CN106048005A - Molecular marker for diagnosing coronary heart disease - Google Patents
Molecular marker for diagnosing coronary heart disease Download PDFInfo
- Publication number
- CN106048005A CN106048005A CN201610382160.6A CN201610382160A CN106048005A CN 106048005 A CN106048005 A CN 106048005A CN 201610382160 A CN201610382160 A CN 201610382160A CN 106048005 A CN106048005 A CN 106048005A
- Authority
- CN
- China
- Prior art keywords
- fam174b
- gene
- heart disease
- coronary heart
- albumen
- 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.)
- Granted
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
-
- 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
-
- 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
- C12Q2600/118—Prognosis of disease development
-
- 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
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Pathology (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- General Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an FAM174B gene capable of serving as a molecular marker for early diagnosis of the coronary heart disease. It is found that a significant difference exists between expression of the FAM174B gene in blood of a normal person and expression of the FAM174B gene in blood of a patient suffering from the coronary heart disease, in other words, whether a subject suffers from the coronary heart disease or not can be judged by detecting expression of the FAM174B gene in blood. The invention further discloses a reagent kit for diagnosing the coronary heart disease according to the correlation between expression of the FAM174B gene and the coronary heart disease. The reagent kit is used for diagnosing the coronary heart disease by detecting expression of the FAM174B gene. The reagent kit can be used for early diagnosis of diseases and has broad clinical application prospects.
Description
Technical field
The present invention relates to diagnosis of coronary heart disease, prediction prognosis field, more particularly it relates to different with detection FAM174B
It is often the diagnosis of coronary heart disease of means, prediction method of prognosis.
Background technology
Coronary heart disease becomes one of principal disease affecting human health with high fatality rate and disability rate, accounts for all fatal
The 40% of sexual behavior part.And the number of annual worldwide endogenous cause of ill cardiovascular disease death increases year by year.According to statistics, flourishing state
500 ten thousand to the year two thousand thirties from 2000 will be increased to 60,000,000 by family's cardiovascular death number.Rapidly send out along with economy in China
Opening up, living standards of the people are greatly improved, and resident living mode changes, and the incidence and mortality of cardiovascular disease increases the most year by year
High.Showing according to " China cardiovascular diseases report 2013 " survey result, the ill rate of Chinese urban and rural residents cardiovascular diseases becomes in rising
Gesture, mortality rate remains high, and the whole nation there are about 3,500,000 people every year and dies from cardiovascular diseases.Therefore, to the cardiovascular headed by coronary heart disease
The early screening of disease, system diagnosis and treatment and related basic research become the great public health problem that current China faces.
People study and find that coronary heart disease is that many factors acts on different a kind of complex disease caused by link for many years.Remove
The conventional risk factors such as the age of early discovery, sex, dyslipidemia, obesity, hypertension, diabetes, bad life style,
Inherited genetic factors is also the principal element causing incidence of coronary heart disease.There is coronary heart disease, diabetes, hypertension, dyslipidemia family history
Crowd's Incidence of CHD substantially increase.The most clone the susceptible or mutant gene relevant to coronary risk factor
More than 200 kinds.
Summary of the invention
An object of the present invention is that providing a kind of diagnoses hat by detection FAM174B gene or protein expression difference
The method of cardiopathia.
The two of the purpose of the present invention are that providing a kind of predicts hat by detection FAM174B gene or protein expression difference
The method of cardiopathia prognosis.
To achieve these goals, present invention employs following technical scheme:
The invention provides the product of detection FAM174B gene or FAM174B albumen in preparation diagnosis of coronary heart disease instrument
Purposes.
Present invention also offers the product of detection FAM174B gene or FAM174B albumen in preparation prediction coronary heart disease prognosis
Purposes in instrument.
Further, the product of described detection FAM174B gene or FAM174B albumen include detect FAM174B gene or
The product of the expression of FAM174B albumen.Described product includes can be in conjunction with the nucleic acid of FAM174B gene or can be in conjunction with
The material (such as antibody) of FAM174B albumen.Described nucleic acid can detect the expression of FAM174B gene；Described material energy
Enough detect the expression of FAM174B albumen.
The product of the detection FAM174B gene of the present invention can play its merit based on the known method using nucleic acid molecules
Can: such as PCR, such as Southern hybridization, Northern hybridization, dot blot, fluorescence in situ hybridization (FISH), DNA microarray, ASO
Method, high-flux sequence platform etc..This product is used can qualitatively, quantitatively or semi-quantitatively to implement to analyze.
The nucleic acid being included in the said goods can be obtained by chemosynthesis, or by containing from biomaterial preparation
Expect the gene of nucleic acid, then use be designed for amplification expectation nucleic acid primer amplification it obtain.
Further, described PCR method is known method, such as, and ARMS (Amplification Refractory
Mutation System, abruptly-changing system is not answered in amplification) method, RT-PCR (reverse transcriptase-PCR) method, nested PCR method etc..Amplification
Nucleic acid can by use dot blotting hybridization method, Surface Plasmon Resonance (SPR method), PCR-RFLP method, in situ RT-PCR
Method, PCR-SSO (sequence specific oligonucleotide) method, PCR-SSP method, AMPFLP (amplifiable fragment length polymorphism) method,
MVR-PCR method and PCR-SSCP (single strand conformation polymorphism) method detect.
Nucleic acid recited above includes the primer expanding FAM174B gene, and the primer that product includes can pass through
Prepared by chemosynthesis, the method that be those skilled in the art will know that by use is suitably designed with reference to Given information, and leads to
Cross chemosynthesis to prepare.
In specific embodiments of the present invention, described nucleic acid is the amplimer used in QPCR experiment, described primer
Sequence such as SEQ ID NO.1 (forward sequence) and SEQ ID NO.2 (reverse sequence) shown in.
Nucleic acid recited above may also include probe, and described probe can be prepared by chemosynthesis, by using this
The method that skilled person knows appropriately designs with reference to Given information, and is prepared by chemosynthesis, or can lead to
Cross the gene containing expectation nucleotide sequence from biomaterial preparation, and use the primer expansion being designed for amplification expectation nucleotide sequence
Increase it to prepare.
The product of the detection FAM174B albumen of the present invention can play its function based on the known method using antibody: example
As, ELISA, radioimmunoassay, immunohistochemical method, western blot etc. can be included.
The product of the detection FAM174B albumen of the present invention includes antibody or its fragment of specific binding FAM174B albumen.
Antibody or its fragment of any structure, size, immunoglobulin class, origin etc. can be used, as long as it combines target protein
?.Antibody or its fragment that the detection product of the present invention includes can be monoclonal or polyclonal.Antibody fragment refers to
Retain antibody to an antibody part (Partial Fragment) of the combination activity of antigen or the peptide containing an antibody part.Antibody fragment can
To include F (ab ')2, Fab ', Fab, scFv (scFv), disulphide bonding Fv (dsFv) or its polymer, dimerization V
District's (double antibody) or the peptide containing CDR.The product of the detection FAM174B albumen of the present invention can include encoding antibody or coding
The nucleic acid of the separation of the aminoacid sequence of antibody fragment, comprises the carrier of this nucleic acid, and carries the cell of this carrier.
Antibody can be by well known to a person skilled in the art that method obtains.Such as, preparation retains target all or in part
The polypeptide of protein or the mammalian cell expression vector of integration their polynucleotide of coding are as antigen.Use antigen is exempted from
After epidemic disease animal, from passing through immune animal adaptive immune cell fused bone myeloma cells to obtain hybridoma.Then from hybridization
Antibody collected by tumor culture.FAM174B albumen or its part antibody to obtaining of antigen finally can be used as by use
Implement antigenic specificity purification and obtain the monoclonal antibody for FAM174B albumen.Polyclonal antibody can be prepared as follows: use
Antigen-immunized animal same as above, collects blood sample from the animal through immunity, isolates serum, then from blood
Use above-mentioned antigen that serum is implemented antigenic specificity purification.By the antibody obtained with ferment treatment or can be obtained by use
The sequence information of antibody obtain antibody fragment.
The combination of label and antibody or its fragment can be implemented by method as commonly known in the art.Such as, may be used
With following fluorescent marker protein or peptide: clean protein or peptide with phosphate buffer, add with DMSO, buffer agent, etc. standard
Standby dyestuff, then mixed solution, place 10 minutes then at room temperature.It addition, labelling can the labelling kit of commodity in use, all
Such as biotin labeling reagent box, such as biotin labeling reagent box-NH2, biotin labeling reagent box-SH
(DojindoLaboratories)；Alkali phosphatase enzyme mark test kit such as alkali phosphatase enzyme mark test kit-NH2, alkalescence phosphorus
Acid enzyme labelling test kit-SH (Dojindo Laboratories)；Peroxidase labelling test kit such as peroxidase mark
Note test kit-NH2, peroxidase labelling test kit-NH2 (Dojindo Laboratories)；Phycobniliprotein labelled reagent
Box such as phycobniliprotein labelling kit-NH2, phycobniliprotein labelling kit-SH, B-phycoerythrin labelling kit-NH2,
B-phycoerythrin labelling kit-SH, R-PE labelling kit-NH2, R-PE labelling kit SH
(DojindoLaboratories)；Fluorescent labeling reagent box such as fluorescein labelling kit-NH2, HiLyte Fluor (TM)
555 labelling kit-NH2, HiLyte Fluor (TM) 647 labelling kit-NH2 (Dojindo Laboratories)；And
DyLight 547 and DyLight647 (Techno Chemical Corp.), Zenon (TM), Alexa Fluor (TM) antibody
Labelling kit, Qdot (TM) antibody labeling test kit (InvitrogenCorporation) and EZ-label protein labeling
Test kit (Funakoshi Corporation).For correct labeling, it is possible to use suitable instrument detects through labelling
Antibody or its fragment.
In the present invention, " prognosis " refer to that patients with coronary heart disease is by the suppression such as surgical procedure or the mistake after alleviating coronary heart disease
Journey or result.In this manual, prognosis can be suppressed or alleviate after coronary heart disease 1 by surgical procedure, 2,3,4,5,6,7,
8,9,10,15,20 years or more long time life state.Prognosis can be by checking biomarker i.e. FAM174B albumen or volume
The gene of code FAM174B albumen is predicted.Prognosis prediction can be performed such that according to biomarker with or without, or rise
Height or reduction, determine that the prognosis of patient is good or bad, or determine the probability of good prognosis or poor prognosis.
In the present invention, " prognosis bona " refers to after being suppressed by surgical procedure etc. for patient or alleviate coronary heart disease,
Patient (such as 3,5,6,7,8,9,10,15,20 years or longer) over a long time does not has critical condition.
In the present invention, " prognosis mala " refer to patient by the suppression such as surgical procedure or after alleviating coronary heart disease in short-term
Fatal condition is there is in phase (such as 1,2,3,4,5 years or shorter).
Prediction prognosis refers to predict process or the result of status of patient, is not meant to predict with the accuracy of 100%
The process of status of patient or result.Prediction prognosis refers to whether the probability determining some process or result increases, and the most unexpectedly
Taste to be compared by situation about not occurring with some process or result and is determined some process of generation or the probability of result.Such as this
For invention, in the patient that in the present invention, the level of FAM174B gene or FAM174B albumen is raised and lowered, and do not show this
The patient of feature compares, and more likely observes particular procedure or result.
Further, the product of described detection FAM174B gene or FAM174B albumen can be detection FAM174B gene or
The reagent of FAM174B albumen, can also be to comprise the test kit of described reagent, chip, reagent paper etc., it is also possible to be to use described examination
The high-flux sequence platform of agent.
Utilize the FAM174B gene in foregoing detection Product checking experimenter's sample or the table of FAM174B albumen
Reaching level, compared with normal person, FAM174B gene or the expression of FAM174B albumen in experimenter's sample reduce, then examine
This experimenter disconnected is the poor prognosis of patients with coronary heart disease or this experimenter.
Sample as the detection product according to the present invention, it is possible to use the tissue sample such as obtained from biopsy experimenter
Or fluid.Sample is not particularly limited, as long as it is suitable to the mensuration of the present invention；Such as, it can include tissue, blood, blood plasma,
Serum, lymph fluid, urine, serous cavity liquid, spinal fluid, synovial fluid, aqueous humor, tear, saliva or its fraction or treated material
Material.
In specific embodiments of the present invention, described sample is from the blood of experimenter.
Present invention also offers the instrument of a kind of diagnosis of coronary heart disease, described instrument can detect in experimenter's sample
FAM174B gene or the expression of FAM174B albumen.Described instrument include can in conjunction with the nucleic acid of FAM174B gene or
Can be in conjunction with the material (such as antibody) of FAM174B albumen.Described nucleic acid can detect the expression of FAM174B gene；Institute
State material and can detect the expression of FAM174B albumen.
Further, the character of described nucleic acid and described material is with noted earlier.
Further, the instrument of described diagnosis of coronary heart disease includes but not limited to chip, test kit, reagent paper or high-flux sequence
Platform；High-flux sequence platform is the instrument of a kind of special diagnosis of coronary heart disease, along with the development of high throughput sequencing technologies, to one
The structure of gene expression profile of individual will become and work the most easily.By contrast Disease and the gene table of normal population
Reaching spectrum, the exception easily analyzing which gene is relevant to disease.Therefore, high-flux sequence is known FAM174B gene
The abnormal purposes that fall within FAM174B gene relevant to coronary heart disease, equally within protection scope of the present invention.
Present invention also offers a kind of instrument predicting coronary heart disease prognosis, described instrument can detect in experimenter's sample
FAM174B gene or the expression of FAM174B albumen, described prediction coronary heart disease prognostic tool includes can be in conjunction with FAM174B
The nucleic acid of gene or can be in conjunction with the material (such as antibody) of FAM174B albumen.Described nucleic acid can detect FAM174B gene
MRNA level in-site；Described material can detect the expression of FAM174B albumen.
Further, the character of described nucleic acid and described material is with noted earlier.
Further, the instrument of described prediction coronary heart disease prognosis includes but not limited to chip, test kit, reagent paper or high flux
Order-checking platform；High-flux sequence platform is the instrument of a kind of special diagnosis of coronary heart disease, along with the development of high throughput sequencing technologies,
The structure of the gene expression profile of one people will be become and work the most easily.By contrast Disease and the base of normal population
Because of express spectra, the exception easily analyzing which gene is relevant to disease.Therefore, high-flux sequence is known FAM174B base
The exception of the cause purposes that fall within FAM174B gene relevant to coronary heart disease, equally within protection scope of the present invention.
The aminoacid that the anti-FAM174B antibody used in detection product, the diagnostic tool of the present invention or its fragment are identified
Number be not particularly limited, as long as antibody can be in conjunction with FAM174B.When antibody is as medicine, preferably
It is capable of identify that aminoacid as much as possible, as long as it can suppress FAM174B function.Antibody or its fragment identification amino acid whose
Number is at least one, more preferably at least three.The immunoglobulin class of antibody is unrestricted, can be IgG, IgM, IgA,
IgE, IgD or IgY.
Further, described experimenter's sample can use the tissue sample or fluid such as obtained from biopsy experimenter.Sample
Originally it is not particularly limited, as long as it is suitable to the mensuration of the present invention；Such as, it can include tissue, blood, blood plasma, serum, lymph
Liquid, urine, serous cavity liquid, spinal fluid, synovial fluid, aqueous humor, tear, saliva or its fraction or treated material.In the present invention
Specific embodiments in, described sample is from the blood of experimenter.
Present invention also offers a kind of diagnosis of coronary heart disease or the method for prediction coronary heart disease prognosis, described method includes walking as follows
Rapid:
(1) sample of experimenter is obtained；
(2) FAM174B gene or the expression of albumen in detection Samples subjects；
(3) the FAM174B gene recorded or the expression of albumen are associated with the whether ill of experimenter.
(4) compared with the control, the expression of FAM174B gene or albumen reduces, then this experimenter is diagnosed as coronary disease
Disease, or this experimenter is confirmed as prognosis mala.
In the context of the present invention, " diagnosis of coronary heart disease " both includes judging that experimenter has suffered from coronary heart disease, also
Including judging whether experimenter exists and suffer from the risk of coronary heart disease.
Advantages of the present invention and beneficial effect:
The molecular marker being found that a kind of diagnosis of coronary heart disease of the present invention, uses this molecular marker can be in coronary heart disease
The early stage occurred can be used as judging, it is provided that the survival rate of patient.
It addition, by the prognosis predicting patient, the present invention can provide significant information to determine treatment side for patient
Case strategy.
Accompanying drawing explanation
Fig. 1 shows the expression utilizing genechip detection FAM174B gene in patients with coronary heart disease with normal person；
Fig. 2 shows the expression utilizing QPCR detection FAM174B gene in patients with coronary heart disease with normal person；
Fig. 3 shows the expression feelings utilizing Western blot detection FAM174B albumen in patients with coronary heart disease with normal person
Condition.
Specific embodiment
The present invention is further detailed explanation with embodiment below in conjunction with the accompanying drawings.Following example are merely to illustrate this
Invention rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in embodiment, generally 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.
The differential expression of embodiment 1 FAM174B gene
1, object of study:
Collect patients with coronary heart disease 5 example, normal person 5 example.
The inclusive criteria of CHD group: according to the diagnostic criteria of the ischemic heart desease that World Health Organization (WHO) formulates, select
Underwent coronary radiography confirms the patients with coronary heart disease having one or more angiostenosis degree >=50%.The inclusion criteria of matched group
For: 1) screen age, sex when Epidemiological study, national all match with CAD group, through questionnaire survey, physical examination,
Cardiac ultrasound and Electrocardioscopy and laboratory inspection do not have the clinical manifestation of coronary heart disease and Major Risk Factors
Examinee；2) the parallel coronary artery revasualization of row health examination of being in hospital gets rid of coronary heart disease age, sex, national and CAD group
Matcher；Meet one person of any of the above to enter to elect matched group as.Informed Consent Form is signed for two groups before including research in.
Rejecting standard: the full person of CAD group-clinical data and merge the one of following disease simultaneously and rejected.
(such as: Congenital Heart patient, dissection of aorta, MOFE, rheumatic heart disease and there is spirit
Obstacle can not the person of cooperation).Matched group: spiritedness obstacle person or check that confirmation has carotid atherosclerotic plaque or narrow person through Doppler, gives
To reject.
2, the extraction of total serum IgE in blood
(1) homogenized (Homogenization)
Requiring object of study at least 12h on an empty stomach, under m seq 7:00～8:00 room temperature, extraction 10ml venous blood is in second
Ethylenediamine tetraacetic acid (EDTA) (EDTA) anticoagulant tube, adds 3 times of volume erythrocyte cracked liquids, room temperature placement 10 minutes after mixing, and 10,000rpm
Centrifugal 1 minute.Thoroughly inhale and abandon supernatant, collect leukocyte cell pellet.The leukocyte cell pellet of every 100-200 μ l blood collecting adds 1ml
TRIzol。
(2) layering (Phase Separation)
A., after sample adds TRIzol, room temperature places 5min, makes sample fully crack.
B. every 1ml TRIzol adds 200 μ l chloroforms, and acutely after vibration mixing, room temperature is placed 3-5min and made its natural split-phase.
(3) RNA precipitate (RNA Precipitation)
A.4 a DEG C 12,000rpm is centrifuged 10-15min.Sample can be divided into three layers: the organic facies of yellow, intermediate layer and colourless
Aqueous phase, RNA, mainly in aqueous phase, transfers to aqueous phase (generally can draw 550 μ l) in new pipe.
B. adding the isopropanol that equal-volume is ice-cold in supernatant, room temperature places 10-20min.4 DEG C of 12,000rpm are centrifuged
10min, abandons supernatant, and RNA precipitate is at the bottom of pipe.
(4) RNA rinsing (RNA Wash)
A.RNA precipitation adds 1ml 75% ethanol (preparing with RNase-free water), gentle vibration centrifuge tube, suspends heavy
Form sediment.Every 1ml TRIzol adds 1ml 75% ethanol.
B.4 DEG C 5,000-8,000rpm is centrifuged 1-2min, abandons supernatant；Of short duration the most centrifugal, carefully inhale with pipettor and abandon
Clearly, room temperature is placed and is dried precipitation in 1-2 minute.
(5) RNA (Redissolving the RNA) is dissolved
Precipitation adds 50-100 μ l RNase-free water, flicks tube wall, fully to dissolve RNA ,-70 DEG C of preservations.
3, RNA mass and purity detecting
RNA mass: represented by RNA integrity, available plain agar sugar gel electrophoresis (deposition condition: 1.2% glue；
0.5 × TBE electrophoretic buffer；150v, 15 minutes) detection integrity.
RNA purity: OD260/OD280 ratio is to weigh the index of protein contamination degree in RNA sample.High-quality
RNA sample, OD260/OD280 value (10mM Tris, pH7.5) is about 2.0.
4, order-checking: above-mentioned RNA is delivered Shanghai Biotechnology Corporation and uses Solexa sequencing technologies to sample
Check order.
5, data analysis
5.1 original data processing
The raw image data that sequenator produces is converted into sequence data through Base Calling, we term it original sequence
Column data, result is with FASTQ stored in file format.Owing to original sequence data may comprise low quality sequence, joint sequence etc.
Contaminating sequences data, it is impossible to be directly used in analysis of biological information, so original sequence data must pass through data process and is converted to
High-quality sequence data, utilizes the spliced mapping algorithm of Tophat (version:2.0.6) software to high-quality sequence
Data carry out genome alignment, and using genome version is Sscrofa10.2.
The screening of 5.2 differential genes
The calculating of gene expression amount makes FPKM method.Expression (FPKM value) according to gene calculates this gene at different samples
Between differential expression multiple.Difference expression gene is defined as FDR≤0.01 and fold difference at 2 times and above gene.
6, result
Result shows (as shown in Figure 1), compared with normal person, and the mRNA water of FAM174B gene in patients with coronary heart disease blood
Putting down and significantly reduce, difference has statistical significance (P < 0.05).
The gene of differential expression in embodiment 2 QPCR experimental verification patients with coronary heart disease and normal person
1, object of study:
Screening criteria is with each 50 examples of embodiment 1, patients with coronary heart disease and normal person.
2, the extraction of total serum IgE in blood
Step is with embodiment 1.
3, reverse transcription
With RT Buffer, l μ g total serum IgE is carried out reverse transcription and synthesize cDNA.Use 25 μ l reaction systems, each sample
Take 1 μ g total serum IgE as template ribonucleic acid, PCR pipe is separately added into following components: DEPC water, 5 × RT Buffer,
10mmol/L dNTP, 0.1mmol/l DTT, 30 μm mol/l Oligo dT, 200U/ μ l M-MLV, template ribonucleic acid.Hatch for 42 DEG C
1h, 72 DEG C of 10min, of short duration centrifugal.
4、QPCR
(1) design of primers
According to the coded sequence design QPCR amplimer of FAM174B gene and GAPDH gene in Genbank, by Shanghai
The synthesis of Sheng Gong biotechnology Services Co., Ltd.Concrete primer sequence is as follows:
FAM174B gene:
Forward primer is 5 '-AGACACGCAAGTATGATA-3 ' (SEQ ID NO.3)；
Reverse primer is 5 '-GAGTCCTCATCTTCATCA-3 ' (SEQ ID NO.4),
GAPDH gene:
Forward primer is 5 '-GAAGGTGAAGGTCGGAGT-3 ' (SEQ ID NO.5)；
Reverse primer is 5 '-CATGGGTGGAATCATATTGGAA-3 ' (SEQ ID NO.6).
(2) PCR reaction system is prepared according to table 1:
Wherein, SYBR Green polymerase chain reaction system is purchased from Invitrogen company.
Table 1 PCR reaction system
| Reagent | Volume |
| Forward primer | 1μl |
| Reverse primer | 1μl |
| SYBR Green polymerase chain reaction system | 12.5μl |
| Template | 2μl |
| Deionized water | Supply 25 μ l |
(3) PCR reaction condition: 95 DEG C of 5min, (95 DEG C of 10s, 60 DEG C of 40s) * 45 circulations.Using SYBR Green as glimmering
Signal thing, is reacted at the Light enterprising performing PCR of Cycler quantitative real time PCR Instrument, is determined by melt curve analysis analysis and electrophoresis
Purpose band, Δ Δ CT method carries out relative quantification.
5, statistical method
Result data is all to represent in the way of mean+SD, uses SPSS13.0 statistical software to unite
Meter is analyzed, and difference between the two uses t inspection, it is believed that when P < has statistical significance when 0.05.
6, result
Result is as in figure 2 it is shown, compared with normal person, in patients with coronary heart disease blood, the mRNA level in-site of FAM174B gene is notable
Reducing, difference has statistical significance (P < 0.05), result is homogenic array experiment.
Embodiment 3 FAM174B protein diversity detection of expression
1, object of study
With embodiment 2.
2, mononuclear cell separates
Aseptic collection venous blood 10ml, injects in the sterile vials containing heparin, shakes up the most gently after adding a cover.Use aseptic suction
Pipe adds isopyknic HBSS (NaCl 8.0g, Na2HPO40.132g, KH2PO40.06g, KCl 0.4g, phenol red 1ml,
NaHCO30.35g, D-Glucose 1.0g, be dissolved in 1000ml distilled water), to reduce erythrocytic cohesion.Draw 8ml lymph thin
Born of the same parents are layered liquid and put in 50ml centrifuge tube, are slowly added to along tube wall by dilute blood, keep interface to understand, do not make both mix mutually,
20 DEG C of 2 000r/min is centrifuged 30min, and careful layering liquid of drawing joins the buffy coat that position is muddy, i.e. lymphocyte with blood plasma
Layer, adds in another centrifuge tube, washs 2 times with the HBSS of 5 times of volumes, successively with 2 000r/min, 1 500r/min in room
The lower centrifugal 10min of temperature, in order to remove the platelet that major part mixes, with 10ml distilled water and cell mass mixing 1min, make residual
Remaining erythrocyte splitting, is then rapidly added equivalent 1.8%NaCl solution, and 2 000r/min are centrifuged, and remove supernatant, after cell counting
Cell is adjusted to 1 × 10 with HBSS solution6Individual/ml is standby.
3, mononuclear cell gross protein extracts
By above-mentioned experiment gained cell suspension, (concentration is 1 × 106Individual/ml) room temperature 1 000r/min is centrifuged 10min, abandons
Add 100 μ l lysis buffers, 4 DEG C of concussion 1h after clear, use ultrasonoscope smudge cells, each 10s, totally 10 times, in 4 DEG C 12
000r/min is centrifuged 1h；Taking supernatant Brandford standard measure albumen, be distributed into 2.5 μ g/ μ l ,-80 DEG C of Refrigerator stores are standby.
4, Western blot detection
The albumen of extraction is carried out SDS-PAGE electrophoresis, carry out afterwards transferring film, closing, one anti-hatch, two anti-hatch, aobvious
Color.
5, statistical procedures
Image J software is used to be analyzed, with β-actin as internal reference, by purpose informal voucher the gray value of protein band
The gray value of band is normalized.Result data is all to represent in the way of mean+SD, uses
SPSS13.0 statistical software carries out statistical analysis, and difference between the two uses t inspection, it is believed that when P < has system when 0.05
Meaning learned by meter.
6, result
Result is as it is shown on figure 3, compared with normal person, in patients with coronary heart disease blood, FAM174B protein content is low, and difference has
Statistical significance (P < 0.05).
The explanation of above-described embodiment is only intended to understand the method for the present invention and core concept thereof.It should be pointed out that, for this
For the those of ordinary skill in field, under the premise without departing from the principles of the invention, it is also possible to the present invention is carried out some improvement
And modification, these improve and modify also by the protection domain falling into the claims in the present invention.
Claims (10)
1. the product of detection FAM174B gene or FAM174B albumen is preparing diagnosis of coronary heart disease or the work of prediction coronary heart disease prognosis
Application in tool.
Application the most according to claim 1, it is characterised in that described detection FAM174B gene or the product of FAM174B albumen
Product include the product detecting the expression of FAM174B gene or FAM174B albumen.
Application the most according to claim 2, it is characterised in that utilize in described Product checking experimenter's sample
FAM174B gene or the expression of FAM174B albumen, compared with normal person, FAM174B gene in experimenter's sample or
The expression of FAM174B albumen reduces, then diagnose the poor prognosis that this experimenter is patients with coronary heart disease or this experimenter.
Application the most according to claim 3, it is characterised in that the source of described experimenter's sample is blood.
5. according to the application according to any one of claim 1-4, it is characterised in that described product includes can be in conjunction with
The nucleic acid of FAM174B gene or can be in conjunction with the material of FAM174B albumen；Described nucleic acid can detect FAM174B gene
Expression；Described material can detect the expression of FAM174B albumen.
Application the most according to claim 5, it is characterised in that described nucleic acid is the special expansion used in real-time quantitative PCR
Increase the primer of FAM174B gene as shown in SEQ ID NO.1 and SEQ ID NO.2.
7. a diagnosis of coronary heart disease or the instrument of prediction coronary heart disease prognosis, it is characterised in that described instrument includes detecting and is subject to
FAM174B gene in examination person's sample or the instrument of the expression of FAM174B albumen.
Instrument the most according to claim 7, it is characterised in that described instrument includes can be in conjunction with the core of FAM174B gene
Acid or can be in conjunction with the material of FAM174B albumen；Described nucleic acid can detect the expression of FAM174B gene；Described thing
Mass-energy enough detects the expression of FAM174B albumen.
Instrument the most according to claim 8, it is characterised in that described nucleic acid is the special expansion used in real-time quantitative PCR
Increase the primer of FAM174B gene as shown in SEQ ID NO.1 and SEQ ID NO.2.
10. according to the instrument according to any one of claim 7-9, it is characterised in that the source of described experimenter's sample is blood
Liquid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610382160.6A CN106048005B (en) | 2016-06-01 | 2016-06-01 | A kind of molecular marker of diagnosis of coronary heart disease |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610382160.6A CN106048005B (en) | 2016-06-01 | 2016-06-01 | A kind of molecular marker of diagnosis of coronary heart disease |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106048005A true CN106048005A (en) | 2016-10-26 |
| CN106048005B CN106048005B (en) | 2019-10-11 |
Family
ID=57171874
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610382160.6A Active CN106048005B (en) | 2016-06-01 | 2016-06-01 | A kind of molecular marker of diagnosis of coronary heart disease |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106048005B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110241208A (en) * | 2019-08-02 | 2019-09-17 | 辽宁中医药大学 | Application of the TREM2 as the molecular marker of early diagnosis coronary heart disease |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105543408A (en) * | 2016-03-16 | 2016-05-04 | 宫蕊 | Marker for early diagnosis of coronary heart disease |
| CN105567861A (en) * | 2016-03-16 | 2016-05-11 | 宫蕊 | Application of IFI27 as coronary heart disease diagnosis marker |
-
2016
- 2016-06-01 CN CN201610382160.6A patent/CN106048005B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105543408A (en) * | 2016-03-16 | 2016-05-04 | 宫蕊 | Marker for early diagnosis of coronary heart disease |
| CN105567861A (en) * | 2016-03-16 | 2016-05-11 | 宫蕊 | Application of IFI27 as coronary heart disease diagnosis marker |
Non-Patent Citations (1)
| Title |
|---|
| KARI A. SARAHAN.ETC: "Four out of eight genes in a mouse chromosome 7 congenic donor region are candidate obesity genes", 《PHYSIOL GENOMICS》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110241208A (en) * | 2019-08-02 | 2019-09-17 | 辽宁中医药大学 | Application of the TREM2 as the molecular marker of early diagnosis coronary heart disease |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106048005B (en) | 2019-10-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107209184B (en) | Marker combinations for diagnosing multiple infections and methods of use thereof | |
| CN105907870A (en) | Use of DHX36 as coronary heart disease diagnostic marker | |
| JP2016526888A (en) | Sepsis biomarkers and their use | |
| CN105567861B (en) | Purposes of the IFI27 as diagnosis of coronary heart disease marker | |
| CN107022637A (en) | A kind of gene marker related to major depressive disorder | |
| CN109097462B (en) | Application of AP1M2 gene as molecular marker for diagnosing glaucoma | |
| CN105821152B (en) | Coronary heart disease biomarker | |
| CN107227358A (en) | Purposes of the NUMB in postmenopausal women's diagnosis of primary osteoporosis or prognosis | |
| CN115948544B (en) | Use of CITED4 and/or METRN in differential diagnosis of the degree of disc degeneration | |
| CN108303547A (en) | A kind of molecular marker for diagnosing chronic obstructive disease of lung | |
| CN106048005B (en) | A kind of molecular marker of diagnosis of coronary heart disease | |
| CN114959005B (en) | Hypertrophic cardiomyopathy diagnosis product based on molecular marker and application thereof | |
| CN110656169B (en) | Diagnostic markers for atrial fibrillation | |
| CN106048006B (en) | SMCHD1 is preparing the application in diagnosis of coronary heart disease product | |
| CN105907871B (en) | Biomarker for diagnosis of coronary heart disease | |
| CN105950745A (en) | NFIL3 serving as diagnostic marker of coronary heart disease | |
| CN113631723B (en) | Application of tuberculosis marker in tuberculosis diagnosis and efficacy evaluation | |
| CN107022633A (en) | Gene marker for diagnosing ankylosing spondylitis | |
| CN107164551A (en) | Purposes of the UBAP2L in type I diabetes of children diagnosis or prognosis | |
| CN106520970B (en) | Marker for diagnosing cerebral apoplexy | |
| CN107012236A (en) | SKIL as diagnosis of coronary heart disease molecular marker | |
| CN110184358A (en) | The OIT3 gene of thyroid cancer early diagnosis and its application | |
| CN110229903A (en) | Molecular marker of the PODN as Diagnosis of Thyroid Carcinoma | |
| CN108559775A (en) | Dengue fever and dengue hemorrhagic fever diagnose diacritics object | |
| CN107058566A (en) | LSMEM1 genes as ankylosing spondylitis diagnostic tool |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information | ||
| 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 Yang Shen biology information technology company limited Address before: 100080 Beijing city Haidian District Shanyuan Street No. 1 cubic court building room 3103 Applicant before: Beijing Yang Shen biology information technology company limited |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20191219 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: Room 1210, Building 3, Ronghua Xintai Building, 10 Ronghua South Road, Yizhuang Economic and Technological Development Zone, Daxing District, Beijing Patentee before: Beijing Yang Shen biology information technology company limited |