CN106048006B - SMCHD1 is preparing the application in diagnosis of coronary heart disease product - Google Patents
SMCHD1 is preparing the application in diagnosis of coronary heart disease product Download PDFInfo
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Abstract
The invention discloses the molecular markers that SMCHD1 gene and its coding albumen can be used as diagnosis of coronary heart disease.May determine that whether subject suffers from coronary heart disease or diagnose subject by the content of SMCHD1 gene and its expression product in detection subject's blood whether there is the risk with coronary heart disease.Research achievement of the invention is clinically to provide a kind of noninvasive, special, sensitive diagnosis of coronary heart disease method.
Description
Technical field
The present invention relates to diagnosiss of coronary heart disease, prediction prognosis field, more particularly it relates to detect SMCHD1 exception
Diagnosis of coronary heart disease, prediction method of prognosis for means.
Background technique
Coronary atherosclerotic heart disease (abbreviation coronary heart disease) is one of the most common type in heart disease, cardiovascular disease
Disease is one of the major disease that the whole world leads to patient's death, second is occupied in numerous lethal causes of disease, disease incidence is about 319/
100000.In disease registration and classification system of the China due to lacking standard, so being unable to get the stream of very accurate Chinese
Row disease learns data.Since 1980, the country began to use the register system of international standard.Dead people in 1 year 2004
It counts and has accounted for the 9% of urban population death, the 22% of cardiovascular disease death toll；Rural area corresponding data is respectively 4% He
13%.The same year has 400,000 patients with coronary artery disease dead, 650,000 new diagnosed case.In world wide, every year nearly 2,500,000
Patient dies of coronary heart disease related disease.With the continuous accurate and urbanization process of statistics, Chinese patients with coronary artery disease is just
More and more.It is expected that cardiovascular disease death toll to the year two thousand twenty can E to it is all death people one third.Problem is more next
It is severeer.
The pathogenesis of coronary heart disease is sufficiently complex, and existing environmental factor also has hereditary feature, it is numerous perspective or return
It is found on the basis of the analysis and research of Gu property, hypertension, smoking, obesity (high cholesterol and diabetes and coronary heart disease in Chinese
Correlation highest is not only because the risk factor that these factors are, more importantly hypertension, smoking and overweight
In China Modernization Process process Chinese due to the most important change of lifestyle change bring.In the research that the sixth of the twelve Earthly Branches has
Main pathogenic verified, that Abnormal Lipid Metabolism, smoking, diabetes and hypertension are, accounts for about in morbidity's factor
Left and right.In addition, being exactly that genetic factors influence.In recent years, molecular diagnosis becomes research hotspot, because molecular diagnosis can be in disease
The early stage of disease can provide the risk probability with coronary heart disease as diagnosis or for subject to patient, to remind risky person
It takes appropriate measures and prevents the generation of coronary heart disease.Therefore, a kind of new specific and high coronary heart disease molecule of sensitivity is found
Diagnosis marker is research emphasis of the invention.
Summary of the invention
One of the objects of the present invention is to provide one kind, and hat is diagnosed by detection SMCHD1 gene or protein expression difference
The method of heart trouble.
The second object of the present invention is to provide one kind by detection SMCHD1 gene or protein expression difference to predict to be preced with
The method of heart trouble prognosis.
To achieve the goals above, present invention employs following technical solutions:
The present invention provides the products of detection SMCHD1 gene or SMCHD1 albumen in preparation diagnosis of coronary heart disease tool
Purposes.
The present invention also provides the products of detection SMCHD1 gene or SMCHD1 albumen to predict coronary heart disease prognosis work in preparation
Purposes in tool.
Further, the product of the detection SMCHD1 gene or SMCHD1 albumen includes detection SMCHD1 gene or SMCHD1
The product of the expression of albumen.The product includes that can combine the nucleic acid of SMCHD1 gene or can combine SMCHD1 egg
White substance (such as antibody).The nucleic acid is able to detect the expression of SMCHD1 gene；The substance is able to detect
The expression of SMCHD1 albumen.
The product of detection SMCHD1 gene of the invention can play its function based on the known method of nucleic acid molecules is used:
As PCR, such as Southern hybridization, Northern hybridization, dot blot, fluorescence in situ hybridization (FISH), DNA microarray, ASO method,
High-flux sequence platform etc..It can qualitatively, quantitatively or semi-quantitatively implement analysis using the product.
Include that nucleic acid in the said goods can be obtained by chemical synthesis, or by containing from biomaterial preparation
It is expected that the gene of nucleic acid, then using primer amplification designed for amplification expectation nucleic acid, it is obtained.
Further, the PCR method is known method, for example, ARMS (Amplification Refractory
Abruptly-changing system is not answered in Mutation System, amplification) method, RT-PCR (reverse transcriptase-PCR) method, nesting PCR method etc..Amplification
Nucleic acid can be by using dot blotting hybridization method, Surface Plasmon Resonance (SPR method), PCR-RFLP method, original position 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 for expanding SMCHD1 gene, and the primer for including in product can be by passing through
Synthesis is learned to prepare, by using those skilled in the art will know that method be suitably designed with reference to Given information, and pass through
It is prepared by chemical synthesis.
In specific embodiments of the present invention, the nucleic acid is amplimer used in QPCR experiment, the primer
Sequence such as SEQ ID NO.1 (positive sequence) and SEQ ID NO.2 (reverse sequence) shown in.
Nucleic acid recited above may also include probe, and the probe can be prepared by chemical synthesis, by using this
The method that field technical staff knows appropriately is designed with reference to Given information, and is prepared by chemical synthesis, or can lead to
The gene for containing desired nucleic acid sequence from biomaterial preparation is crossed, and is expanded using the primer designed for amplification expectation nucleic acid sequence
Increase it to prepare.
The product of detection SMCHD1 albumen of the invention can play its function: example based on the known method of antibody is used
It such as, may include ELISA, radioimmunoassay, immunohistochemical method, western blot etc..
The product of detection SMCHD1 albumen of the invention includes the antibody or its segment for specifically binding SMCHD1 albumen.It can
To use the antibody or its segment of any structure and size, immunoglobulin class, origin etc., as long as it is in conjunction with target protein
It can.The antibody or its segment for including in testing product of the invention can be monoclonal or polyclonal.Antibody fragment refers to guarantor
Stay peptide of the antibody to the active antibody of the combination of antigen a part of (Partial Fragment) or containing antibody a part.Antibody fragment can be with
Including F (ab ')2, Fab ', Fab, scFv (scFv), disulphide bonding Fv (dsFv) or its polymer, the area dimerization V
(double antibody) or peptide containing CDR.The product of detection SMCHD1 albumen of the invention may include encoding antibody or encoding antibody
The isolated nucleic acid of the amino acid sequence of segment, the carrier comprising the nucleic acid, and carry the cell of the carrier.
Antibody can be obtained by the way that well known to a person skilled in the art methods.For example, preparation retains target all or in part
The mammalian cell expression vector of their polynucleotides of the polypeptide or integration coding of protein is as antigen.Exempted from using antigen
After epidemic disease animal, from the immune animal adaptive immune cell of process and myeloma cell is merged to obtain hybridoma.Then from hybridization
Tumor culture collects antibody.It finally can be real by using antibody of the SMCHD1 albumen for being used as antigen or part thereof to acquisition
Antigentic specificity purifying is applied to obtain the monoclonal antibody for SMCHD1 albumen.Polyclonal antibody can be prepared as follows: with
Identical antigen-immunized animal above collects blood sample from by immune animal, serum is isolated from blood, is then made
Implement antigentic specificity to serum with above-mentioned antigen to purify.It can be by the antibody that is obtained with enzymatic treatment or by using acquisition
The sequence information of antibody obtains antibody fragment.
The combination of marker and antibody or its segment can be implemented by method as commonly known in the art.For example, can
With following fluorescent marker protein or peptide: clean protein or peptide with phosphate buffer, addition DMSO, buffer, etc. standards
Standby dyestuff, then mixed solution, then at being placed at room temperature for 10 minutes.In addition, the labelling kit of commercialization can be used in label, it is all
Such as biotin labeling reagent box, such as biotin labeling reagent box-NH2, biotin labeling reagent box-SH
(DojindoLaboratories)；Alkali phosphatase enzyme mark kit such as alkali phosphatase enzyme mark kit-NH2, alkaline phosphorus
Sour enzyme labelling kit-SH (Dojindo Laboratories)；Peroxidase labelling kit such as peroxidase mark
Remember kit-NH2, peroxidase labelling 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-NH2,647 labelling kit-NH2 of HiLyte Fluor (TM) (Dojindo Laboratories)；And
DyLight 547 and DyLight647 (Techno Chemical Corp.), Zenon (TM), Alexa Fluor (TM) antibody
Labelling kit, Qdot (TM) antibody labeling kit (Invitrogen Corporation) and EZ- marker protein mark
Remember kit (Funakoshi Corporation).For correct labeling, suitable instrument can be used to detect by label
Antibody or its segment.
In the present invention, " prognosis " refers to mistake of the patients with coronary heart disease after inhibiting by surgical procedure etc. or alleviating coronary heart disease
Journey or result.In the present specification, prognosis can be by surgical procedure inhibit or alleviate coronary heart disease after 1,2,3,4,5,6,7,
8,9,10,15,20 years or more long when life state.Prognosis can be by checking biomarker, that is, SMCHD1 albumen or coding
The gene of SMCHD1 albumen is predicted.Prognosis prediction can be performed such that according to biomarker with or without, or increase or
It reduces, determines 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 inhibiting for patient by surgical procedure etc. or alleviating coronary heart disease,
Patient's long-term (such as 3,5,6,7,8,9,10,15,20 years or longer) does not have critical condition.The most preferred state of prognosis bona
It is survival for a long time without disease.
In the present invention, " prognosis mala " refers to patient after inhibiting or alleviating coronary heart disease by surgical procedure etc. in short-term
Fatal condition occurs in phase (such as 1,2,3,4,5 year or shorter).
Prediction prognosis is referred to the process of prediction status of patient or as a result, is not meant to be predicted with 100% accuracy
The process or result of status of patient.Prediction prognosis refers to whether a possibility that determining certain processes or result increases, and simultaneously unexpectedly
Taste by determining a possibility that certain processes or result occurs or not compared with certain processes or result.Such as this
For invention, in the present invention in the patient that is raised and lowered of the level of SMCHD1 gene or SMCHD1 albumen, and the spy is not shown
The patient of sign compares, and more likely observes particular procedure or result.
Further, it is described detection SMCHD1 gene or SMCHD1 albumen product can be detection SMCHD1 gene or
The reagent of SMCHD1 albumen is also possible to include kit, chip, test paper of the reagent etc., is also possible to using the examination
The high-flux sequence platform of agent.
Utilize the expression of SMCHD1 gene or SMCHD1 albumen in mentioned-above testing product detection subject's sample
Level is compared with normal people, and the expression of SMCHD1 gene or SMCHD1 albumen in subject's sample increases, then diagnosis should
Subject is patients with coronary heart disease or the poor prognosis of the subject.
As the sample according to testing product of the invention, the tissue sample for example obtained from biopsy subject can be used
Or fluid.Sample is not particularly limited, as long as it is suitable for measurement of the invention；For example, it may include tissue, blood, blood plasma,
Serum, lymph, urine, serous cavity liquid, spinal fluid, synovia, aqueous humor, tear, saliva or its fraction or treated material
Material.
In specific embodiments of the present invention, blood of the sample from subject.
The present invention also provides a kind of tool of diagnosis of coronary heart disease, the tool is able to detect SMCHD1 in subject's sample
The expression of gene or SMCHD1 albumen.The tool includes that can combine the nucleic acid of SMCHD1 gene or can combine
The substance (such as antibody) of SMCHD1 albumen.The nucleic acid is able to detect the expression of SMCHD1 gene；The substance can
Detect the expression of SMCHD1 albumen.
Further, the property of the nucleic acid and the substance is the same as noted earlier.
Further, the tool of the diagnosis of coronary heart disease includes but is not limited to chip, kit, test paper or high-flux sequence
Platform；High-flux sequence platform is a kind of tool of special diagnosis of coronary heart disease, with the development of high throughput sequencing technologies, to one
The building of personal gene expression profile will become very easily work.By the gene table for comparing Disease and normal population
Up to spectrum, the exception for being easy to analyze which gene is related to disease.Therefore, the different of SMCHD1 gene is known in high-flux sequence
The often purposes for also belonging to SMCHD1 gene related to coronary heart disease, equally within protection scope of the present invention.
The present invention also provides a kind of tool for predicting coronary heart disease prognosis, the tool is able to detect in subject's sample
The expression of SMCHD1 gene or SMCHD1 albumen, the prediction coronary heart disease prognostic tool include that can combine SMCHD1 base
The nucleic acid of cause can be in conjunction with the substance (such as antibody) of SMCHD1 albumen.The nucleic acid is able to detect SMCHD1 gene
MRNA level in-site；The substance is able to detect the expression of SMCHD1 albumen.
Further, the property of the nucleic acid and the substance is the same as noted earlier.
Further, the tool of the prediction coronary heart disease prognosis includes but is not limited to chip, kit, test paper or high throughput
Microarray dataset；High-flux sequence platform is a kind of tool of special diagnosis of coronary heart disease, with the development of high throughput sequencing technologies,
Very easily work will be become to the building of the gene expression profile of a people.By the base for comparing Disease and normal population
Because of express spectra, the exception for being easy to analyze which gene is related to disease.Therefore, SMCHD1 gene is known in high-flux sequence
The exception purposes for also belonging to SMCHD1 gene related to coronary heart disease, equally within protection scope of the present invention.
The amino acid that anti-SMCHD1 antibody used in testing product of the invention, diagnostic tool or its segment are identified
Number is not particularly limited, as long as antibody can combine SMCHD1.When antibody is as therapeutic agent, preferably its energy
Amino acid as much as possible is enough identified, as long as it can inhibit SMCHD1 function.The number of antibody or the amino acid of its segment identification
It 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.
Other properties of anti-SMCHD1 antibody used in testing product of the invention, diagnostic tool are the same as noted earlier.
Further, the tissue sample or fluid for example obtained from biopsy subject can be used in subject's sample.Sample
Originally it is not particularly limited, as long as it is suitable for measurement of the invention；For example, it may include tissue, blood, blood plasma, serum, lymph
Liquid, urine, serous cavity liquid, spinal fluid, synovia, aqueous humor, tear, saliva or its fraction or treated material.In the present invention
Specific embodiment in, blood of the sample from subject.
The present invention also provides a kind of diagnosis of coronary heart disease or the methods for predicting coronary heart disease prognosis, and the method includes walking as follows
It is rapid:
(1) sample of coronary disease disease subject is obtained；
(2) expression of SMCHD1 gene or albumen in Samples subjects is detected；
(3) it associates whether by the expression of the SMCHD1 gene or albumen that measure with the illness of subject.
(4) compared with the control, the expression of SMCHD1 gene or albumen increases, then the subject is diagnosed as coronary disease
Disease or the subject are confirmed as prognosis mala.
In the context of the present invention, " diagnosis of coronary heart disease " both include judge subject whether suffered from coronary heart disease or
Including judging that subject whether there is the risk with coronary heart disease.
The advantages of the present invention:
The molecular marker for having found a kind of diagnosis of coronary heart disease of the invention, can be in coronary heart disease using the molecular marker
The early stage of generation can be used as judging, provide the survival rate of patient.
In addition, the present invention is capable of providing significant information to determine treatment side for patient by the prognosis of prediction patient
Case strategy.
Detailed description of the invention
Fig. 1 shows the expression using genechip detection SMCHD1 gene in patients with coronary heart disease and normal person；
Fig. 2 shows the expression using QPCR detection SMCHD1 gene in patients with coronary heart disease and normal person；
Fig. 3 shows the expression feelings using Western blot detection SMCHD1 albumen in patients with coronary heart disease and normal person
Condition.
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.
The differential expression of 1 SMCHD1 gene of embodiment
1, research object:
Collect patients with coronary heart disease 5, normal person 5.
CHD group is included in standard: according to the diagnostic criteria for the ischemic heart disease that the World Health Organization formulates, selection
Underwent coronary radiography confirms the patients with coronary heart disease for having one or more hemadostewnosis degree >=50%.The inclusion criteria of control group
Are as follows: 1) screened in epidemiological survey the age, gender, it is national match with CAD group, by questionnaire survey, physical examination,
Cardiac ultrasound and ECG examination and laboratory check the not clinical manifestation of coronary heart disease and Major Risk Factors
Examinee；2) the parallel coronary artery revasualization of row health examination of being hospitalized excludes coronary heart disease and age, gender, national and CAD group
Matcher；Meet one person of any of the above to enter to be selected as control group.Two groups are signed informed consent form before being included in research.
Rejecting standard: the infull person of CAD group-clinical data and the one for merging following disease simultaneously are rejected.
(such as: Congenital Heart patient, dissection of aorta, multiple organ failure, rheumatic heart disease and have spirit
Obstacle is not able to cooperate person).Control group: having mentally disturbed or checking through Doppler confirms there is carotid plaques or narrow person, gives
To reject.
2, in blood total serum IgE extraction
(1) homogenized (Homogenization)
It is required that at least 12h at room temperature in next morning 7:00~8:00 extracts 10ml venous blood in second to research object on an empty stomach
Ethylenediamine tetraacetic acid (EDTA) (EDTA) anticoagulant tube, is added 3 times of volume erythrocyte cracked liquids, and 10 minutes are placed at room temperature for after mixing, and 10,000rpm
Centrifugation 1 minute.It thoroughly inhales and abandons supernatant, collect leukocyte cell pellet.1ml is added in the leukocyte cell pellet of every 100-200 μ l blood collection
TRIzol。
(2) it is layered (Phase Separation)
A. after TRIzol is added in sample, it is placed at room temperature for 5min, cracks sample sufficiently.
B. 200 μ l chloroforms are added in every 1ml TRIzol, and 3-5min is placed at room temperature for after shaking vigorously and mix well makes its natural split-phase.
(3) RNA precipitate (RNA Precipitation)
A.4 DEG C 12,000rpm is centrifuged 10-15min.Sample can be divided into three layers: the organic phase of yellow, middle layer and colourless
Water phase, RNA mainly in water phase, water phase (can usually draw 550 μ l) are transferred in new pipe.
B. isometric ice-cold isopropanol is added in supernatant, is placed at room temperature for 10-20min.4 DEG C of 12,000rpm centrifugations
10min abandons supernatant, and RNA precipitate is in tube bottom.
(4) RNA rinses (RNA Wash)
75% ethyl alcohol of 1ml (being prepared with RNase-free water) is added in a.RNA precipitating, mildly vibrates centrifuge tube, it is heavy to suspend
It forms sediment.75% ethyl alcohol of 1ml is added in every 1ml TRIzol.
B.4 DEG C 5,000-8,000rpm is centrifuged 1-2min, abandons supernatant；Of short duration rapid centrifugation is carefully inhaled in abandoning with pipettor
Clearly, 1-2 minutes are placed at room temperature for and dries precipitating.
(5) RNA (Redissolving the RNA) is dissolved
50-100 μ l RNase-free water is added in precipitating, flicks tube wall, sufficiently to dissolve RNA, -70 DEG C of preservations.
3, RNA mass and purity detecting
RNA mass: being indicated by RNA integrality, can use plain agar sugar gel electrophoresis (deposition condition: 1.2% glue；
0.5 × TBE electrophoretic buffer；150v, 15 minutes) detection integrality.
RNA purity: OD260/OD280 ratio is the index for measuring protein contamination degree in RNA sample.High quality
RNA sample, OD260/OD280 value (10mM Tris, pH7.5) is 2.0 or so.
4, it is sequenced: above-mentioned RNA is delivered into Shanghai Biotechnology Corporation using Solexa sequencing technologies to sample
It is sequenced.
5, data are analyzed
5.1 original data processing
The raw image data that sequenator generates is converted into sequence data through Base Calling, and we term it original sequences
Column data, as a result with FASTQ stored in file format.Since original sequence data may include low quality sequence, joint sequence etc.
Contaminating sequences data, cannot be directly used to analysis of biological information, so original sequence data must pass through data processing and be converted to
High quality sequence data, using the spliced mapping algorithm of Tophat (version:2.0.6) software to high quality sequence
Data carry out genome alignment, use genome version for Sscrofa10.2.
The screening of 5.2 differential genes
The calculating of gene expression amount makes FPKM method.The gene is calculated in different samples according to the expression quantity (FPKM value) of gene
Between differential expression multiple.Difference expression gene is defined as FDR≤0.01 and gene of the fold difference at 2 times or more.
6, result
(as shown in Figure 1) as the result is shown, is compared with normal people, the mRNA level in-site of SMCHD1 gene in patients with coronary heart disease blood
Significant to increase, difference has statistical significance (P < 0.05).
The gene of differential expression in 2 QPCR experimental verification patients with coronary heart disease of embodiment and normal person
1, research object:
Screening criteria is with embodiment 1, patients with coronary heart disease and normal person each 50.
2, in blood total serum IgE extraction
Step is the same as embodiment 1.
3, reverse transcription
Reverse transcription synthesis cDNA is carried out to l μ g total serum IgE with RT Buffer.Using 25 μ l reaction systems, each sample
It takes 1 μ g total serum IgE as template ribonucleic acid, following components is separately added into PCR pipe: DEPC water, 5 × RT Buffer,
10mmol/L dNTP, 0.1mmol/l DTT, 30 μm of mol/l Oligo dT, 200U/ μ l M-MLV, template ribonucleic acid.42 DEG C of incubations
1h, 72 DEG C of 10min, of short duration centrifugation.
4、QPCR
(1) design of primers
QPCR amplimer is designed according to the coded sequence of SMCHD1 gene and GAPDH gene in Genbank, is given birth to by Shanghai
The synthesis of work biotechnology Services Co., Ltd.Specific primer sequence is as follows:
SMCHD1 gene:
Forward primer is 5 '-ATTCATTGTCTGCTACTTCTC-3 ' (SEQ ID NO.3)；
Reverse primer is 5 '-TCCTCTTCCATTATCTATCACT-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.
1 PCR reaction system of table
(3) PCR reaction condition: 95 DEG C of 5min, (95 DEG C of 5s, 60 DEG C of 45s) * 45 circulations.Using SYBR Green as glimmering
Signal object carries out PCR reaction on Light Cycler fluorescence quantitative PCR instrument, is determined by melt curve analysis analysis and electrophoresis
Purpose band, Δ Δ CT method carry out relative quantification.
5, statistical method
Result data is indicated in a manner of mean+SD, is united using SPSS13.0 statistical software
Meter analysis, difference between the two is examined using t, it is believed that has statistical significance as P < 0.05.
6, result
As a result as shown in Fig. 2, being compared with normal people, the mRNA level in-site of SMCHD1 gene significantly increases in patients with coronary heart disease blood
Add, difference has statistical significance (P < 0.05), as a result homogenic array experiment.
3 SMCHD1 protein diversity detection of expression of embodiment
1, research object
With embodiment 2.
2, monocyte separates
Aseptic collection venous blood 10ml, injection are contained in the sterile vials of heparin, are gently shaken up immediately after capping.With sterile suction
Isometric HBSS (NaCl 8.0g, Na is added in pipe2HPO40.132g, KH2PO40.06g, KCl 0.4g, phenol red 1ml,
NaHCO30.35g, D-Glucose 1.0g are dissolved in 1000ml distilled water), to reduce the cohesion of red blood cell.It is thin to draw 8ml lymph
Born of the same parents are layered liquid and set in 50ml centrifuge tube, and dilute blood is slowly added to along tube wall, and interface is kept to understand, mix the two mutually,
20 DEG C of 2 000r/min is centrifuged 30min, the careful buffy coat for drawing layering liquid and blood plasma handover position muddiness, i.e. lymphocyte
Layer is added in another centrifuge tube, is washed 2 times with the HBSS of 5 times of volumes, successively with 2 000r/min, 1 500r/min in room
Temperature is lower to be centrifuged 10min, and the blood platelet largely mixed to remove is made residual with 10ml distilled water and cell mass mixing 1min
Remaining erythrocyte splitting, is then rapidly added equivalent 1.8%NaCl solution, and 2 000r/min centrifugation removes supernatant, after cell count
Cell is adjusted to 1 × 10 with HBSS solution6A/ml is spare.
3, monocyte gross protein extracts
By cell suspension obtained by above-mentioned experiment, (concentration is 1 × 106A/ml) room temperature 1 000r/min be centrifuged 10min, in abandoning
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 12
000r/min is centrifuged 1h；Supernatant Brandford standard measure albumen is taken, 2.5 μ g/ μ l are distributed into, -80 DEG C of refrigerators save backup.
4, Western blot is detected
The albumen of extraction is subjected to SDS-PAGE electrophoresis, transferring film is carried out later, closing, primary antibody incubation, secondary antibody incubation, shows
Color.
5, statistical procedures
The gray value of protein band is analyzed using Image J software, 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 software is next for statistical analysis, and difference between the two is examined using t, it is believed that has system as P < 0.05
Meter learns meaning.
6, result
As a result as shown in figure 3, being compared with normal people, SMCHD1 protein content is high in patients with coronary heart disease blood, and difference has
Statistical significance (P < 0.05).
The explanation of above-described embodiment is only intended to understand method and its core concept of the 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 scope of the claims in the present invention.
Claims (10)
1. the product of detection SMCHD1 gene or SMCHD1 albumen is preparing diagnosis of coronary heart disease or is predicting the tool of coronary heart disease prognosis
In application.
2. application according to claim 1, which is characterized in that the product of the detection SMCHD1 gene or SMCHD1 albumen
The product of expression including detection SMCHD1 gene or SMCHD1 albumen.
3. application according to claim 2, which is characterized in that utilize the SMCHD1 in the product testing subject sample
The expression of gene or SMCHD1 albumen, is compared with normal people, SMCHD1 gene or SMCHD1 albumen in subject's sample
Expression increase, then diagnose the poor prognosis that the subject is patients with coronary heart disease or the subject.
4. application according to claim 3, which is characterized in that the source of subject's sample is blood.
5. application described in any one of -4 according to claim 1, which is characterized in that the product includes that can combine SMCHD1
The nucleic acid of gene can be in conjunction with the substance of SMCHD1 albumen；The nucleic acid is able to detect the expression of SMCHD1 gene；
The substance is able to detect the expression of SMCHD1 albumen.
6. application according to claim 5, which is characterized in that the nucleic acid is specifically expanded used in real-time quantitative PCR
Increase the primer of SMCHD1 gene as shown in SEQ ID NO.1 and SEQ ID NO.2.
7. application according to claim 1, which is characterized in that the tool includes being able to detect in subject's sample
The tool of the expression of SMCHD1 gene or SMCHD1 albumen.
8. application according to claim 7, which is characterized in that the tool includes can be in conjunction with the nucleic acid of SMCHD1 gene
Or it can be in conjunction with the substance of SMCHD1 albumen；The nucleic acid is able to detect the expression of SMCHD1 gene；The substance energy
Enough detect the expression of SMCHD1 albumen.
9. application according to claim 8, which is characterized in that the nucleic acid is specifically expanded used in real-time quantitative PCR
Increase the primer of SMCHD1 gene as shown in SEQ ID NO.1 and SEQ ID NO.2.
10. the application according to any one of claim 7-9, which is characterized in that the source of subject's sample is blood
Liquid.
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