CN108796068A - DOCK10 genes for early diagnosing myocardial infarction - Google Patents
DOCK10 genes for early diagnosing myocardial infarction Download PDFInfo
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- CN108796068A CN108796068A CN201810717286.3A CN201810717286A CN108796068A CN 108796068 A CN108796068 A CN 108796068A CN 201810717286 A CN201810717286 A CN 201810717286A CN 108796068 A CN108796068 A CN 108796068A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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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
- G01N2800/32—Cardiovascular disorders
- G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
Abstract
The invention discloses myocardial infarction diagnosis related genes.The present invention screens the gene that expression is had differences in myocardial infarction patient and normal person by RNA-seq, proves that the content of DOCK10 genes and DOCK10 albumen in myocardial infarction patient blood is lower than normal person by large sample QPCR verifications.It accordingly can be using DOCK10 genes and DOCK10 albumen as the biomarker of diagnosing myocardial infarction.The achievement in research of the present invention provides a kind of noninvasive method clinically to carry out the early diagnosis of myocardial infarction, is suitable for clinical expansion.
Description
Technical field
The invention belongs to molecular diagnosis field, it is related to the DOCK10 genes for early diagnosing myocardial infarction.
Background technology
Myocardial infarction is a global public health problem, seriously threatens health (J.Ross, Jr., the A of the public
50-year research journey.From laboratory to clinic,Circulation journal:
official journal of the Japanese Circulation Society,73(2009)3-12；D.Lloyd-
Jones,R.Adams,M.Carnethon,G.De Simone,T.B.Ferguson,K.Flegal,E.Ford,K.Furie,
A.Go,K.Greenlund,N.Haase,S.Hailpern,M.Ho,V.Howard,B.Kissela,S.Kittner,
D.Lackland,L.Lisabeth,A.Marelli,M.McDermott,J.Meigs,D.Mozaffarian,G.Nichol,
C.O'Donnell,V.Roger,W.Rosamond,R.Sacco,P Sorlie,R.Stafford,J.Steinberger,
T.Thom,S.Wasserthiel-Smoller,N.Wong,J.Wylie-Rosett,Y.Hong,C.American Heart
Association Statistics,S.).Several method can be effectively applied to the treatment of myocardial infarction, such as percutaneously
Percutantnoeus coronary intervention or coronary revascularization, these treatments can protect the local necrosis cardiac muscle that blood flows through, reduce necrosis
Region.The clinical effectiveness of acute myocardial infarction patients after treatment shows that the infarct of left compartment muscle is improved.However, at present
Treatment for acute myocardial infarction AMI is still limited, main cause be treatment time window very it is narrow (R.Suresh,
X.Li,A.Chiriac,K.Goel,A.Terzic,C.Perez-Terzic,T.J.Nelson,ranscriptome from
circulating cells suggests dysregulated pathways associated with long-term
recurrent events following first-time myocardial infarction,Journal of
Molecular and cellular cardiology, 74 (2014) 13-21.), therefore there is an urgent need to understand that the acute heart occurs
Main molecules mechanism when flesh infarct.
Myocardial infarction is a complicated physiology course, and ischemic leads to the expression Cascaded amplification of the certain genes of cardiac muscle cell,
Apoptosis or the necrosis of cardiac muscle cell are in turn resulted in, therefore extends the therapeutic time window of myocardial infarction, inhibits the gene table of catchment
Reach, can effectively prevent cardiac muscle cell apoptosis or necrosis generation (M.Ashburner, C.A.Ball, J.A.Blake,
D.Botstein,H.Butler,J.M.Cherry,A.P.Davis,K.Dolinski,S.S.Dwight,J.T.Eppig,
M.A.Harris,D.P.Hill,L.Issel-Tarver,A.Kasarskis,S.Lewis,J.C.Matese,
J.E.Richardson,M.Ringwald,G.M.Rubin,G.Sherlock,Gene ontology:tool for the
unification of biology.The Gene Ontology Consortium,Nature genetics,25(2000)
25-29).The gene of early stage response after myocardial ischemia is analyzed, the gene expression atlas of research myocardial ischemia gradient at any time discloses
Gene Activity when myocardial ischemia, based on energy research and clinical diagnosis new foundation is provided.Various physiological processes are along with the heart
Appearance of flesh infarct, such as inflammation, abnormal calcium intake, cell cycle exception, peptide secretion, oxidative stress, Apoptosis etc., mesh
When some related genes express unclear after the preceding precise time that these processes occur and generation myocardial ischemia, also
It waits for further exploring.
More and more researchs find that specific gene can become the biomarker of diagnosing myocardial infarction, such as following
The patent disclosure of application number：201710592045.6,201510923782.0,201810019374.6,
201710592044.1、201510727115.5、201510727744.8、201710591408.4、201710592593.9、
201510727102.8.Most of disease is all that the disease of polygenes regulation and control will be a large amount of in order to improve the accuracy rate of diagnosis of disease
Gene association diagnosis becomes inevitable, it is therefore desirable to excavate more with the relevant molecular marker of myocardial infarction so as to clinical application.
Invention content
In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide one kind can be used for myocardial infarction early diagnosis
Molecular marker.Compared to the diagnostic method of traditional myocardial infarction, carry out having for diagnosing myocardial infarction using gene marker
Promptness, specificity and sensitivity, for risk height, are taken to make patient that can know disease risks in disease early stage
Corresponding prevention and treatment measure.
To achieve the goals above, the present invention adopts the following technical scheme that：
The present invention provides application of the product of detection DOCK10 gene expressions in the tool for preparing diagnosing myocardial infarction.
Further, the product of detection DOCK10 gene expressions mentioned above includes：Pass through RT-PCR, real-time quantitative
PCR, immune detection, in situ hybridization, chip or high-flux sequence detection of platform DOCK10 gene expression doses are to diagnose myocardium stalk
Dead product.
Further, the product with RT-PCR diagnosing myocardial infarctions includes at least a pair of of specific amplified DOCK10 genes
Primer；The product with real-time quantitative PCR diagnosing myocardial infarction includes at least the primer of a pair of of specific amplified DOCK10 genes；
The product with immune detection diagnosing myocardial infarction includes：The antibody combined with DOCK10 protein-specifics；The original position
Hybridization diagnosing myocardial infarction product include：With the probe of the nucleic acid array hybridizing of DOCK10 genes；It is described to diagnose the heart with chip
The product of flesh infarct includes：Protein chip and genetic chip；Wherein, protein chip includes being combined with DOCK10 protein-specifics
Antibody, genetic chip include the probe with the nucleic acid array hybridizing of DOCK10 genes.
In specific embodiments of the present invention, the product with real-time quantitative PCR diagnosing myocardial infarction includes at least
The sequence of the primer of a pair of of specific amplified DOCK10 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 DOCK10 gene expressions can be applied to platform realization pair
The detection of the expression of DOCK10 genes.With the development of high throughput sequencing technologies, to the structure of the gene expression profile of a people
It builds to become and very easily work.By comparing the gene expression profile of Disease and normal population, it is easy which is analyzed
The exception of gene is related to disease.Therefore, know that the exception of DOCK10 genes is related to myocardial infarction in high-flux sequence
Belong to the purposes of DOCK10 genes, equally within protection scope of the present invention.
The present invention also provides a kind of tool of diagnosing myocardial infarction, 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 for detecting being directed to for DOCK10 gene transcription levels
The oligonucleotide probe of DOCK10 genes；The protein-chip includes solid phase carrier and is fixed on the DOCK10 of solid phase carrier
The specific antibody of albumen；The genetic chip can be used for detecting multiple genes including DOCK10 genes (for example, and the heart
The relevant multiple genes of flesh infarct) expression.The protein-chip can be used for detecting including DOCK10 albumen
The expression of multiple protein (such as with the relevant multiple protein of myocardial infarction).By by multiple with myocardial infarction mark
Will object detects simultaneously, is greatly improved the accuracy rate of myocardial infarction diagnosis.
Wherein, the kit includes gene detecting kit and protein immunization detection kit；The genetic test examination
Agent box includes the reagent for detecting DOCK10 gene transcription levels；The protein immunization detection kit includes DOCK10 albumen
Specific antibody.Further, the reagent includes using RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or core
Piece method detects reagent needed for DOCK10 gene expression dose processes.Preference, the reagent include being directed to DOCK10 bases
The primer and/or probe of cause.It is easy to design according to the nucleotide sequence information of DOCK10 genes and can be used for detecting DOCK10
The primer and probe of gene expression dose.
Probe with the nucleic acid array hybridizing of DOCK10 genes can be DNA, RNA, DNA-RNA chimera, PNA or other
Derivative.There is no limit for the length of the probe, as long as completing specific hybrid, being specifically bound with purpose nucleotide sequence,
Any length can.The length of the probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the probe
Length can be grown to 60,80,100,150,300 base-pairs or longer or even whole genes.Since different probe lengths is to miscellaneous
Efficiency, signal specificity is handed over to have different influences, the length of the probe is typically at least 14 base-pairs, and longest does not surpass generally
30 base-pairs are crossed, it is best with 15-25 base-pair with the length of purpose nucleotide sequence complementation.The probe self-complementary sequence
Row are most preferably less than 4 base-pairs, in order to avoid influence hybridization efficiency.
The high-flux sequence platform includes the reagent for detecting DOCK10 gene expression doses.
The test paper includes test paper carrier and the oligonucleotides that is fixed on test paper carrier, and the oligonucleotides can detect
The transcriptional level of DOCK10 genes.
Further, the specific antibody of the DOCK10 albumen includes monoclonal antibody, polyclonal antibody.The DOCK10
The specific antibody of albumen 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 DOCK10 albumen.For protein water
When the preparation of flat antibody well known to a person skilled in the art, and the present invention may use any method it is described anti-to prepare
Body.
In specific embodiments of the present invention, the primer sequence for DOCK10 genes is as follows：Forward primer sequence
Row are as shown in SEQ ID NO.1, and reverse primer is as shown in SEQ ID NO.2.
Include but not limited to blood, tissue for the DOCK10 genes of diagnosing myocardial infarction and its source of expression product
Liquid, urine, saliva, spinal fluid etc. can obtain the body fluid of genomic DNA.In specific embodiments of the present invention, for examining
The DOCK10 genes of disconnected myocardial infarction and its source of expression product are blood.
In the context of the present invention, " DOCK10 genes " includes any function of DOCK10 genes and DOCK10 genes
The polynucleotides of equivalent.DOCK10 genes (Chromosome 2, NC_000002.12 (224765090..225042689,
Complement)) sequence can inquire in international public GenBank GeneBank.
In the context of the present invention, DOCK10 gene expression products include the portion of DOCK10 albumen and DOCK10 albumen
Divide peptide.The partial peptide of the DOCK10 albumen contains and the relevant functional domain of myocardial infarction.
" DOCK10 albumen " includes any functional equivalent of DOCK10 albumen and DOCK10 albumen.The function is equivalent
Object includes DOCK10 albumen conservative variation protein or its active fragment or its reactive derivative, allelic variant, natural
Mutant, induced mutants, can be with the encoded protein of DNA of the DNA hybridization of DOCK10 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 for changing single amino acids or small percentage or to amino acid sequence it is individual add,
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 adding the protein of an amino acid or more amino acid modification is melting for DOCK10 albumen
Hop protein.For the peptide or protein with DOCK10 protein fusions, there is no limit as long as the fusion protein of gained retains
The biological activity of DOCK10 albumen.
In the context of the present invention, " diagnosing myocardial infarction " had both included judging whether subject has suffered from cardiac muscle stalk
Extremely, also include the risk that judges subject and whether there is with myocardial infarction.
The advantages of the present invention：
Present invention firstly discovers that DOCK10 gene expressions are related to myocardial infarction, pass through and detect DOCK10 in subject
Expression, it can be determined that whether subject suffers from myocardial infarction or judges that subject whether there is the risk with myocardial infarction,
To instruct clinician to provide prevention scheme or therapeutic scheme to subject.
Present invention finds a kind of new molecular marked compound-DOCK10 genes, compare traditional detection means, gene diagnosis
More in time, more special, sensitiveer, the early diagnosis of myocardial infarction can be realized, to reduce the death rate of myocardial infarction.
Description of the drawings
Fig. 1 shows the differential expression in myocardial infarction patient and normal person using QPCR detection DOCK10 genes；
Fig. 2, which is shown, utilizes differential expression of the immune-blotting method DOCK10 albumen in myocardial infarction patient 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.Test method without specific conditions 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 normal condition proposed by manufacturer.
Embodiment 1 screens the gene of differential expression in myocardial infarction patient and normal person
1, research object
The myocardial infarction inpatient for choosing 6 hospitalizes is research object, wherein 3 males, 3 women are average
Age is 56.00 scholars 8.75；Control group is 7 Healthy Peoples；Every patient for being invited to that research is added and healthy person are signed above
Informed consent form.
2, the collection and storage of sample
Patient was admitted to hospital the same day, before row coronary angiography in acquisition 8mL fresh steriles arterial blood to EDTA anti-freezing purple head tubes.If not
It uses at once, sample can be put in 4 DEG C of refrigerators and preserve 2h.
3, Ficoll methods detach PBMCs
Following steps are completed in super-clean bench：
(1) physiological saline dilutes blood sample in equal volume, and (human lymphocyte separating liquid is purchased from Tianjin to isometric Ficoll liquid
The oceans Hao biological products Science and Technology Ltd.) it is added in 50mL centrifuge tubes, 45 DEG C of angle centrifuge tubes are tilted, no RNA enzyme pipette tips pair are used
Blood sample after dilution is drawn, and is slowly added into separation ullage along tube wall, blood sample is able in Ficoll separating liquids
Tiling, it is ensured that liquid level is not disturbed, and tightens pipe lid.
(2) it is put into horizontal centrifuge, room temperature 800g centrifuges 20min, and needs slow raising speed.
(3) it is sucked out and discards serum layer, draw serum layer along tube wall using no RNA enzyme pipette tips is rich in separating liquid intersection
The tunica albuginea confluent monolayer cells of PBMCs are placed in new 10mL without in RNA enzyme EP pipes, and physiological saline is added to 10mL, mixing, room temperature 1700r/
Min centrifuges 15min.
(4) supernatant is abandoned, it is seen that tube bottom precipitates, as PBMCs, then 1mL physiological saline is added into pipe, and precipitation piping and druming is mixed
It is even, 1.5mL is transferred to without in RNA enzyme EP pipes, and room temperature 1500r/min centrifuges l0min, washs cell again.
(5) supernatant is abandoned, 1mL Trizol are added and blow and beat mixing, if not extraction RNA at once, can be placed on -80 DEG C of refrigerators and preserves.
4, the RNA in PBMCs is extracted
Total RNAs extraction use phenol chloroform one-step method, carry out all step operations on ice, need to wear no RNA enzyme mask and
Gloves, operating procedure are as follows：
(1) PBMCs for being dissolved in Trizol, slow mechanism dissolved are taken out from -80 DEG C of refrigerators.
(2) chloroform of 200 μ l is added into every pipe, acutely vibrates 15sec, 4 DEG C of 12000r/min centrifuge 15min.
(3) it is added to the 1.5mL newly got ready without upper phase (being sure not to touch middle level and lower layer) is drawn in enzyme EP pipes
500 μ l isopropanols of volume overturn mixing, are stored at room temperature 10min, and 4 DEG C of 12000r/min centrifuge 15min.
(4) supernatant is abandoned, 75% ethyl alcohol of 1mL (configuration of DEPC water) is added, overturns mixing for several times, washing precipitation, subsequent 40C
7500r/min centrifuges 10min.
(5) supernatant is abandoned, under the premise of without impinging on precipitation, liquid in pipe is sucked out as possible, opening is stood in subsequent super-clean bench
After drying precipitate, 15-20 μ l 1/1000 are added without RNA enzyme water dissolution RNA in 15-20min.
(6) RNA purity is detected：The dissolved RNA samples of 2 μ L are added after microplate reader self-test, zeroing to be detected.Directly obtain
Take RNA concentration.The OD260/OD280 of 1.8-2.2 illustrates that the RNA purity of extraction is higher, can use.
5, high-throughput transcript profile sequencing
(1) RNA-seq reads position
Low-quality read is removed to obtain cleaning read first, then utilize TopHat v1.3.1 will clean segment and
UCSC H.sapiens reference genes groups (hg19) are matched, the index of H.sapiens UCSC hg19 editions built in advance
It is downloaded from TopHat homepages, and as with reference to genome, when being matched with genome using TopHat, allows each read (acquiescence
To 20) having multiple matching sites, most 2 mispairing.TopHat establishes possible according to exon region and GT-AG shear signals
Shearing site library navigates to the read for not navigating to genome on genome according to these shearing site libraries.We use
The system default parameter of TopHat methods.
(2) transcript abundance is assessed
The read file matched is by Cufflinks v1.0.3 processing, and Cufflinks v1.0.3 are by RNA-seq pieces
Hop count mesh is standardized the relative abundance for calculating transcript.FPKM values refer to being matched in every 1,000,000 sequencing segment specific
The segment number of the exon region of gene 1kb long.The confidence interval of FPKM estimated values is calculated by Bayesian inference method.
The GTF comment files for the reference that Cufflinks is used download (Homo_ from Ensembl databases
sapiens.GRCh37.63.gtf)。
(3) detection of difference expression gene
It is transferred to Cuffdiff by the Ensembl GTF files of download and by the matched original documents of TopHat,
Cuffdiff re-evaluates the gene expression abundance for the transcript listed in GTF files using original matching files, detects difference table
It reaches.The only q values < 0.01 in Cuffidff outputs, test display is considered as successfully more just differential expression.
6, result
RNA-seq is the results show that compared with normal population, and the gene of high expression is 347 in myocardial infarction patient blood,
The gene of low expression is 254, and difference all has statistical significance (P<0.05).
The gene of differential expression in embodiment 2QPCR experimental verifications myocardial infarction patient and normal person
1, research object：
Screening criteria is the same as embodiment 1, myocardial infarction patient and each 35 of normal person.
2, in blood total serum IgE extraction
Step is the same as embodiment 1.
3、RT-PCR
(1)RT
RT reaction systems (20 μ l)：
RT response procedures：
42℃ 15min
85℃ 5s
4℃ ---
(2)qPCR
PCR reaction systems (20 μ l)：
PCR response procedures：
Amplification program：
1 95 DEG C of 10min of stage
2 95 DEG C of 10s of stage
55℃ 10s
Repeat 40 cycles
3 multiple holes of each sample, internal reference GAPDH are set.
(3) primer
The primer sequence of DOCK10 genes and GAPDH genes is as follows：
GAPDH genes
Forward primer：5'-CCACATCGCTCAGACACCAT-3'(SEQ ID NO.3)；
Reverse primer：5'-GGCAACAATATCCACTTTACCAGAGT-3'(SEQ ID NO.4).
DOCK10 genes
Forward primer：5'-GTCTGGATTCGCTGGATAA-3'(SEQ ID NO.1)；
Reverse primer：5'-CGTGTAGGTTGTTCTCTGA-3'(SEQ ID NO.2).
(4) interpretation of result
Ct values are corresponding recurring number when inner fluorescent tube intensity reaches exponential growth phase by background.
Formula be sample Ct values-internal reference (GAPDH) Ct values=△Ct；Sample △ Ct values-negative control △ Ct=△△Ct；Sample
Relative value=2 product mRNA-△△Ct。
(5) data statistics
All data are shown with mean+SD (Mean ± SD).Variance analysis multi-group data group difference.P<
0.05 is to have significant difference.
4, result
The results show that compared with normal person's average level, have in 35 myocardial infarction patients in 33 blood samples of patients
The mRNA level in-site of DOCK10 genes is remarkably decreased.Statistical result is as shown in Figure 1, compared with normal person, myocardial infarction patient blood
The mRNA level in-site of middle DOCK10 genes significantly reduces, and difference has statistical significance (P<0.05), as a result RNA-seq is tested.
3 immunoblot experiment of embodiment verifies the expression product of difference expression gene in myocardial infarction patient and normal person
1, research object：With embodiment 2.
2, monocyte detaches
Myocardial infarction patient 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 is added with 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 set in 50ml centrifuge tubes, dilute blood is slowly added to along tube wall, keep interface to understand, not
So that the two is mutually mixed, 30min, the careful canescence for drawing layering liquid and blood plasma handing-over position muddiness are centrifuged in 20 DEG C of 2000r/min
Layer, i.e. buffy coat are added in another centrifuge tube, and 2 times are washed with the HBSS of 5 times of volumes, successively with 2000r/min,
1500r/min centrifuges 10min at room temperature, the blood platelet largely mixed to remove, with 10ml distilled waters and cell mass
1min is mixed, residual red blood cells is made to crack, 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.
3, monocyte gross protein extracts
By cell suspension (a concentration of 1 × 10 obtained by above-mentioned experiment6A/ml) room temperature 1 000r/min centrifuge 10min, abandon
It is added 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.
4, Western blot are detected
With conventional method.
5, 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 indicated in a manner of mean+SD, is used
SPSS13.0 statistical softwares are next for 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.
6, result
The results show that compared with normal person's average level, have in 35 myocardial infarction patients in 33 blood samples of patients
DOCK10 protein levels are remarkably decreased.Statistical result is as shown in Fig. 2, compared with normal person, in myocardial infarction patient blood
DOCK10 protein levels significantly reduce, 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 also be fallen into the protection domain of the claims in the present invention.
Sequence table
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<120>DOCK10 genes for early diagnosing myocardial infarction
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<170> SIPOSequenceListing 1.0
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<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
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gtctggattc gctggataa 19
<210> 2
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<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
cgtgtaggtt gttctctga 19
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
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ccacatcgct cagacaccat 20
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<212> DNA
<213>Artificial sequence (Artificial Sequence)
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ggcaacaata tccactttac cagagt 26
Claims (10)
1. detecting application of the product of DOCK10 gene expressions in the tool for preparing diagnosing myocardial infarction.
2. application according to claim 1, which is characterized in that the product includes：Pass through reverse transcription PCR, real-time quantitative
PCR, immune detection, in situ hybridization, chip or high-flux sequence detection of platform DOCK10 gene expression doses are to diagnose myocardium stalk
Dead product.
3. application according to claim 2, which is characterized in that the product with reverse transcription PCR diagnosing myocardial infarction is extremely
Few includes the primer of a pair of of specific amplified DOCK10 genes；It is described at least to be wrapped with the product of real-time quantitative PCR diagnosing myocardial infarction
Include the primer of a pair of of specific amplified DOCK10 genes；The product with immune detection diagnosing myocardial infarction includes：With DOCK10
The antibody that protein-specific combines；The product in situ hybridization diagnosing myocardial infarction includes：With the nucleic acid of DOCK10 genes
The probe of sequence hybridization；The product with chip diagnosing myocardial infarction includes：Protein chip and genetic chip；Wherein, albumen
Chip includes the antibody combined with DOCK10 protein-specifics, and genetic chip includes the nucleic acid array hybridizing with DOCK10 genes
Probe.
4. application according to claim 3, which is characterized in that the product with real-time quantitative PCR diagnosing myocardial infarction
The primer of a pair of of the specific amplified DOCK10 genes included at least is as shown in SEQ ID NO.1 and SEQ ID NO.2.
5. a kind of tool for diagnosing myocardial infarction, which is characterized in that the tool can be by detecting DOCK10 in sample
The expression of gene carrys out diagnosing myocardial infarction.
6. tool according to claim 5, which is characterized in that the tool includes chip, kit, test paper or high throughput
Microarray dataset.
7. tool 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 DOCK10 genes of DOCK10 gene transcription levels；The protein-chip includes that solid phase carries
Body and be fixed on solid phase carrier DOCK10 albumen specific antibody；The kit includes gene detecting kit and egg
White immunity detection reagent；The gene detecting kit includes the reagent for detecting DOCK10 gene transcription levels；It is described
Protein immunization detection kit includes the specific antibody of DOCK10 albumen；The test paper includes turning for detecting DOCK10 genes
Record horizontal reagent；The high-flux sequence platform includes the reagent for detecting DOCK10 gene transcription levels.
8. tool according to claim 7, which is characterized in that it is described detection DOCK10 gene transcription levels reagent include
For the primer and/or probe of DOCK10 genes.
9. tool according to claim 8, spy are characterized in that, the primer sequence for DOCK10 genes is as follows：
Forward primer sequence is as shown in SEQ ID NO.1, and reverse primer is as shown in SEQ ID NO.2.
10. according to the tool described in any one of claim 5-9, which is characterized in that the sample is blood.
Priority Applications (3)
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| CN201810717286.3A CN108796068A (en) | 2018-07-03 | 2018-07-03 | DOCK10 genes for early diagnosing myocardial infarction |
| PCT/CN2019/094268 WO2020007270A1 (en) | 2018-07-03 | 2019-07-01 | Marker and diagnostic method for non-invasive diagnosis of myocardial infarction |
| US17/247,534 US11718880B2 (en) | 2018-07-03 | 2020-12-15 | Marker and diagnosis method for noninvasive diagnosis of myocardial infarction |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2020007270A1 (en) * | 2018-07-03 | 2020-01-09 | 北京泱深生物信息技术有限公司 | Marker and diagnostic method for non-invasive diagnosis of myocardial infarction |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2020007270A1 (en) * | 2018-07-03 | 2020-01-09 | 北京泱深生物信息技术有限公司 | Marker and diagnostic method for non-invasive diagnosis of myocardial infarction |
| US11718880B2 (en) | 2018-07-03 | 2023-08-08 | Beijing Medintell Biomed Co., Ltd | Marker and diagnosis method for noninvasive diagnosis of myocardial infarction |
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