CN108796069A - Diagnosis marker-ING1 the genes of myocardial infarction - Google Patents
Diagnosis marker-ING1 the genes of myocardial infarction Download PDFInfo
- Publication number
- CN108796069A CN108796069A CN201810718258.3A CN201810718258A CN108796069A CN 108796069 A CN108796069 A CN 108796069A CN 201810718258 A CN201810718258 A CN 201810718258A CN 108796069 A CN108796069 A CN 108796069A
- Authority
- CN
- China
- Prior art keywords
- ing1
- myocardial infarction
- genes
- chip
- product
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
-
- 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
Abstract
The invention discloses the diagnostic tools that ING1 genes can be used as myocardial infarction.The present invention is known by high-flux sequence and QPCR researchs, ING1 genes content in the blood of myocardial infarction patient raising more notable than normal person, by western blot experiments have shown that the raising more notable than normal person of ING1 albumen content in the blood of myocardial infarction patient.Therefore ING1 genes and ING1 albumen can be as the molecular markers of diagnosing myocardial infarction.It is had the advantage that with the content of gene in blood or albumen to characterize myocardial infarction：Sample of tissue detection is avoided, alleviates the pain of subject, while the characterizing method can be used for the screening of healthy population, effectively prevents the generation of myocardial infarction.
Description
Technical field
The invention belongs to molecular diagnosis fields, are related to the diagnosis marker-ING1 genes of 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,Circulationjournal:
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 ING1 gene expressions in the tool for preparing diagnosing myocardial infarction.
Further, the product of detection ING1 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 ING1 gene expression doses are with the production of diagnosing myocardial infarction
Product.
Further, the product with RT-PCR diagnosing myocardial infarctions includes at least drawing for a pair of of specific amplified ING1 genes
Object；The product with real-time quantitative PCR diagnosing myocardial infarction includes at least the primer of a pair of of specific amplified ING1 genes；It is described
Product with immune detection diagnosing myocardial infarction includes：The antibody combined with ING1 protein-specifics；It is described to be examined in situ hybridization
The product of disconnected myocardial infarction includes：With the probe of the nucleic acid array hybridizing of ING1 genes；It is described with chip diagnosing myocardial infarction
Product includes：Protein chip and genetic chip；Wherein, protein chip includes the antibody combined with ING1 protein-specifics, gene
Chip includes the probe with the nucleic acid array hybridizing of ING1 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 ING1 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 ING1 gene expressions can be applied to the platform and realize to ING1 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.Which by comparing the gene expression profile of Disease and normal population, it is easy to analyze the different of gene
It is often related to disease.Therefore, know that the exception of ING1 genes is related to myocardial infarction in high-flux sequence and also belong to ING1 bases
The purposes of cause, 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 ING1 gene transcription levels
The oligonucleotide probe of ING1 genes；The protein-chip includes solid phase carrier and is fixed on the ING1 albumen of solid phase carrier
Specific antibody；The genetic chip can be used for detecting multiple genes including ING1 genes (for example, and myocardial infarction
Relevant multiple genes) expression.The protein-chip can be used for detecting multiple albumen including ING1 albumen
The expression of matter (such as with the relevant multiple protein of myocardial infarction).By by multiple markers with myocardial infarction simultaneously
Detection, 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 ING1 gene transcription levels；The protein immunization detection kit includes the spy of ING1 albumen
Heterogenetic antibody.Further, the reagent includes using RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or chip side
Method detects reagent needed for ING1 gene expression dose processes.Preference, the reagent include the primer for ING1 genes
And/or probe.It is easy to design according to the nucleotide sequence information of ING1 genes and can be used for detecting ING1 gene expression doses
Primer and probe.
Probe with the nucleic acid array hybridizing of ING1 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, and longest is usually no more than
30 base-pairs, it is best 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 for detecting ING1 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 ING1 genes.
Further, the specific antibody of the ING1 albumen includes monoclonal antibody, polyclonal antibody.The ING1 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 ING1 albumen.Antibody for 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 ING1 genes is as follows：Forward primer sequence
As shown in SEQ ID NO.1, reverse primer is as shown in SEQ ID NO.2.
For the ING1 genes of diagnosing myocardial infarction and its source of 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 the heart
The ING1 genes of flesh infarct and its source of expression product are blood.
In the context of the present invention, " ING1 genes " includes any functional equivalent of ING1 genes and ING1 genes
Polynucleotides.ING1 genes (Chromosome 13, NC_000013.11 (110712623..110721074)) sequence can be
It is inquired in international public GenBank GeneBank.
In the context of the present invention, ING1 gene expression products include the partial peptide of ING1 albumen and ING1 albumen.
The partial peptide of the ING1 albumen contains and the relevant functional domain of myocardial infarction.
" ING1 albumen " includes any functional equivalent of ING1 albumen and ING1 albumen.The functional equivalent includes
ING1 albumen conservative variation protein or its active fragment or its reactive derivative, allelic variant, lure at natural mutation
Lead mutant, can be with the encoded protein of DNA of the DNA hybridization of ING1 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 the protein for adding an amino acid or more amino acid modification is the fusion of ING1 albumen
Albumen.For the peptide or protein with ING1 protein fusions, there is no limit as long as the fusion protein of gained retains ING1 albumen
Biological activity.
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 ING1 gene expressions are related to myocardial infarction, pass through the table for detecting ING1 in subject
It reaches, it can be determined that whether subject suffers from myocardial infarction or judges that subject whether there is the risk with myocardial infarction, from
And clinician is instructed to provide prevention scheme or therapeutic scheme to subject.
Present invention finds a kind of new molecular marked compound-ING1 genes, compare traditional detection means, gene diagnosis is 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 ING1 genes；
Fig. 2, which is shown, utilizes differential expression of the immune-blotting method ING1 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:ColdSpring 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
51℃ 10s
Repeat 45 cycles
3 multiple holes of each sample, internal reference GAPDH are set.
(3) primer
The primer sequence of ING1 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).
ING1 genes
Forward primer：5'-CAAGGAACCTCAAGTCAT-3'(SEQ ID NO.1)；
Reverse primer：5'-CTCACATACAGCAGGAAG-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, there is ING1 in 32 blood samples of patients in 35 myocardial infarction patients
The mRNA level in-site of gene significantly raises.Statistical result is as shown in Figure 1, compared with normal person, ING1 in myocardial infarction patient blood
The mRNA level in-site of gene significantly increases, 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, there is ING1 in 32 blood samples of patients in 35 myocardial infarction patients
Protein level significantly raises.Statistical result is as shown in Fig. 2, compared with normal person, ING1 albumen water in myocardial infarction patient blood
HUD, which writes, to be increased, 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
<110>Beijing Yang Shen biology information technologies Co., Ltd
<120>Diagnosis marker-ING1 the genes of myocardial infarction
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
caaggaacct caagtcat 18
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
ctcacataca gcaggaag 18
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
ccacatcgct cagacaccat 20
<210> 4
<211> 26
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
ggcaacaata tccactttac cagagt 26
Claims (10)
1. detecting application of the product of ING1 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 ING1 gene expression doses are with diagnosing myocardial infarction
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 ING1 genes；The product with real-time quantitative PCR diagnosing myocardial infarction includes at least
The primer of a pair of of specific amplified ING1 genes；The product with immune detection diagnosing myocardial infarction includes：It is special with ING1 albumen
The antibody that the opposite sex combines；The product in situ hybridization diagnosing myocardial infarction includes：With the nucleic acid array hybridizing of ING1 genes
Probe；The product with chip diagnosing myocardial infarction includes：Protein chip and genetic chip；Wherein, protein chip includes
The antibody combined with ING1 protein-specifics, genetic chip include the probe with the nucleic acid array hybridizing of ING1 genes.
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 ING1 genes included at least is as shown in SEQ ID NO.1 and SEQ IDNO.2.
5. a kind of tool for diagnosing myocardial infarction, which is characterized in that the tool can be by detecting ING1 bases in sample
The expression of cause 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 ING1 genes of ING1 gene transcription levels；The protein-chip include solid phase carrier with
And it is fixed on the specific antibody of the ING1 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 ING1 gene transcription levels；The protein immunization
Detection kit includes the specific antibody of ING1 albumen；The test paper includes the examination for detecting ING1 gene transcription levels
Agent；The high-flux sequence platform includes the reagent for detecting ING1 gene transcription levels.
8. tool according to claim 7, which is characterized in that the reagent of the detection ING1 gene transcription levels includes needle
To the primer and/or probe of ING1 genes.
9. tool according to claim 8, spy are characterized in that, the primer sequence for ING1 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 tool described in any one of claim 5-9, which is characterized in that the sample is blood.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810718258.3A CN108796069A (en) | 2018-07-03 | 2018-07-03 | Diagnosis marker-ING1 the genes of 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 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810718258.3A CN108796069A (en) | 2018-07-03 | 2018-07-03 | Diagnosis marker-ING1 the genes of myocardial infarction |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108796069A true CN108796069A (en) | 2018-11-13 |
Family
ID=64074340
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810718258.3A Pending CN108796069A (en) | 2018-07-03 | 2018-07-03 | Diagnosis marker-ING1 the genes of myocardial infarction |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108796069A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN111286536A (en) * | 2020-04-24 | 2020-06-16 | 山东大学齐鲁医院(青岛) | Gene marker for diagnosing myocardial infarction and application thereof |
| CN112760373A (en) * | 2021-03-23 | 2021-05-07 | 山东第一医科大学第二附属医院 | Product for detecting myocardial infarction |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105177171A (en) * | 2015-10-30 | 2015-12-23 | 北京泱深生物信息技术有限公司 | Application of AKR1B10 to preparation of acute myocardial infarction diagnosis and treatment preparation |
| CN105349642A (en) * | 2015-10-30 | 2016-02-24 | 北京泱深生物信息技术有限公司 | Acute myocardial infarction marker and application thereof |
| CN105400880A (en) * | 2015-12-11 | 2016-03-16 | 天津市人民医院 | Acute myocardial infarction early diagnosis marker |
| CN105567862A (en) * | 2016-03-16 | 2016-05-11 | 宫蕊 | Application of CDK18 in preparing products for diagnosing coronary heart disease |
| CN107312852A (en) * | 2017-07-19 | 2017-11-03 | 北京泱深生物信息技术有限公司 | Myocardial infarction diagnosis mark compositions |
-
2018
- 2018-07-03 CN CN201810718258.3A patent/CN108796069A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105177171A (en) * | 2015-10-30 | 2015-12-23 | 北京泱深生物信息技术有限公司 | Application of AKR1B10 to preparation of acute myocardial infarction diagnosis and treatment preparation |
| CN105349642A (en) * | 2015-10-30 | 2016-02-24 | 北京泱深生物信息技术有限公司 | Acute myocardial infarction marker and application thereof |
| CN105400880A (en) * | 2015-12-11 | 2016-03-16 | 天津市人民医院 | Acute myocardial infarction early diagnosis marker |
| CN105567862A (en) * | 2016-03-16 | 2016-05-11 | 宫蕊 | Application of CDK18 in preparing products for diagnosing coronary heart disease |
| CN107312852A (en) * | 2017-07-19 | 2017-11-03 | 北京泱深生物信息技术有限公司 | Myocardial infarction diagnosis mark compositions |
Non-Patent Citations (2)
| Title |
|---|
| GARKAVTSEV,I.等: "Homo sapiens growth inhibitor p33ING1 (ING1) mRNA, complete cds", 《GENBANK DATABASE》 * |
| 何向民等: "ING1抑癌基因在胃癌中表达的研究", 《中华消化杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN111286536A (en) * | 2020-04-24 | 2020-06-16 | 山东大学齐鲁医院(青岛) | Gene marker for diagnosing myocardial infarction and application thereof |
| CN112760373A (en) * | 2021-03-23 | 2021-05-07 | 山东第一医科大学第二附属医院 | Product for detecting myocardial infarction |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR101211280B1 (en) | Novel markers for diagnosing brain disease caused by brain injury and use thereof | |
| EP2904118B1 (en) | Urine exosome mrnas and methods of using same to detect diabetic nephropathy | |
| WO2015069933A1 (en) | Circulating cell-free dna for diagnosis of transplant rejection | |
| CN105603101B (en) | Detect application of the system of 8 miRNA expression quantity in diagnosis or auxiliary diagnosis of hepatoma product is prepared | |
| US10266895B2 (en) | Exosomes and microvesicles in intestinal luminal fluids and stool and use of same for the assessment of inflammatory bowel disease | |
| CN108796069A (en) | Diagnosis marker-ING1 the genes of myocardial infarction | |
| CN110198711A (en) | Method for detecting cancer | |
| CN108624680B (en) | The application of RAE1 gene or albumen as the biomarker of diagnosing myocardial infarction | |
| CN105349641B (en) | Acute myocardial infarction AMI related gene SERPINB13 and its application | |
| CN114182009A (en) | Application of plasma exosome CircOGDH as acute ischemic stroke diagnostic biomarker | |
| CN103820556B (en) | A kind of pituitary tumor Disease-causing gene CCNB1 and application thereof | |
| CN108796067B (en) | The diagnosis new function of MAEA gene in blood | |
| CN105567862B (en) | CDK18 is preparing the purposes in diagnosis of coronary heart disease product | |
| CN108753956A (en) | Application of the KDSR genes in preparing myocardial infarction diagnosis tool | |
| CN108753957A (en) | The IVD genes of diagnosing myocardial infarction and its application | |
| CN108796068A (en) | DOCK10 genes for early diagnosing myocardial infarction | |
| CA3232274A1 (en) | Drain fluid for diagnostics | |
| CN105969889B (en) | The purposes of MUC3B gene SNP site | |
| CN108048554A (en) | The molecular marker that THBD genes are diagnosed as parkinsonism | |
| EP4036233A1 (en) | Method for detecting brain tumor | |
| CN107475376A (en) | Predict label and kit of the ED-SCLC to chemotherapy drug susceptibility | |
| CN114959001A (en) | Product for diagnosing acute myocardial infarction and application thereof | |
| CN110229891A (en) | The product of non-invasive diagnosis Male Osteoporosis | |
| WO2020007270A1 (en) | Marker and diagnostic method for non-invasive diagnosis of myocardial infarction | |
| CN110988346A (en) | Marker for auxiliary diagnosis of lung cancer and detection method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information |
Address after: Room 1210, Building 3, Ronghua Xintai Building, 10 Ronghua South Road, Yizhuang Economic and Technological Development Zone, Daxing District, Beijing Applicant after: BEIJING MEDINTELL BIOMED Co.,Ltd. Address before: 100080 Beijing city Haidian District Shanyuan Street No. 1 cubic court building room 3103 Applicant before: BEIJING MEDINTELL BIOMED Co.,Ltd. |
|
| CB02 | Change of applicant information | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20191212 Address after: Room 2503, Qianshan building, building D2, Qingdao International Innovation Park Phase II, No. 1, Keyuan Weiyi Road, Laoshan District, Qingdao, Shandong Province Applicant after: Qingdao Yangshen biomedical Co.,Ltd. Address before: Room 1210, Building 3, Ronghua Xintai Building, 10 Ronghua South Road, Yizhuang Economic and Technological Development Zone, Daxing District, Beijing Applicant before: BEIJING MEDINTELL BIOMED Co.,Ltd. |
|
| TA01 | Transfer of patent application right | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20181113 |
|
| RJ01 | Rejection of invention patent application after publication |