probe self-complementary sequences
Most preferably less than 4 base-pairs, in order to avoid influence hybridization efficiency.
The high-flux sequence platform includes the reagent of detection THBD gene expression doses.
The test paper includes test paper carrier and the oligonucleotides being fixed on test paper carrier, and the oligonucleotides can detect
The transcriptional level of THBD genes.
Further, the specific antibody of the THBD albumen includes monoclonal antibody, polyclonal antibody.The THBD albumen
Specific antibody include complete antibody molecule, antibody any segment or modification (for example, chimeric antibody, scFv, Fab, F
(ab ') 2, Fv etc..As long as the segment can retain the binding ability with THBD albumen.For the antibody of protein level
Preparation when well known to a person skilled in the art and the present invention may use any method to prepare the antibody.
In specific embodiments of the present invention, the primer sequence for THBD genes is as follows：Forward primer sequence
As shown in SEQ ID NO.1, reverse primer is as shown in SEQ ID NO.2.
Source for diagnosing the THBD genes of parkinsonism and its expression product include but not limited to blood, tissue fluid,
Urine, saliva, spinal fluid etc. can obtain the body fluid of genomic DNA.In specific embodiments of the present invention, for diagnosing pa
The THBD genes of the gloomy disease of gold and its source of expression product are blood.
In the context of the present invention, " THBD genes " includes any functional equivalent of THBD genes and THBD genes
Polynucleotides.THBD genes (Chromosome 20, NC_000020.11 (23045633..23049664,
Complement)) sequence can inquire in international public GenBank GeneBank.
In the context of the present invention, THBD gene expression products include the partial peptide of THBD albumen and THBD albumen.
The partial peptide of the THBD albumen contains and the relevant functional domain of parkinsonism.
" THBD albumen " includes any functional equivalent of THBD albumen and THBD albumen.The functional equivalent includes
THBD albumen conservative variation protein or its active fragment or its reactive derivative, allelic variant, natural mutation lure
Lead mutant, can be with the encoded protein of DNA of the DNA hybridization of THBD under high or low stringent condition.
It is known that, conventionally, the modification of one or more amino acid does not interfere with the function of protein in a protein.
Those skilled in the art can approve the amino acid that changes single amino acids or small percentage or to indivedual additions of amino acid sequence,
Missing, insertion, replacement are conservative modifications, and the change of wherein protein generates the protein with identity function.Function phase is provided
As the Conservative substitution tables of amino acid be well known in the art.
Example by the protein for adding an amino acid or more amino acid modification is the fusion of THBD albumen
Albumen.For the peptide or protein with THBD protein fusions, there is no limit as long as the fusion protein of gained retains THBD albumen
Biological activity.
In the context of the present invention, " diagnosis parkinsonism " had both included judging whether subject has suffered from Parkinson
Disease also includes judging that subject whether there is the risk with parkinsonism.
The advantages of the present invention：
Present invention firstly discovers that THBD gene expressions are related to parkinsonism, by the table for detecting THBD in subject
It reaches, it can be determined that whether subject suffers from parkinsonism or judges that subject whether there is the risk with parkinsonism, from
And clinician is instructed to provide prevention scheme or therapeutic scheme to subject.
Present invention finds a kind of new molecular marked compound-THBD genes, and compared to traditional detection means, gene diagnosis is more
In time, it is more special, sensitiveer, the early diagnosis of parkinsonism can be realized, so as to reduce the death rate of parkinsonism.
Description of the drawings
Fig. 1, which is shown, utilizes differential expression of the genechip detection THBD genes in Parkinson's disease patients and normal person；Fig. 2
Differential expression of the display using QPCR detection THBD genes in Parkinson's disease patients and normal person.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this
It invents rather than limits the scope of the invention.The experimental method of actual conditions is not specified in embodiment, usually according to conventional strip
Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring HarborLaboratory
Press, 1989) condition described in or according to the condition proposed by manufacturer.
Embodiment 1 screens the gene of differential expression in Parkinson's disease patients and normal person
1st, research object：
Collect primary PD patient 10, man 5, female 5, age 32-85 Sui, the course of disease -20 years 5 months.PD enters a group mark
It is accurate：Diagnostic criteria meets PD clinical criterias (with reference to " Jiang Yuping, Wang Jian, Ding Zhengtong, etc. Primary ventricular hemorrhage is examined
Disconnected standard, 2005, Chinese Clinical Neuscience, 2006,14:40”).Exclusion criteria：(1) essential tremor；(2) secondary pa
The gloomy syndrome of gold；(3) advanced dementia, dysarthrosis person；(4) other mental disease persons are suffered from.This studies oneself and passes through hospital's human relations
The reason committee ratifies and all patients sign informed consent form.
Normal group：Choose the healthy volunteer 10 at age 32-80 Sui, each 5 of men and women.
Age, the not statistically significant (P of gender differences between two groups>0.10), it is comparable.
2nd, in blood total serum IgE extraction
(1) homogenized (Homogenization)
Fresh blood (peripheral blood) is directly taken, 3 times of volume erythrocyte cracked liquids is added in, 10 points is placed at room temperature for after mixing
Clock, 10,000rpm centrifugations 1 minute.It thoroughly inhales and abandons supernatant, collect leukocyte cell pellet.Per the leucocyte of 100-200 μ l blood collections
Precipitation adds in 1ml TRIzol.
(2) it is layered (Phase Separation)
A. after sample adds in TRIzol, 5min is placed at room temperature for, sample is made fully to crack.
B. 200 μ l chloroforms are added in per 1ml TRIzol, 3-5min is placed at room temperature for after shaking vigorously and mix well makes its natural split-phase.
(3) RNA precipitate (RNA Precipitation)
A.4 DEG C 12,000rpm centrifugations 10-15min.Sample can be divided into three layers：The organic phase of yellow, interlayer and colourless
Water phase, RNA mainly in water phase, water phase (can usually draw 550 μ l) are transferred in new pipe.
B. isometric ice-cold isopropanol is added in supernatant, is placed at room temperature for 10-20min.4 DEG C of 12,000rpm centrifugations
10min abandons supernatant, and RNA precipitate is in tube bottom.
(4) RNA rinses (RNA Wash)
75% ethyl alcohol of 1ml (being prepared with RNase-free water) is added in a.RNA precipitations, mildly vibrates centrifuge tube, it is heavy to suspend
It forms sediment.75% ethyl alcohol of 1ml is added in per 1ml TRIzol.
B.4 DEG C 5,000-8,000rpm centrifugation 1-2min, abandon supernatant；Of short duration quick centrifugation is carefully inhaled with pipettor and abandoned
Clearly, 1-2 minutes are placed at room temperature for and dries precipitation.
(5) RNA (Redissolving the RNA) is dissolved
50-100 μ l RNase-free water is added in precipitation, flicks tube wall, fully to dissolve RNA, -70 DEG C of preservations.
3rd, RNA mass and purity detecting
RNA mass：It is represented by RNA integralities, plain agar sugar gel electrophoresis (deposition condition can be used：1.2% glue；
0.5 × TBE electrophoretic buffers；150v, 15 minutes) detection integrality.
RNA purity：OD260/OD280 ratios are the indexs for weighing protein contamination degree in RNA sample.High quality
RNA sample, OD260/OD280 values (10mM Tris, pH7.5) are 2.0 or so.
4th, gene chip hybridization and scanning
After the linearized amplification of total serum IgE, cy3-UTP is marked, and the cRNAs after fluorescent marker uses RNEASY Mini Kit
Purifying, fragmentation processing is carried out with the RNA Fragmentation Reagents of Amhion to the cRNAs marked.Using U.S.
People's full genome chip of expression spectrum (4x 44K genes) of Agilent companies of state, 65 DEG C of hybridization 17h in chip hybridization stove, then
Elution, dyeing, finally with Agilent DNA MicroarrayScanner scanner scannings.
5th, chip data processing and analysis
Chip after hybridization imports data to analysis software, for two groups of ratios after chip scanner reads data point
Natural logrithm absolute value be more than 2.0 or the gene less than 0.5 as difference expression gene.
6th, statistical procedures
Data analysis is carried out using 13.0 statistical softwares of SPSS, group difference compares using one-way analysis of variance method, P<
0.05 difference has significant.
7th, result
The results show (as shown in Figure 1), compared with normal person, the mRNA level in-site of THBD genes in Parkinson's disease patients blood
Significantly rise, difference have statistical significance (P<0.05).
The gene of differential expression in 2 QPCR experimental verifications Parkinson's disease patients of embodiment and normal person
1st, research object：
Screening criteria is the same as embodiment 1, Parkinson's disease patients and each 50 of normal person.
2nd, in blood total serum IgE extraction
Step is the same as embodiment 1.
3rd, reverse transcription
Reverse transcription synthesis cDNA is carried out to l μ g total serum IgEs with RT Buffer.Using 25 μ l reaction systems, each sample
1 μ g total serum IgEs is taken to be separately added into following components in PCR pipe as template ribonucleic acid：DEPC water, 5 × RT Buffer,
10mmol/L dNTP, 0.1mmol/l DTT, 30 μm of mol/l Oligo dT, 200U/ μ l M-MLV, template ribonucleic acid.42 DEG C of incubations
1h, 72 DEG C of 10min, of short duration centrifugation.
4、QPCR
(1) design of primers
QPCR amplimers are designed according to the coded sequence of THBD genes and GAPDH genes in Genbank, work is given birth to by Shanghai
Biotechnology Services Co., Ltd synthesizes.Specific primer sequence is as follows：
THBD genes：
Forward primer is 5 '-CTCAATGCCAGTCAGATC-3 ' (SEQ ID NO.1)；
Reverse primer is 5 '-GTTCAGTAGCAAGGAAATG-3 ' (SEQ ID NO.2),
GAPDH genes：
Forward primer is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ' (SEQ ID NO.3)；
Reverse primer is 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO.4).
(2) PCR reaction systems are prepared according to table 1：
Wherein, SYBR Green PCRs system is purchased from Invitrogen companies.
1 PCR reaction systems of table
| Reagent | Volume |
| Forward primer | 1μl |
| Reverse primer | 1μl |
| SYBR Green PCR systems | 12.5μl |
| Template | 2μl |
| Deionized water | Supply 25 μ l |
(3) PCR reaction conditions：95 DEG C of 10min, (95 DEG C of 10s, 60 DEG C of 40s) * 45 Xun Huans.Using SYBR Green as
Fluorescent marker carries out PCR reactions on Light Cycler fluorescence quantitative PCR instruments, true by melt curve analysis analysis and electrophoresis
Determine purpose band, Δ Δ CT methods carry out relative quantification.
5th, statistical method
Result data is represented in a manner of mean+SD, is united using SPSS13.0 statistical softwares
Meter analysis, difference between the two is examined using t, it is believed that works as P<There is statistical significance when 0.05.
6th, result
The results are shown in Figure 2, and compared with normal person, the mRNA level in-site of THBD genes significantly increases in Parkinson's disease patients blood
Add, difference has statistical significance (P<0.05), as a result homogenic array experiment.
3 immunoblot experiment of embodiment verifies the expression product of difference expression gene in Parkinson's disease patients and normal person
1st, clinical subjects：With embodiment 2.
2nd, monocyte separates
Parkinson's disease patients and normal person extracting vein blood 10ml, injection are contained in the sterile vials of heparin, light immediately after capping
Jog is even.Isometric HBSS (NaCl 8.0g, Na are added in aseptic straw2HPO40.132g, KH2PO40.06g, KCl
0.4g, phenol red 1ml, NaHCO30.35g, D-Glucose 1.0g are dissolved in 1000ml distilled waters), to reduce the cohesion of red blood cell.
It draws 8ml lymphocytes separating solutions to put in 50ml centrifuge tubes, dilute blood is slowly added to along tube wall, interface is kept to understand, not
The two is made mutually to mix, 30min, the careful canescence for drawing layering liquid and blood plasma handing-over position muddiness are centrifuged in 20 DEG C of 2000r/min
In another centrifuge tube of addition, 2 times are washed with the HBSS of 5 times of volumes for layer, i.e. buffy coat, successively with 2000r/min,
1500r/min centrifuges 10min at room temperature, to remove the blood platelet largely mixed, with 10ml distilled waters and cell mass
1min is mixed, cracks residual red blood cells, is then rapidly added equivalent 1.8%NaCl solution, supernatant is removed in 2000r/min centrifugations,
Cell is adjusted to 1 × 10 with HBSS solution after cell count6A/ml is spare.
3rd, monocyte gross protein extracts
By cell suspension obtained by above-mentioned experiment, (concentration is 1 × 106A/ml) room temperature 1 000r/min centrifugation 10min, abandon
Add in 100 μ l lysis buffers after clear, 4 DEG C of concussion 1h, with ultrasonoscope smudge cells, each 10s, totally 10 times, in 4 DEG C
12000r/min centrifuges 1h；Supernatant Brandford standard measure albumen is taken, is distributed into 2.5 μ g/ μ l, -80 DEG C of refrigerators preserve standby
With.
4th, Western blot are detected
Total protein of cell Brandford standard measures take to mix with sample buffer in right amount and boil 5min, cool down 5min；
30pg albumen is taken to be loaded to 15% polyacrylamide gel prepared, electrophoresis is carried out, starts to be set to 80V constant pressures, see
120V is increased to after Marker；Glue after electrophoresis is taken out, using the half-dried transferring systems of Bio.Rad in 100V transferase 45s 0min；Turn
It after film, is washed once with 1xPBS, immerses confining liquid, 40C is overnight；Confining liquid is outwelled, adds in Western cleaning solutions washing 5-
10min adds in primary antibody shaking table room temperature hybridization 2h；According to proper proportion with Western secondary antibody diluteds in Block buffer
In, it is incubated 60min；Film washing liquid is washed 3 times, each 10min；Developed using ECL reagents, fixing detection protein expression.
5th, statistical procedures
The gray value of protein band is analyzed using Image J softwares, using β-actin as internal reference, by purpose informal voucher
The gray value of band is normalized.Result data is represented in a manner of mean+SD, is used
SPSS13.0 statistical softwares carry out statistical analysis, and difference between the two is examined using t, it is believed that works as P<There is system when 0.05
Meter learns meaning.
6th, result
The results show that using the albumen relative expression levels in normal person's group blood as 1, THBD eggs in parkinsonism group blood
White level is 4.14 ± 1.12, is significantly raised, and difference has statistical significance (P<0.05).
The explanation of above-described embodiment is only intended to understand the method and its core concept of the present invention.It should be pointed out that for this
For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention
And modification, these improvement and modification will be also fallen into the protection domain of the claims in the present invention.
Sequence table
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Claims (10)
1. detect application of the product of THBD gene expressions in the instrument for preparing diagnosis parkinsonism.
2. application according to claim 1, which is characterized in that the product includes：By RT-PCR, real-time quantitative PCR,
Immune detection, in situ hybridization, chip or high-flux sequence detection of platform THBD gene expression doses are to diagnose the production of parkinsonism
Product.
3. application according to claim 2, which is characterized in that the product with RT-PCR diagnosis parkinsonisms at least wraps
Include the primer of a pair of of specific amplified THBD genes；The product with real-time quantitative PCR diagnosis parkinsonism includes at least a pair
The primer of specific amplified THBD genes；The product with immune detection diagnosis parkinsonism includes：With THBD protein-specifics
With reference to antibody；The product in situ hybridization diagnosis parkinsonism includes：With the spy of the nucleic acid array hybridizing of THBD genes
Pin；The product with chip diagnosis parkinsonism includes：Protein chip and genetic chip；Wherein, protein chip include with
The antibody that THBD protein-specifics combine, genetic chip include the probe with the nucleic acid array hybridizing of THBD genes.
4. application according to claim 3, which is characterized in that the product with real-time quantitative PCR diagnosis parkinsonism
The primer of a pair of of the specific amplified THBD genes included at least is as shown in SEQ ID NO.1 and SEQID NO.2.
5. a kind of instrument for being used to diagnose parkinsonism, which is characterized in that the instrument can be by detecting THBD bases in sample
The expression of cause diagnoses parkinsonism.
6. instrument according to claim 5, which is characterized in that the instrument includes chip, kit, test paper or high throughput
Microarray dataset.
7. instrument according to claim 6, which is characterized in that the chip includes genetic chip, protein-chip；It is described
Genetic chip includes solid phase carrier and is fixed on the oligonucleotide probe of solid phase carrier, and the oligonucleotide probe includes being used for
Detect the oligonucleotide probe for THBD genes of THBD gene transcription levels；The protein-chip include solid phase carrier with
And it is fixed on the specific antibody of the THBD albumen of solid phase carrier；The kit includes gene detecting kit and protein immunization
Detection kit；The gene detecting kit includes the reagent for detecting THBD gene transcription levels；The protein immunization
Detection kit includes the specific antibody of THBD albumen；The test paper includes detecting the examination of THBD gene transcription levels
Agent；The high-flux sequence platform includes the reagent for detecting THBD gene transcription levels.
8. instrument according to claim 7, which is characterized in that the reagent of the detection THBD gene transcription levels includes pin
To the primer and/or probe of THBD genes.
9. instrument according to claim 8, spy are characterized in that, the primer sequence for THBD genes is as follows：Just
To primer sequence as shown in SEQ ID NO.1, reverse primer is as shown in SEQ ID NO.2.
10. according to the instrument any one of claim 5-9, which is characterized in that the sample is blood.
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| CN201711483559.4A CN108048554B (en) | 2017-12-29 | 2017-12-29 | The molecular marker that THBD gene is diagnosed as parkinsonism |
| PCT/CN2018/123794 WO2019129029A1 (en) | 2017-12-29 | 2018-12-26 | Gene as marker for diagnosing and treating parkinson's disease and use thereof |
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| CN201711483559.4A CN108048554B (en) | 2017-12-29 | 2017-12-29 | The molecular marker that THBD gene is diagnosed as parkinsonism |
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Effective date of registration: 20191210 Address after: Room 2503, Qianshan building, building D2, Qingdao International Innovation Park Phase II, No. 1, Keyuan Weiyi Road, Laoshan District, Qingdao, Shandong Province Patentee after: Qingdao Yangshen biomedical Co., Ltd Address before: 100080 Beijing city Haidian District Shanyuan Street No. 1 cubic court building room 3103 Patentee before: Beijing Yang Shen biology information technology company limited |