CN105349642B - A kind of acute myocardial infarction AMI marker and its application - Google Patents
A kind of acute myocardial infarction AMI marker and its application Download PDFInfo
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Abstract
The present invention relates to a kind of acute myocardial infarction AMI marker and its applications, more particularly relate to RFX2 and its agonist and are preparing the application in acute myocardial infarction AMI diagnosis and treatment drug.Inventor is based on high-flux sequence result and filters out RFX2 gene, molecular biology method verifying, it was confirmed that RFX2 low expression in acute myocardial infarction AMI peripheral blood and cardiac muscular tissue.The present invention provides the potential diagnosis and treatment target spots of acute myocardial infarction AMI, have important clinical value.
Description
Technical field
The present invention relates to biomedicine fields, and in particular to a kind of acute myocardial infarction AMI marker and its application, more specifically
Be related to RFX2 and its agonist preparing the application in acute myocardial infarction AMI diagnosis and treatment drug.
Background technique
Disease incidence of the acute myocardial infarction AMI (AMI) in recent years gradually appears the situation of rising, and tends to rejuvenation,
It is one of the Etiological for leading to human death now.This disease is common in the overweight people of American-European countries, and China comes this in recent years
The trend obviously risen is also presented in the incidence of disease.Main clinical manifestation is to ache after having fierce lasting breastbone when this onste
Bitterly, through rest or buccal nitroglycerin drug after cannot slow down, while with serum cardiac necrosis marker increase and
The differentiation of dynamic ECG.The generation of acute myocardial infarction AMI and Coronary Atherosclerotic Plaque rupture initiation blood vessel blockage are close
Correlation, angiemphraxis cause cardiac muscle occur ischemic and it is downright bad, constitute acute myocardial infarction AMI at this time.The treatment of the disease at present is not
Disconnected progress, makes the prognosis of patient be greatly improved including drug therapy and traumatic treatment method.But still there are many patient's drugs
Therapeutic effect is not good enough, while losing the opportunity of operation interventional therapy again.For such some patients, in addition to conventional drug
Outside equal Palliatives symptomatic treatment, need to seek some new treatment methods.
This research composes variance analysis by the peripheral blood rna expression of AMI, has which gene to take part in AMI to study specifically
Process lays the foundation for the diagnosis of AMI in future early gene.Inventor is to 6 acute myocardial infarction AMI case groups and 6 control group blood
Sample carries out high-flux sequence, carries out genescreen in conjunction with bioinformatics method, selects in the apparent gene of differential expression
The candidate gene RFX2 of low expression out, there is no RFX2 and the relevant reports of acute myocardial infarction AMI in existing research, further, hair
Bright people has carried out Molecular, it was confirmed that RFX2 high expression in acute myocardial infarction AMI group.The present invention provides a kind of new urgency
Property myocardial infarction monitoring therapy target, have important clinical value.
Summary of the invention
The purpose of the present invention is to provide a kind of Diagnosis of Acute Myocardial Infarction preparation, the Diagnosis of Acute Myocardial Infarction preparation
Detect the expression product of RFX2 gene and/or gene.Further, the expression product of the RFX2 gene and/or gene exists
Low expression in acute myocardial infarction AMI tissue or peripheral blood.
Further, the Diagnosis of Acute Myocardial Infarction preparation is using PCR kit for fluorescence quantitative, genetic chip, immune side
Method detects the expression of RFX2 gene in acute myocardial infarction AMI tissue or peripheral blood.Preferably, the PCR kit for fluorescence quantitative
In the primer containing a pair of of specific amplification RFX2 gene；It include miscellaneous with the nucleic acid sequence of RFX2 gene in the genetic chip
The probe of friendship.It is furthermore preferred that the upstream primer containing specific detection RFX2 gene and downstream are drawn in PCR kit for fluorescence quantitative
Object, upstream primer sequence are SEQ ID NO.1, and downstream primer sequence is SEQ ID NO.2.
Further, the diagnostic preparation of the acute myocardial infarction AMI using immunization method detection acute myocardial infarction AMI tissue or
The expression product of RFX2 gene in peripheral blood.Preferably, the immunization method is that ELISA is detected and/or colloidal gold detects.Into one
The ELISA method of step, the detection RFX2 albumen is to use ELISA detection kit.City can be used in antibody in the kit
The RFX2 monoclonal antibody or polyclonal antibody sold.Further, the kit includes: the solid phase load for being coated with RFX2 antibody
Body, enzyme labelled antibody, the substrate of enzyme, protein standard substance, negative controls, dilution, cleaning solution, enzyme reaction terminate liquid etc..
Further, the colloidal gold method of the detection RFX2 albumen is using detection kit, and city can be used in the antibody
The RFX2 monoclonal antibody or polyclonal antibody sold.Further, the gold-immunochromatographyreagent reagent for assay box uses colloid gold immune layer
Analysis technology or colloidal gold percolation.Further, detection zone (T) spray on the gold-immunochromatographyreagent reagent for assay box nitrocellulose filter
Point has anti-RFX2 antibody, quality control region (C) specking to have Immunoglobulin IgG.
The purpose of the present invention is to provide above-mentioned Diagnosis of Acute Myocardial Infarction preparations in preparation Diagnosis of Acute Myocardial Infarction work
Application in tool.
The purpose of the present invention is to provide a kind of preparation for treating acute myocardial infarction AMI, contain promotion RFX2 in the preparation
The reagent or compound of transcription or the expression of gene.Further, the expression product of the RFX2 gene and/or gene is in urgency
Low expression in property myocardial infarction tissue or peripheral blood.
Those skilled in the art are known to promote the expression of gene and its expression product usually can be using one of following methods
And/or several: the promoter of activating molecules marker, the albumen of activating molecules marker expression or the factor import promotion molecule
The carrier of marker transcription or expression.
Preferably, the carrier containing transcription or the expression for promoting RFX2 gene in the preparation of the treatment acute myocardial infarction AMI
And/or it activates the promoter of RFX2 gene and/or activates the albumen or the factor of RFX2 gene expression.
The purpose of the present invention is to provide the preparations of above-mentioned treatment acute myocardial infarction AMI to prepare acute myocardial infarction
Application in drug or reagent.
To achieve the above object, the present invention passes through high-flux sequence combination bioinformatics method first and screens candidate base
The relationship of RFX2 gene and acute myocardial infarction AMI: RFX2 gene is demonstrated because of RFX2 gene, and then by molecular biology method
There is good correlation with acute myocardial infarction AMI, can be used for preparing acute myocardial infarction AMI auxiliary diagnosis preparation, have important
Clinical value.
Fluorescence quantitative PCR method is that PCR product is marked by the probe of the specificity of fluorescent dye or fluorescent marker
Tracking, real-time online monitoring reaction process can analyze product in conjunction with corresponding software, calculate sample to be tested template
Initial concentration.The appearance of quantitative fluorescent PCR greatly simplifies the process of quantitative detection, and is truly realized absolute quantitation.
The appearance of a variety of detection systems keeps the selectivity of experiment stronger.Automatic operation improves work efficiency, rapid reaction, repetition
The good, high sensitivity of property, high specificity, result are clear.
Genetic chip is also known as DNA microarray (DNA microarray), can be divided into three kinds of main Types: 1) be fixed on poly-
The nucleic acid probe or cDNA segment on object substrate (nylon membrane, nitrocellulose membrane etc.) surface are closed, the target of isotope labelling is usually used
Gene is hybrid with it, and is detected by radiography technology.2) DNA probe array on a glass is fixed with point sample method,
Hybridized by the target gene with fluorescent marker and is detected.3) oligonucleotide probe directly synthesized on the hard surfaces such as glass
Array hybridizes with the target gene of fluorescent marker and is detected.Genetic chip is as a kind of advanced, extensive, high-throughput detection
Technology, applied to the diagnosis of disease, advantage has the following aspects: first is that the sensitivity and accuracy of height；Second is that quickly
It is easy；Third is that a variety of diseases can be detected simultaneously.
Known antigen or antibody are adsorbed on surface of solid phase carriers by enzyme-linked immunosorbent assay (ELISA), make enzyme mark
The technology that the antigen-antibody reaction of note is carried out in solid phase surface.The technology can be used for detecting macromolecular antigen and specific antibody
Deng having many advantages, such as that quick, sensitive, easy, carrier is easy to standardize.ELISA detection kit is according to testing goal and operation
Step can be divided into indirect method, double-antibody method, competition law, double site one-step method, prize law survey IgM antibody, using Avidin and
The ELISA of biotin.Horseradish peroxidase (HRP) or alkaline phosphatase may be selected in chromogenic substrate in ELISA detection kit
Enzyme (AP).
Common immune colloid gold detection technique: (1) immune colloid gold light microscopic decoration method cell suspension smear or tissue are cut
Piece can be dyed with the antibody of colloid gold label, can also be enhanced with silver-colored developer solution and be marked on the basis of colloid gold label,
So that the silver atoms being reduced is deposited on marked gold particle surface, the sensibility of colloid gold label can be remarkably reinforced.(2) it is immunized
Colloidal gold staining method for electron microscopy with the antibody of colloid gold label or antiantibody and negative staining Virus Sample or can be organized in conjunction with ultra-thin section,
Then negative staining is carried out.It can be used for observation and the viral diagnosis of morphology of virus.(3) dot immunogold filtration assay application miillpore filter is made
Antigen or antibody point are first added sample to be examined on film by carrier after closing, corresponding with the antibody test of colloid gold label after washing
Antigen or antibody.(4) antigen of specificity or antibody are fixed on film by colloidal gold immunity chromatography with ribbon, colloidal gold
Labelled reagent (antibody or monoclonal antibody) is adsorbed on bonding pad, when sample to be examined is added in the sample pad of test strips one end
Afterwards, it moves forward, reacts to each other after dissolving the colloid gold label reagent on bonding pad through capillary action, it is fixed when being moved to
When the region of antigen or antibody, the conjugate of object and gold marked reagent to be checked occurs specific binding therewith again and is trapped, and assembles
It is taken in detection, colour developing result can be observed by the naked eye.The method has developed into diagnosis test paper, and use is very convenient.
Acceptable carrier is the carrier usually utilized in preparation in pharmacy of the invention, which includes lactose
(lactose), dextrose (dextrose), sucrose (sucrose), sorbierite (sorbitol), mannitol (mannitol), shallow lake
Powder, acacia gum, calcium phosphate, alginates (alginate), gel (gelatin), calcium silicates, microcrystalline cellulose, polyethylene
Pyrrolidones (polyvinylpyrrolidone), cellulose (cellulose), water, syrup, methylcellulose (methyl
Cellulose), methyl hydroxybenzoate (methyl hydroxybenzoate), propyl hydroxy propyl benzoate (propyl
Hydroxybenzoate), talcum, magnesium stearate (stearic acid magnesium) and mineral oil (mineral oil)
Deng, but it is not limited to this.
Pharmaceutically acceptable carrier of the invention can also include lubricant, wetting agent, sweet tea in addition to the above ingredients
Taste agent, flavouring agent, emulsifier, suspending agent, preservative etc..The suitable carrier and preparation permitted in pharmacy is recorded in thunder in detail
Bright Deng Shi pharmacy pandect.
Pharmaceutical composition of the invention can by it is oral or it is non-oral be administered, when as non-oral administration, can lead to
Cross intravenous injection, intranasal injection, locally injecting, intraventricular injection, spinal cavity injection is subcutaneously injected, intraperitoneal injection, percutaneously
The modes such as administration are administered.
The suitable dosage of pharmaceutical composition of the invention according to preparation ways, administration mode, patient year
The factor of age, weight, gender, morbid state, food, administration time, administration route, drainage rate and draw property etc and can be with
Carry out a variety of prescriptions, in general, skilled practitioner can be easily determined by and prescription to it is desired treatment or prevention effectively to
Pharmaceutical quantities.
Person of an ordinary skill in the technical field can be easy to implement pharmaceutical composition of the invention according to the present invention
Method, carried out using carrier receptible in pharmacy and/or excipient it is formulation, so as in the form of unit dose
It is prepared by preparation or interior be mounted in multicapacity container.At this point, dosage form be solution in oiliness or aqueous medium, suspension or
Emulsion form is either also possible to extract, powder agent, granule, tablet or capsule form, can also include dispersion
Agent or stabilizer.
The purpose of the present invention is to provide a kind of gene detecting kit for detecting acute myocardial infarction AMI, the kit inspection
Cls gene RFX2, using special upstream primer and downstream primer, upstream primer sequence is SEQ ID NO.1, downstream primer sequence
It is classified as SEQ ID NO.2.
Further, which is suitable for presently, there are all types fluorescence quantitative gene extender in the market, clever
Sensitivity is high, it is quantitative quick and precisely, stability it is good, have a good application prospect.
Further, above-mentioned PCR kit for fluorescence quantitative component includes: specific primer, internal control primer, quantitative fluorescent PCR
Reaction solution.The internal reference is GAPDH.
The kit also includes RNA extraction agent.
It is an object of the present invention to provide a kind of acute myocardial infarction AMI protein detection kit, the detection kit detection
RFX2 albumen.Further, the kit further includes other detection reagents.
It is an object of the present invention to provide it is a kind of detect acute myocardial infarction AMI genetic chip, the genetic chip include with
The probe of the nucleic acid array hybridizing of RFX2 gene.
Detailed description of the invention
Fig. 1 is the figure of the relative expression quantity of RFX2mRNA in RT-PCR measurement peripheral blood
Specific embodiment
Present invention will be further explained below with reference to specific examples, for explaining only the invention, and should not be understood as to this
The limitation of invention.It will be understood by those skilled in the art that: without departing from the principle and spirit of the present invention may be used
To carry out a variety of change, modification, replacement and modification to these embodiments, the scope of the present invention is limited by claim and its equivalent
It is fixed.In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition or according to item proposed by manufacturer
Part examinations.
The collection of 1 case of embodiment
Take in December, 2012 to during in June, 2014 at the initial patients of acute myocardial infarction of hospital's Internal Medicine-Cardiovascular Dept. 6,
Acute myocardial infarction to admission time is 0.5-10 hours.The standard of being included in meet announced in ESC conference in 2012 it is acute
Myocardial infarction is included in standard.Oneself signs informed consent form to patient in group.Normal healthy controls are the healthy body at Hospital Physical Examination center
6 people of inspection person, and match with experimental group patient age, gender etc., related disease is excluded referring concurrently to the exclusion criteria of AMI group.
Health volunteer knows and agrees to participate in the research.
It is included in standard:
Referring to the new edition myocardial infarction whole world unified definition announced in the ESC conference of Munich, Germany for 2012
Myocardial infarction diagnosis standard, and all patient in group's admission times are in morbidity 12 hours.
Diagnostic criteria is i.e.: serum cardiac biochemical marker (predominantly serum cardiac troponin I) occur it is significantly raised (and at least
More than the 99% normal reference value upper limit), while being associated with the clinical evidence of at least one of following myocardial ischemia:
1. the clinical manifestation of myocardial ischemia；
2. there is the ECG alteration of kainogenesis in electrocardiogram, the change or emerging including emerging ST-T
Left bundle branch block (LBBB) figure；
3. the formation that Electrocardiogram Feature is pathologic Q wave；
4. radiological evidence, including new myocardial activity loses or new regional myocardial Abnormal Wall Motion；
5. confirming that there are thrombus in coronary artery by the objective evidence of coronary angiography inspection or postmortem.
Exclusion criteria:
1. age<20 year old,>90 years old；
2. previously once there is the patient that coronary artery bracket is implanted into or coronary artery bypass grafting is postoperative；
3. have high heart trouble, expand other various cardiomyopathys such as heart trouble, have old myocardial infarction, valvulopathy, myocarditis,
The history of heart disease persons such as pericarditis, infectious endocarditis, dissection of aorta；
4. having systemic immune system disease or immunologic function disorder, low person；
5. occurring fever, cough, expectoration, nausea,vomiting,diarrhea, abdominal pain, frequent micturition, urgent urination, urodynia etc. in recent January
The infection of the other systems such as respiratory tract, alimentary canal or uropoiesis, and have the patient of other chronic infectious diseases；
6. having anemia, primary disease in the blood system person；
7. there is tumour medical history person；
8. chronic hepatitis, cirrhosis, serious hepatic and kidney function obstacle person；
9. having cerebral apoplexy, lung infraction, peripheral vascular disease person in the recent period；
10. organ or Patients Following Bone Marrowtransplantation.
AMI group: it is admitted to hospital respectively at patient and acquires median basilic vein blood 3m1 at that time.
Control group: empty stomach median basilic vein blood 3m1 is also acquired.
2 high-flux sequence of embodiment and analysis
Human peripheral blood carries out RNA extraction, and agarose gel electrophoresis after RNA is extracted can be mentioned from electrophoresis result with preliminary judgement
Whether the RNA sample taken is up-to-standard, if can be used for further transcript profile sequencing.And then pass through NanoDrop1000 points
Light photometer detects the extraction situation of RNA sample, the sample requirement of RNA-seq sequencing: OD260/OD280 1.8-2.2.Sample
Sequencing company is sent to after qualification to be sequenced, microarray dataset is the 2500 high-flux sequence platform of HiSeq of Illumina company,
Carry out high-throughput transcript profile deep sequencing, sequencing company provide data analysis result combination document we screened differential expression
Gene RFX2.
RFX2 expression conditions in 3 patients of acute myocardial infarction group of embodiment and control group peripheral blood
One, material and method
1, material
48 patients of acute myocardial infarction group peripheral bloods and 45 control group peripheral bloods are chosen, it is grouped and is compiled
Number.Two groups are included in standard and exclusion criteria referring to embodiment 1.
2, method
2.1 patients of acute myocardial infarction and the extraction for compareing crowd's peripheral blood RNA
Leucocyte separation
(1) take 2m1 anticoagulation cirumferential blood (blood sampling time is no more than 3h)；
(2) isometric sterile PB S is added to be sufficiently mixed in peripheral blood, forms cell suspension；
(3) 4m1 lymphocyte separation medium is added in another centrifuge tube；
(4) draw 4m1 cell suspension be gently added to along tube wall lymphocyte separation medium surface (pay attention to not with lymphocyte
Separating liquid mixing).It is centrifuged 1500rpm 20min；
(5) boundary layer (tunica albuginea) is gently sucked out with suction pipe to enter in another centrifuge tube.Sterile cold PBS is washed 2 times, is washed for last 1 time
Washing can move into cell suspension in EP pipe, and supernatant is removed in centrifugation, for extracting RNA.
RNA is extracted
(1) first add lml Trizol in EP pipe, if freeze-stored cell is directly added into Trizol, be not required to thaw, piping and druming cracking
After be stored at room temperature 5-l0min；
(2) 0.2m1 chloroform is added, acutely shakes 15s, is stored at room temperature 2-3min, 12000 turns of centrifugation 15min at 4 DEG C；
(3) the supernatant water 600ul that makes an appointment carefully is sucked out and moves into another centrifuge tube (being careful not to be extracted into albumin layer), is added 500,
1 isopropanol, is mixed by inversion, and is stored at room temperature 10min；
(4) 4 DEG C of 12000g are centrifuged l 0min, abandon supernatant, bottom visible white substance；
(5) the rotation washing of the cold ethyl alcohol of lml 75% is added, cleans isopropanol；
(6) 4 DEG C of 7500g are centrifuged 5min, and dry in the air 5-l0min after removal ethyl alcohol, translucent, with the dissolution of 20u1DEPC water
RNA.3u1RNA sample is taken, the electrophoresis in 1.5% Ago-Gel；Lu1RNA sample in UV spectrophotometer measuring concentration,
It is considered as RNA sample qualification in 1.8-2.0 with A260/280.
2.2 reverse transcriptions synthesize cDNA
UsingIII Reverse Transcriptase (invitrogen, article No. 18080-044) into
Row cDNA reverse transcription, experimental implementation are carried out by product description, and concrete operations are as follows:
Using Reverse Transcriptase kit, converse record is carried out to l μ g total serum IgE with RT Buffer and synthesizes cDNA.Using 25 μ l
Reaction system, each sample take 1 μ g total serum IgE as template ribonucleic acid.It is spare that -20 DEG C of refrigerators are put in the cDNA preservation of acquisition.
2.3 Real-Time PCR
2.3.1 instrument and analysis method
With 7500 type fluorescence quantitative PCR instrument of ABI, the relative quantitative assay of data is carried out using 2- Δ Δ CT method.
2.3.2 design of primers
Using online primer-design software, gene order is referring to NCBI:NM_000635.3 (RFX2), interior participation in the election GAPDH,
It is synthesized after design of primers by invitrogen company.Specific primer sequence is as follows:
1 primer sequence of table
Operating process is as follows:
(1) reaction system: Power is usedGreen PCR Master Mix (invitrogen, article No.
4367659) it is expanded, experimental implementation is carried out by product description.Amplification program are as follows: 95 ° of 5min, (95 DEG C of 15sec, 57 DEG C
45sec) × 35 circulation.
2 RealTime reaction system of table
| Component | Additional amount |
| 2×mix | 10μl |
| Upstream primer (10uM) | 0.5μl |
| Downstream primer (10uM) | 0.5μl |
| Template | 2μl |
| Sterile purified water is added | To 25 μ l |
(2) primer screening
After each sample cDNA is mixed, 5 times of gradient dilutions are carried out as template, sample respectively takes 2 μ l to make template after dilution,
It is expanded respectively with target gene primer and reference gene primer, while in 60-95 DEG C of progress melt curve analysis analysis, according to expansion
Increasing Efficiency height and the unimodal principle of solubility curve carry out primer screening.
(3) sample RealTimePCR is detected
2 μ l will be taken to make template after 10 times of each sample cDNA dilutions, respectively with target gene primer and reference gene primer into
Row amplification.Simultaneously in 60-95 DEG C of progress solubility curve analysis.
Two, experimental result
Real-time quantitative PCR amplification curve inflection point understands that amplification curve entirety collimation is good, shows the amplification effect of each reaction tube
Rate is close, and the limit is flat and present without raising up, and exponent phase slope is larger, illustrates that amplification efficiency is higher；Sample amplified production is molten
Solution curve be all it is unimodal, illustrate that amplified production only has one, be specific amplification；According to the relative quantification formula of qRT-PCR: 2-
Δ Ct × 100% compares expression of the RFX2 gene in acute myocardial infarction AMI group and control group.As the result is shown: qRT-PCR
Stable amplification result, wherein expression of the RFX2 gene in acute myocardial infarction AMI group is only about 0.16 times of control group, is seen
Described in Fig. 1, the confluence analysis RFX2 gene that result above demonstrates high-throughput transcript profile expression data is suffered from acute myocardial infarction AMI
The result of low expression in person.
4 acute myocardial infarction in mice model of embodiment
Experimental animal: wild type (wild-type) male mice, strain C57B/L6, week old 10-12w, weight 18-
22g, SPF grades of raisings.
Grouping: control group, that is, sham-operation group；Acute myocardial infarction AMI group, every group of 15 mouse are put to death after anesthesia in postoperative 4 weeks.
Modeling:
1) preoperative weighing is anesthetized with ether to the anesthesia reaction such as absent corneal reflex occurs in mouse, muscular strength declines, collapses from physical exhaustion,
Then it is lain on the back and is fixed in mouse plate.
2) breathing machine ventilation is connected after trachea cannula: tidal volume: 2-3ml, frequency: 120cycles/min.
3) 70% alcohol disinfecting pareordia skin, shaves off hair.
4) pareordia skin is cut off, blunt separation muscle cuts off the wall of the chest in the three or four intercostal space.
5) at the auricle lower edge 3-4mm of left side, with the swaged needle of 7-0, left coronary artery descending anterior branch is ligatured.Ligature position with
Lower regional myocardial, which whitens, to be proved to ligature successfully.Control group does not ligature.
6) thoracic cavity, layer-by-layer suture chest muscle, last continuous suture chest are closed along the 3rd the 4th rib cage with the silk thread of 6-0
Skin.
7) squeezing chest exclusion Thoracic gas makes lung recruitment, pulls out ventilator, is placed on 40 DEG C of heat preservation platforms and waits revival.
8) the postoperative continuous penicillin 10000U of intraperitoneal injection in three days, prevention infection, conventional drinking-water illumination.
To guarantee the homogeneity of murine myocardial infarction model and the comparativity of each group starting point infarct size, all operations
Operation is completed by well-trained special messenger.
Embodiment 5WB method detects the expression of RFX2 in Acute myocardial tissue
One, protein example is prepared and is quantified
It after cardiac muscular tissue is washed with deionized water, shreds, is transferred in homogenizer, addition extracting solution (9mol/L urea+
40g/L CHAPS+5.0g/L DTT+1.4g/L PMFS), ice-water bath homogenate is transferred in centrifuge tube, ice-water bath ultrasound
Appropriate Dnase and Rnase, 4 DEG C of placement 15min are added in 10min, and then 15000r/min is centrifuged 30min, abandon precipitating, use
Bradford method measures total protein concentration, and the packing of remaining protein solution is placed in -80 DEG C of refrigerator cryo-conservations.
Two, SDS- polyacrylamine gel electrophoresis (SDS-PAGE)
1. protein example is denaturalized:
A) according to determination of protein concentration as a result, the total protein extract of phase homogenous quantities is added in each gel well.
The ratio of 0.25 microlitre of albumen sample-loading buffer is added according to every 1 microlitre of protein sample, mixed protein sample and albumen loading are slow
Fliud flushing (5 ×).
B) 100 DEG C or boiling water bath heating 3-5 minutes, with abundant albuminate.
C) it after being cooled to room temperature, is directly loaded in SDS-PAGE glue well.
2. prepared by offset plate:
The gel of 0.75mm thickness is prepared using the miniature vertical plate electrophoresis device of Bio-Rad company, book installs as directed
After glass plate, the separation gel of 5ml 10% is first prepared in small beaker, is formulated as follows:
Table 3 separates glue formula
Then plus the covering of lml distilled water encapsulating immediately after mixing places about 30min after glue polymerization, with distillation at room temperature
Washing 2-3 times, then blotted with filter paper.Then the concentration glue of 2m15% is prepared, is formulated as follows:
Glue formula is concentrated in table 4
| Component | Dosage |
| 30% acrylamide solution | 0.33ml |
| Tris-HCl (1.0M, pH6.8) | 0.25ml |
| 10%SDS | 0.02ml |
| 10%AP | 0.02ml |
| TEMED | 0.002ml |
| Sterilize ddH2O | It is supplemented to 2ml |
Encapsulating, insertion sample comb avoid generating bubble immediately after mixing, after being gelled admittedly, take out sample comb, rear to use distillation
Water and 1 × protein electrophoresis buffer successively rinse sample well.
Three, loading and electrophoresis
By gel slab on electrophoretic apparatus, l × protein electrophoresis buffer, l × protein electrophoresis in outer groove are filled it up in inside groove
Buffer should be more than platinum filament, in order loading.Protein quality standard protein gradient is added in the swimming lane of end.It is blue when electrophoresis
The bottom end that dyestuff reaches glue can nearby stop electrophoresis.
Four, Western blotting
1. first carrying out PAGE gel according to the method described above is separated by electrophoresis albumen.
2. impregnating NC film, filter paper, foam rubber cushion with transfer buffer in advance.Gel is taken out after SDS-PAGE, is removed dense
Contracting glue rinses the several seconds in Tris/ glycine buffer, is subsequently placed in transfer buffer and impregnates 15-30min.Electricity is opened to turn
One piece of dedicated foam rubber cushion impregnated with transfer buffer is padded in print folder, every side, then respectively puts the filter paper that one block of transfer liquid is impregnated with,
Filter paper and sea pad size it is identical or with NC film, gel size is identical, and gel is lain on cathode side filter paper, finally will
NC film is lain on gel, removes bubble removing, clips electricity transfer folder.Electricity transfer liquid is filled it up in electrophoresis tank, is inserted into electricity transfer folder, it will be electric
Swimming slot is put into refrigerator and (to be put into pre-cooling in refrigerator before electricity transfer liquid), connects electrode, turn-on current transfers the NC film of folder
Cope with the anode of electrophoresis tank.
3. closing: being rinsed with 1 × TBS primary.The alipoidic milk power TBS Block buffer containing 5% is added, is placed in shaken cultivation
It is closed in case；
4. primary antibody hybridizes: abandoning confining liquid, primary antibody (the Anti-RFX2 antibody for using primary antibody diluted is added
(ab86170)) hybridization solution is placed in 4 DEG C of hybridized overnights, is hybridized in shaken cultivation case within second day；
5. recycling primary antibody hybridization solution, washed film 3 times with TBST；
6. abandoning TBST, it is added and uses the diluted secondary antibody of Block buffer (Goat Anti-Rabbit IgG, HRP
Conjugated (CW0103)) hybridization solution, it is placed in shaken cultivation case and is hybridized；
7. abandoning two corresponding anti-solution, washed film 3 times with TBST；
8.ECL chemiluminescence and Image Acquisition and analysis: according to highly sensitive chemical luminescence detection kit, (health is century
Article No. CW0049B), specific steps are referring to specification.
9. carrying out data normalization using β-Actin as internal reference, using RFX2 in control group tissue as sample for reference, calculate
The relative expression levels of RFX2 albumen in experimental group.
Five, experimental result
4 weeks execution sham-operation groups and acute myocardial infarction AMI group after modeling respectively, observation discovery sham-operation group heart surface
Smoothly, geometry is regular, each ventricular wall thickness uniformity, and elasticity and toughness keep good, heart and ventricular chamber volume ratio
It is relatively normal, it is inflammatory hyperplasia under the external membrane of heart at threading, it is seen that slight pale asphyxia, remaining heart surface are in light red color；Acute myocardial
Infarct group heart surface is rough, and shape is in general large size, loses original geometry, heart size and left chamber obviously expand
Greatly, fibrosis forms cicatricial tissue to infarcted region cardiac muscle, and the thinning outside bulge of infarcted region cardiac muscle, elasticity and toughness all disappear, obstructs
Dead zone cardiac muscle is in pale asphyxia, forms notable difference with non-infarcted region, non-infarcted region locular wall obviously thickens.WB is the results show that acute
Compared with sham-operation group, RFX2 protein expression is substantially reduced (P < 0.01) myocardial infarction group, and difference has conspicuousness.
The present invention filters out acute myocardial infarction AMI related gene RFX2, binding molecule biological experiment using high-flux sequence
Confirm that RFX2 is acute myocardial infarction AMI diagnosis and treatment marker.
Claims (5)
1. application of the Diagnosis of Acute Myocardial Infarction preparation in preparation Diagnosis of Acute Myocardial Infarction tool, the acute myocardial infarction AMI
Diagnostic preparation detects the expression product of RFX2 gene and/or RFX2 gene.
2. application according to claim 1, which is characterized in that using PCR kit for fluorescence quantitative, genetic chip, be immunized
Method detects the expression of RFX2 gene in acute myocardial infarction AMI tissue or peripheral blood.
3. application according to claim 2, which is characterized in that contain specific detection in PCR kit for fluorescence quantitative
The upstream primer and downstream primer of RFX2 gene, upstream primer sequence are SEQ ID NO.1, and downstream primer sequence is SEQ ID
NO.2。
4. application according to claim 1, which is characterized in that using immunization method detection acute myocardial infarction AMI tissue or outside
The expression product of RFX2 gene in all blood.
5. application as claimed in claim 4, which is characterized in that immunization method is ELISA detection method and/or colloidal gold detection method.
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| CN106801101B (en) * | 2017-02-23 | 2020-12-04 | 吉林大学 | Application of MED6 gene as acute myocardial infarction risk prediction marker |
| CN107271681A (en) * | 2017-06-05 | 2017-10-20 | 中国人民解放军沈阳军区总医院 | Applications of the blood plasma S100A12 in ST sections of elevation myocardial infarction early diagnosis |
| CN108680692A (en) * | 2018-05-16 | 2018-10-19 | 天津市第三中心医院 | The diagnosis marker of inferior wall myocardial infarction and/or Anterior wall myocardial infarction |
| CN108753956A (en) * | 2018-07-03 | 2018-11-06 | 北京泱深生物信息技术有限公司 | Application of the KDSR genes in preparing myocardial infarction diagnosis tool |
| CN108796069A (en) * | 2018-07-03 | 2018-11-13 | 北京泱深生物信息技术有限公司 | Diagnosis marker-ING1 the genes of myocardial infarction |
| 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 |
| CN108913769A (en) * | 2018-07-26 | 2018-11-30 | 泰山医学院 | Early diagnose the molecular marker of myocardial infarction |
| CN110556158B (en) * | 2019-08-30 | 2022-02-15 | 山西农业大学 | Screening method of anti-myocardial fibrosis drug |
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