CN105648094B - Coronary heart disease and coronary heart disease complication with diabetes detection target and its application - Google Patents
Coronary heart disease and coronary heart disease complication with diabetes detection target and its application Download PDFInfo
- Publication number
- CN105648094B CN105648094B CN201610151586.0A CN201610151586A CN105648094B CN 105648094 B CN105648094 B CN 105648094B CN 201610151586 A CN201610151586 A CN 201610151586A CN 105648094 B CN105648094 B CN 105648094B
- Authority
- CN
- China
- Prior art keywords
- heart disease
- coronary heart
- frg1
- gene
- diabetes
- 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.)
- Active
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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- 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
- C12Q2600/158—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
- G01N2800/04—Endocrine or metabolic disorders
- G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Pharmacology & Pharmacy (AREA)
- Biochemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Physics & Mathematics (AREA)
- Urology & Nephrology (AREA)
- Animal Behavior & Ethology (AREA)
- Microbiology (AREA)
- Epidemiology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Pathology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Cell Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to coronary heart disease and coronary heart disease complication with diabetes detection target and its applications.Invention based on to simple coronary heart disease, Patients with coronary artery disease complicated with diabetes mellitus and healthy human peripheral blood high-flux sequence and analysis, pick out candidate gene FRG1, further, experiments prove that FRG1 gene has good correlation with coronary heart disease and coronary heart disease complication with diabetes, and gene FRG1 can also be used to distinguish both diseases, provide reference for clinical prevention and treatment, furthermore, invention devises the efficient RNA interfering for FRG1, and the development for successive treatment preparation provides basis.Coronary heart disease and coronary heart disease complication with diabetes auxiliary diagnosis target provided by the invention has good clinical value.
Description
Technical field
The present invention relates to biomedicine fields, and in particular to coronary heart disease and coronary heart disease complication with diabetes detect target and its answer
With more particularly relating to coronary heart disease and coronary heart disease complication with diabetes detection target FRG1 and its application.
Background technique
Coronary heart disease (coronary heart disease, CHD) the i.e. abbreviation of coronary atherosclerotic heart disease refers to
Luminal stenosis or occlusion caused by atherosis occur for coronary artery, lead to heart caused by myocardial ischemia-anoxemia or necrosis
Disease, also referred to as ischemic heart disease.There are about millions of people to die of cardiovascular disease every year in China, especially with coronary heart disease pair among this
The threat of people's health is maximum.About atherosclerosis, oneself is through establishing Other Risk Factors now, wherein with diabetes spy
Not Tu Chu, diabetic's Incidence of CHD compared with non-diabetic people is often higher by several times, and disease progression is very rapid, about
75% diabetic finally dies of coronary heart disease.Between twenty and fifty people with the increase of the illness rate of diabetes, in patient groups
Increase year by year, it is more serious to the harm of society and family.Although for many years to about atherosclerosis basis and clinic
Research has been made significant headway, it is found that its morbidity is closely related with heredity, inflammation, immune, metabolism etc., but its specific machine
System does not illustrate completely yet, in addition, the study of incident mechanism of coronary heart disease complication with diabetes is less, clinical at present there is an urgent need to find hat
The molecular diagnostic markers of heart trouble, coronary heart disease complication with diabetes carry out early prevention, early treatment, especially merge glycosuria to coronary heart disease
Patient, early diagnosis will effectively improve survival.
To solve the problems, such as that current coronary heart disease and coronary heart disease complication with diabetes early molecule diagnostic flag are rare, inventor couple
2 simple patients with coronary heart disease, 6 Patients with coronary artery disease complicated with diabetes mellitus and 10 Healthy People control peripheral blood samples carry out high-throughput
Sequencing analyzes the screening for carrying out target gene in conjunction with bioinformatics method, picks out candidate gene FRG1, further, pass through
Experiment confirms that FRG1 gene and coronary heart disease and coronary heart disease complication with diabetes have good correlation, and can be used to distinguish
Both diseases provide reference for clinical prevention and treatment, and further, invention devises the efficient RNA interfering for FRG1, are subsequent
The development for treating preparation provides basis.Coronary heart disease and coronary heart disease complication with diabetes auxiliary diagnosis target provided by the invention has very
Good clinical value.
Summary of the invention
The purpose of the present invention is to provide detection FRG1 gene and/or its express albumen preparation preparation coronary heart disease and/
Or the application in coronary heart disease complication with diabetes diagnostic preparation.
Further, the diagnostic preparation of coronary heart disease and/or coronary heart disease complication with diabetes includes using fluorescence quantifying PCR method, base
Because of the expression of FRG1 gene and/or FRG1 albumen in chip method detection peripheral blood.
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.
The product for detecting FRG1 gene in coronary heart disease and/or coronary heart disease complication with diabetes for fluorescence quantifying PCR method contains
There is the primer of a pair of of specific amplification FRG1 gene；The genetic chip includes the spy with the nucleic acid array hybridizing of FRG1 gene
Needle.
Further, the diagnostic preparation of coronary heart disease and/or coronary heart disease complication with diabetes includes detecting FRG1 egg with immunization method
White expression.It is preferred that FRG1 protein expression is in the immunologic detection method detection coronary heart disease and coronary heart disease complication with diabetes
Western blot and/or ELISA and/colloidal gold detection method.
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.
Further, the ELISA method for detecting FRG1 albumen is to use ELISA detection kit.Antibody in the kit
Commercially available FRG1 monoclonal antibody can be used.Further, the kit includes: the solid phase load for being coated with FRG1 monoclonal antibody
Body, ELIAS secondary antibody, the substrate of enzyme, protein standard substance, negative controls, dilution, cleaning solution, enzyme reaction terminate liquid etc..
Further, the colloidal gold method for detecting FRG1 albumen is using detection kit, and the antibody can be used commercially available
FRG1 monoclonal antibody.Further, the gold-immunochromatographyreagent reagent for assay box is seeped using colloidal gold immunochromatographimethod technology or colloidal gold
Filter method.Further, detection zone (T) specking on the gold-immunochromatographyreagent reagent for assay box nitrocellulose filter has anti-FRG1 monoclonal
Antibody, quality control region (C) specking have Immunoglobulin IgG.
The purpose of the present invention is to provide a kind of detection coronary heart disease and/or the quantitative fluorescent PCRs of coronary heart disease complication with diabetes
Kit, which is characterized in that the kit detects gene FRG1, and using special upstream primer and downstream primer, upstream is drawn
Object sequence is SEQ ID NO.12, and downstream primer sequence is SEQ ID NO.13.
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.Wherein the specific primer includes upstream primer and downstream primer, and upstream primer sequence is SEQ ID NO.12,
Downstream primer sequence is SEQ ID NO.13.The internal control primer is β-actin internal control primer, and upstream primer sequence is SEQ ID
NO.14, downstream primer sequence are SEQ ID NO.15.
The kit also includes RNA extraction agent.
It is an object of the present invention to provide a kind of coronary heart disease and/or coronary heart disease complication with diabetes detection kit, the inspections
Test agent box detects FRG1 albumen.Further, the kit further includes other detection reagents.
It is an object of the present invention to provide a kind of genetic chip for detecting coronary heart disease and coronary heart disease complication with diabetes, the bases
Because chip includes the probe with the nucleic acid array hybridizing of FRG1 gene.
The purpose of the present invention is to provide FRG1 genes and/or its protein inhibitor to prepare resisting coronary heart disease and/or coronary disease
Application in sick complication with diabetes preparation.
To achieve the above object, the present invention passes through high-flux sequence combination bioinformatics method first and screens candidate base
Because of FRG1, the relationship of FRG1 Yu coronary heart disease and coronary heart disease complication with diabetes: FRG1 are then demonstrated by molecular biology method
There is good correlation with coronary heart disease and coronary heart disease complication with diabetes, can be used for preparing resisting coronary heart disease and coronary heart disease merges glycosuria
Sick preparation and/or coronary heart disease and coronary heart disease complication with diabetes diagnostic preparation have important clinical value.
Further, resisting coronary heart disease and/or coronary heart disease complication with diabetes preparation refer to the expression that can inhibit FRG1 gene
Preparation.The expression of the known suppressor of those skilled in the art usually can be using one of following methods and/or several: by swashing
The suppressor of FRG1 gene living, activate FRG1 gene inhibition of gene expression albumen, FRG1 inhibited using RNA perturbation technique
Gene expression, activation promote the microRNA of FRG1 gene mRNA degradation, import point for promoting the degradation of FRG1 gene coded protein
Son inhibits to promote the factor of FRG1 gene expression and the expression of albumen.Pass through the suppressor of activation FRG1 gene, activation suppression
The albumen of FRG1 gene expression processed, the siRNA for importing inhibition FRG1 gene expression, activation promote FRG1mRNA degradation
MicroRNA, the expression for importing the molecule for promoting FRG1 protein degradation, the factor of inhibition promotion FRG1 gene expression and albumen.
RNA interference (RNAi) refers to that exogenous and endogenous double-stranded RNA induces the mRNA of homologous target gene in vivo
Selective degradation, the phenomenon that leading to posttranscriptional gene silencing, be it is a kind of efficiently, specifically blocked using small double-stranded RNA it is internal
The expression of certain specific gene, promotes mRNA to degrade, and cells show is made to go out the technology of specific gene missing phenotype.SiRNA design
After the completion can be using direct synthesis technique or building SiRNA expression vector, the siRNA prepared can be coprecipitated by calcium phosphate
The Mechanical Methods, cationic-liposome such as shallow lake method, electroporation, DEAE- glucan and polybrene method, microinjection or particle gun
The approach such as reagent method transfect cell.
Further, the siRNA target sequence for inhibiting FRG1 gene expression is selected from one of following sequence and/or several
Kind: SEQ ID NO.1, SEQ ID NO.4, SEQ ID NO.7.It is preferred that siRNA target sequence is SEQ ID NO.1, SEQ ID
NO.3。
Further, the siRNA sequence for inhibiting FRG1 gene expression is selected from one of following sequence and/or several:
SEQ ID NO.2,SEQ ID NO.3,SEQ ID NO.5,SEQ ID NO.6,SEQ ID NO.8,SEQ ID NO.9.It is preferred that
SiRNA sequence is SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.8, SEQ ID NO.9.
The purpose of the present invention is to provide a kind of resisting coronary heart disease and/or coronary heart disease complication with diabetes preparation, resisting coronary heart disease and/
Or coronary heart disease complication with diabetes preparation inhibits the expression of FRG1 gene.Further, resisting coronary heart disease and/or coronary heart disease complication with diabetes
Contain the siRNA for inhibiting FRG1 gene expression in preparation.
The purpose of the present invention is to provide the preparations of detection FRG1 gene and/or FRG1 albumen in difference coronary heart disease and coronary disease
Application in sick complication with diabetes.
Further, the preparation fluorescence quantifying PCR method of FRG1 gene is detected, method for gene chip detects FRG1 gene
And/or the expression of FRG1 albumen.
For the primer containing a pair of of specific amplification FRG1 gene in the preparation of fluorescence quantifying PCR method；Genetic chip
It include the probe with the nucleic acid array hybridizing of FRG1 gene in the preparation of method.
Further, the preparation for detecting FRG1 albumen detects the expression of FRG1 albumen with immunization method.It is preferred that the immune inspection
Survey method is western blot and/or ELISA and/colloidal gold detection method.
Detailed description of the invention
Fig. 1 is FRG1 gene relative expression in coronary heart disease, coronary heart disease complication with diabetes peripheral blood and healthy human peripheral blood
Spirogram
Fig. 2 is each group FRG1mRNA relative expression levels figure after RNA interference
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 acquisition of 1 sample of embodiment and extraction
(totally 10 people, 2 simple patients with coronary heart disease, 6 Patients with coronary artery disease complicated with diabetes mellitus, clinical information are detailed in case group
Table 1 and control group (totally 10 people) require at least 12h on an empty stomach, at room temperature in next morning 7:00-8:00, extract 10ml venous blood
In ethylenediamine tetra-acetic acid (EDTA) anticoagulant tube, peripheral blood mononuclear cells PBMCs is extracted, 1ml Trizol reagent is added
(Invitrogen company), mixes well, -80 DEG C of preservation samples, to extract for RNA.All blood samples and pathological examination are answered
True and reliable, research is ratified through Ethics Committee, patient's informed consent.
1 case group patient clinical information of table
Extract total serum IgE in patient and healthy control group peripheral blood mononuclear cells (PBMCs), it is desirable that: RNA purity:
OD260/280≤1.8,28S/18S≤1；RNA integrality: Zhi≤7.0 RIN.Method: RNA concentration and method for detecting purity:
NanoDrop2000；RNA integrality detection method: Agilent 2100 (RNA6000Nano kit), agarose gel electrophoresis
(Ago-Gel concentration: 1% agarose gel；Voltage: 5V/cm；Time: 20min).
2 high-flux sequence of embodiment and analysis
MRNA library construction: eukaryote mRNA 3 ' end has the structure of ployA tail, using with Oligo (dT)
Enrichment with magnetic bead mRNA after high temperature fragmentation, using it as template, synthesizes cDNA.By magnetic beads for purifying, end are repaired, 3 ' ends add
Base A, after adding sequence measuring joints, PCR amplification is carried out, constructs the library mRNA.According to RNA pattern detection as a result, to 2 simple coronary diseases
Patient (GX) and 6 Patients with coronary artery disease complicated with diabetes mellitus (GX_TN) and Normal group carry out mRNA library construction.
Sequencing: being sequenced mRNA with llumina Hiseq2500/Miseq second generation high throughput sequencing technologies, leads to
Past connector such as goes low quality, depollutes complete the processing of data at the processes, obtains final data.
Expression data analysis: by the analysis to CHD group and Normal group sequencing result, 488 differences are screened
Expressing gene (P < 0.05), wherein 400 gene upregulations, 88 gene deregulations.By to coronary heart disease complication with diabetes group and just
The analysis of normal control group sequencing result, screens 439 difference expression genes (P < 0.05), wherein 370 gene upregulations, 69
Gene deregulation.By the analysis to coronary heart disease and CHD group complication with diabetes group sequencing result, 388 differential expressions are screened
Gene (P < 0.05).Based on high-throughput transcript profile sequencing result, we are by comparing simple CHD group VS Normal group, hat
Heart trouble complication with diabetes group VS Normal group, CHD group VS coronary heart disease complication with diabetes foot, three groups relatively in occur simultaneously
Difference expression gene 50, be comprehensively compared choose standby be based on FRG1 carry out Late Stage Verification.
3 coronary heart disease of embodiment, Patients with coronary artery disease complicated with diabetes mellitus, the expression one of FRG1 gene, material in healthy human peripheral blood
Material and method
1, material
It collects outside 65 patients with coronary heart disease peripheral bloods, 52 Patients with coronary artery disease complicated with diabetes mellitus peripheral bloods and 44 Healthy Peoples
All blood, is grouped it and numbers.
2, method
The extraction of 2.1 peripheral blood total serum IgEs
UsingReagent carries out sample rna extraction, and experimental implementation is carried out by product description.
RNA quality judging standard: the OD260/OD280 value of RNA sample is between 1.8-2.2；Total serum IgE electrophorogram has clearly
Clear 28S, 18S band；70 DEG C of water-baths keep the temperature 1 hour after electrophorogram and the map no significant difference before water-bath heat preservation.
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, following components are separately added into PCR pipe:
5 × RT Buffer, 5 μ l, 10mmol/l dNTP, 1.25 μ l, 0.1mmol/l DTT 2.5 μ l, 30 μm of mol/l
Aqua sterilisa is added to 25 μ l of total system in 2 μ l, 200U/ μ l MMLV of OligodT 1.25 μ l, 1 μ g of template ribonucleic acid.
42 DEG C be incubated for 1 hour, 72 DEG C 10 minutes, of short duration centrifugation.It is spare that -20 DEG C of refrigerators are put in cDNA preservation.
2.3Real-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- △ △ CT method.
2.3.2 design of primers
Using online primer-design software, template sequence NM_004477 is closed by invitrogen company after design of primers
At.Specific primer sequence is as follows:
2 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 10min, (95 DEG C of 15sec, 60 DEG C
60sec) × 35 circulation.
Table 3RealTime reaction system
| 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) 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, than
Compared with expression of the FRG1 gene in coronary heart disease and Patients with coronary artery disease complicated with diabetes mellitus peripheral blood and healthy human peripheral blood.
(it is specifically shown in Fig. 1) as the result is shown: expression water of the FRG1 in coronary heart disease, Patients with coronary artery disease complicated with diabetes mellitus peripheral blood
The flat expression being apparently higher than in healthy human peripheral blood, respectively about the 14 of normal healthy controls times and about 5.5 times, result above is tested
The confluence analysis FRG1 of high-throughput transcript profile expression data has been demonstrate,proved in coronary heart disease and Patients with coronary artery disease complicated with diabetes mellitus peripheral blood
Highly expressed result.In addition, FRG1 differential expression between patients with coronary heart disease group and two groups of Patients with coronary artery disease complicated with diabetes mellitus group is bright
Aobvious, the former is about 2.5 times of the latter, and display FRG1 is the molecule that can effectively distinguish coronary heart disease, coronary heart disease complication with diabetes
Marker gene.
Embodiment 4RNAi inhibits FRG1 gene expression
One, material
SiRNA building and synthesis
According to FRG1 gene in GenBank (NCBI Reference Sequence:NM_004477) sequence design phase
The siRNA answered.Synesis Company's synthesis is sent to after design.
The design and synthesis of siRNA:
According to purpose mRNA sequence, 3 RNA interference target sequences and negative control (table 4) are designed.
4 siRNA transcription templates sequence of table
Two, experimental method
(1) RNA perturbation technique inhibits the expression of FRG1 gene in HEK293 cell
1, cell grouping and transient transfection
(1) cell is grouped
C group: blank control group；C1 group: nonspecific siRNA group is transfected；S1, S2, S3 group: specificity is transfected
SiRNA group.
(2) it transfects
According to LipofectamineTMThe step of 2000Transfection Reagent is provided carries out.
1. before transfection for 24 hours, the cell pancreatin of logarithmic growth phase is digested and is counted, dense with DMEM culture medium adjustment cell
Degree is 1 × 105/ ml takes 2m1 to be inoculated in six orifice plates, is placed in 37 DEG C, 5%CO2It cultivates in incubator, is merged in cell up to 80%
When for transfecting.With the DMEM culture medium culture 3-4h without serum before transfection.
2. preparing transfection liquid:
A liquid: 250u1 serum free medium dilutes 4.0ugDNA, mild to mix；
B liquid: 250u1 serum free medium dilutes 10u1Lipofectamine, mild to mix, and is placed at room temperature for 5min；
3. transfection: A liquid is mixed with B liquid, and compound is directly added in every hole by incubation at room temperature 20min, is shaken
Culture plate mixes gently.In CO2Liquid is changed in incubator after 37 DEG C of heat preservations 24-48h, 6h, the culture medium containing serum is added.
2, the variation of transfection front and back FRG1 gene expression is detected using Real-time PCR method
1. the building of standard curve: being chosen at 1 bottle of HEK293 cell normally cultivated in 50mI culture bottle, extract RNA, survey
Determine RNA concentration and purity, carry out reverse transcription reaction, ten times of the DNA profiling dilutions that reaction is generated obtain being equivalent to 104-
100The DNA profiling of copies/ul is separately added into FRG1 primer and internal reference actin primer, prepares 25u1 reaction system, uses
Real-time PCR amplification instrument carries out pcr amplification reaction.Obtain the standard curve of FRG1 and actin.
2. the variation of Real-time PCR method detection transfection front and back FRG1 gene expression: the RNA of group of cells is extracted,
RNA concentration and purity are measured, reverse transcription reaction is carried out, every group of DNA profiling carries out the Real-time of FRG1 and actin simultaneously
PCR reaction, experiment is in triplicate.
3. carrying out agarose gel electrophoresis to PCR product.
Three, experimental result
Using the standard curve of Real-time PCR method building FRG1 and actin, related coefficient is respectively 0.9962,
0.9947, linear relationship is good, meets the requirements.Compare the expression of each group FRG1 gene with the method for double standard curves.Blank pair
Expression according to group, nonspecific transfection group gene is substantially similar, no significant difference.FRG1-siRNA1,FRG1-
Play the role of inhibiting FRG1 gene expression, FRG1-siRNA1 and FRG1- after tri- groups of transfections of siRNA2, FRG1-siRNA3
The effect of siRNA3 becomes apparent from, and inhibits efficiency up to 74% and 77%, and the inhibiting effect of FRG1-siRNA2 is 32%, with blank
Control group, nonspecific transfection group are compared, and difference is statistically significant, P < 0.05 (Fig. 2).
Invention filters out coronary heart disease, coronary heart disease complication with diabetes pathogenic related gene FRG1 using high-flux sequence, in conjunction with
Molecular cytobiology experimental verification, it was confirmed that FRG1 has good correlation with coronary heart disease, coronary heart disease complication with diabetes disease.
The present invention provides new target for coronary heart disease and coronary heart disease complication with diabetes clinic diagnosis, before having good clinical application
Scape.
Claims (5)
1. detecting application of the preparation of FRG1 gene in preparation diagnosis of coronary heart disease preparation.
2. application according to claim 1, which is characterized in that diagnosis of coronary heart disease preparation using fluorescence quantifying PCR method or
The expression of method for gene chip detection FRG1 gene.
3. application according to claim 2, which is characterized in that the diagnostic preparation of fluorescence quantifying PCR method contains a pair of of spy
The primer of specific amplification FRG1 gene；The diagnostic preparation of method for gene chip contains the spy with the nucleic acid array hybridizing of FRG1 gene
Needle.
4. detecting application of the preparation of FRG1 gene in preparation difference coronary heart disease and coronary heart disease complication with diabetes preparation.
5. application according to claim 4, which is characterized in that the preparation fluorescence quantifying PCR method of detection FRG1 gene
Or the expression of method for gene chip detection FRG1 gene.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610151586.0A CN105648094B (en) | 2016-03-16 | 2016-03-16 | Coronary heart disease and coronary heart disease complication with diabetes detection target and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610151586.0A CN105648094B (en) | 2016-03-16 | 2016-03-16 | Coronary heart disease and coronary heart disease complication with diabetes detection target and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105648094A CN105648094A (en) | 2016-06-08 |
| CN105648094B true CN105648094B (en) | 2019-05-17 |
Family
ID=56493934
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610151586.0A Active CN105648094B (en) | 2016-03-16 | 2016-03-16 | Coronary heart disease and coronary heart disease complication with diabetes detection target and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105648094B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103014165A (en) * | 2012-12-20 | 2013-04-03 | 宁波大学 | Kit capable of being used for detecting methylation degree of PLA2G7 gene promoter region relevant to coronary heart disease and application of kit |
-
2016
- 2016-03-16 CN CN201610151586.0A patent/CN105648094B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103014165A (en) * | 2012-12-20 | 2013-04-03 | 宁波大学 | Kit capable of being used for detecting methylation degree of PLA2G7 gene promoter region relevant to coronary heart disease and application of kit |
Non-Patent Citations (2)
| Title |
|---|
| 冠心病合并2 型糖尿病患者的小凹蛋白-1表达的变化及其意义;王琳等;《心脑血管病防治》;20140831;第14卷(第4期);摘要 |
| 外周血单核细胞TLR-4在冠心病合并糖尿病中的表达及其意义;黄钟声等;《中国心血管病研究》;20131031;第11卷(第10期);第775-778页 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105648094A (en) | 2016-06-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105779618B (en) | A kind of new diagnosis and treatment target gene of Dendritic cell and its application | |
| CN103003442B (en) | A method of passing through microrna expression proficiency assessment people's allograft situation | |
| JPWO2014003053A1 (en) | Pancreatic cancer detection method and detection kit | |
| CN113481286B (en) | MiRNA-208a amplification primer pair based on strand exchange amplification and detection kit thereof | |
| Loudig et al. | Illumina whole-genome complementary DNA–mediated annealing, selection, extension and ligation platform: assessing its performance in formalin-fixed, paraffin-embedded samples and identifying invasion pattern–related genes in oral squamous cell carcinoma | |
| CN105063194B (en) | The diagnosis marker of Parkinson a kind of and its application | |
| CN105483275B (en) | The new application of mir-1299 and its maturation miRNA | |
| CN105838820B (en) | The new application of transcription factor FOXD1 | |
| CN105288659B (en) | The application of TENM1 gene and its expression product in diagnosis and treatment papillary adenocarcinoma | |
| CN108034707B (en) | SPAG7 gene is preparing the application in diagnosis of dementia preparation | |
| CN107937515B (en) | A kind of diagnosis and treatment gene target of Alzheimer and its application | |
| CN105233290B (en) | The application of C22orf26 genes and its expression product in Parkinson's diagnosis and treatment reagent is prepared | |
| CN105648094B (en) | Coronary heart disease and coronary heart disease complication with diabetes detection target and its application | |
| CN104984363B (en) | Applications of the ZMYM1 in Parkinson's diagnosis and treatment reagent is prepared | |
| CN105648093B (en) | MAFF is diagnosing and is distinguishing the application in coronary heart disease and coronary heart disease complication with diabetes | |
| CN111979315A (en) | Application of annular TP63 as lung squamous carcinoma diagnosis or treatment target | |
| CN107779503A (en) | The related difference expression gene of Alzheimer and its application | |
| CN106399485A (en) | Genes highly expressed in tongue squamous carcinoma para-carcinoma tissue and applications of genes | |
| CN105648079B (en) | A kind of osteoporosis blood testing target spot and its application | |
| KR20210024986A (en) | Urinary exosome-derived miRNA gene biomarkers for diagnosis of antibody-mediated rejection in kidney allografts and use thereof | |
| US11497817B2 (en) | Senile dementia treatment formulation and application thereof | |
| CN105597109B (en) | The diagnosis and treatment molecular labeling of primary osteosarcoma | |
| KR20210024918A (en) | Urinary exosome-derived miRNA gene biomarkers for diagnosis of T cell-mediated rejection in kidney allografts and use thereof | |
| CN105506171A (en) | LYRM2 (LYR motif-containing 2) gene and new use of expression product thereof | |
| CN105506172B (en) | The osteoporosis Blood diagnosis reagent of the GFOD2 containing detection and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |