CN110283906A - Mankind's early atherosclerosis detection molecules marker and its application - Google Patents
Mankind's early atherosclerosis detection molecules marker and its application Download PDFInfo
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- CN110283906A CN110283906A CN201910740318.6A CN201910740318A CN110283906A CN 110283906 A CN110283906 A CN 110283906A CN 201910740318 A CN201910740318 A CN 201910740318A CN 110283906 A CN110283906 A CN 110283906A
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- eif4e
- marked compound
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- 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
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- 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
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4703—Regulators; Modulating activity
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- 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
- G01N2800/323—Arteriosclerosis, Stenosis
Abstract
The invention discloses a kind of mankind's early atherosclerosis to diagnose molecular marked compound, and the molecular marked compound is eIF4E.The invention also discloses the methods for detecting the molecular marked compound, RAW264.7 cell are detected by protein immunoblotting and real-time quantitative fluorescence PCR, respectively by lipopolysaccharides and IL-4 induced polarization at eIF4E albumen and mrna expression amount in cell after M1 and M2 type.The present invention has found that eIF4E has facilitation to atherosclerosis by mouse model, is detected and is found by patient blood, and eIF4E starts expression early stage in Atheromatosis people and increases, and with the development of the state of an illness, and gradually expresses raising.So the molecular marked compound that eIF4E can be used as early detection human atherosclerosis is applied.
Description
Technical field
The present invention relates to a kind of mankind's early atherosclerosis detection molecules marker and its applications, belong to biotechnology
Field.
Background technique
Atherosclerosis is the harmful chronic inflammation cardiovascular disease of a kind of pair of human health, and causes the elderly dead
One of most common reason died.It is characterized in that in artery plaque the macrophage of cholesterol hyperemia lasting presence.These
Macrophage " foam cells " causes patch to be expanded by raising additional leucocyte and vascular smooth muscle cells, and by thin
The significant promotion Plaque instability of the secretion of extracellular matrix degrading proteinase and cytotoxic factor.Cause clinical events (the i.e. heart
Muscle infarction and apoplexy) atherosclerotic plaque be characterized in that macrophage content is higher.Macrophage be in patch most
Inflammatory cell type abundant, in lesion, signal (such as cell factor or modification of the macrophage perception from its microenvironment
Lipoprotein), their the phenotype polarization of these signal decidings.The phenotype polarization of macrophage be one change over time it is dynamic
State process, mainly there is M1 and M2 phenotype, and M1 type macrophage generates proinflammatory cytokine etc. and accelerates progression of atherosclerosis；M2
Type macrophage, which generates, presses down scorching factor inhibition progression of atherosclerosis.Still lack a kind of early detection human arterial congee at present
The method of sample hardening.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the deficiencies of existing technologies, it is athero- to provide a kind of mankind's early stage artery
Detection molecules marker is hardened, it being capable of early detection human atherosclerosis.
In order to solve the above technical problems, the present invention provides a kind of mankind's early atherosclerosis diagnosis molecular marked compound,
It is characterized in that the molecular marked compound is eIF4E, nucleotide sequence is as shown in SEQ ID No.1.
The present invention also provides a kind of for detecting the primer pair of above-mentioned molecular marked compound, which is characterized in that the primer pair
Nucleotide sequence are as follows:
Upstream primer: 5 '-AGGACGGTGGCTGATCACA-3 ', as shown in SEQ ID NO.2；
Downstream primer: 5 '-TCTCTAGCCAGAAGCGATCGA-3 ', as shown in SEQ ID NO.3.
The present invention also provides a kind of for detecting the kit of above-mentioned molecular marked compound, which is characterized in that comprising above-mentioned
Primer pair.
The present invention also provides a kind of methods for detecting above-mentioned molecular marked compound, which is characterized in that is printed by Western Immuno
Mark and real-time quantitative fluorescence PCR detect RAW264.7 cell by lipopolysaccharides and IL-4 induced polarization into cell after M1 and M2 type
Middle eIF4E albumen and mrna expression amount.
Further, the real-time quantitative fluorescence PCR method particularly includes: extract cell sample and tissue with Trizol method
Then sample total serum IgE carries out qPCR with premix using Reverse Transcriptase kit reverse transcription at cDNA, using comparing Ct value method
Relative quantification is calculated, and relative expression is standardized as internal reference.
The present invention also provides a kind of application of above-mentioned molecular marked compound in early detection human atherosclerosis.
Advantageous effects of the invention: eukaryotic translation initiation factor 4E (eIF4E) is normally synthesized in dependence protein
Initial phase from important regulating and controlling effect, it is huge that eIF4E inhibits albumen that can influence by various Chemokines and access
The activity of phagocyte.The present invention has found that eIF4E has facilitation to atherosclerosis by mouse model, passes through patient blood
Detection discovery, eIF4E start expression early stage in Atheromatosis people and increase, and with the development of the state of an illness, and gradually express
It increases.So the molecular marked compound that eIF4E can be used as early detection human atherosclerosis is applied.
Detailed description of the invention
Fig. 1 be Western-blot detection LPS(200ng/ μ L) and IL-4(20ng/ μ L) induce for 24 hours after, eIF4E is in M1
With protein expression situation (1: not inducing RAW264.7 group in M2 type macrophage；2: by the RAW264.7 group after LPS induction for 24 hours；
3: by the RAW264.7 group after IL-4 induction for 24 hours；Compared with not inducing RAW264.7 group, * P < 0.05, * * P < 0.01；N=3)；
Fig. 2 is qPCR detection mRNA differential expression (1: not inducing RAW264.7 group in M1 and M2 type macrophage eIF4E；2:
By the RAW264.7 group after LPS induction for 24 hours；3: by the RAW264.7 group after IL-4 induction for 24 hours；With do not induce RAW264.7 group ratio
Compared with * P < 0.05, * * P < 0.01；N=3)；
Fig. 3 is Western-blot detection eIF4E in ApoE-/-With albumen in Normal group C57BL/6J mouse abdominal aorta
Differential expression (1:C57BL/6J control group；2:ApoE-/-Group；Compared with C57BL/6J control group, * P < 0.05, * * P < 0.01；
N=3)；
Fig. 4 is qPCR detection eIF4E in ApoE-/-With mRNA differential expression in control group C57BL/6J mouse abdominal aorta (1:
C57BL/6J control group；2:ApoE-/-Group；Compared with C57BL/6J control group, * P < 0.05, * * P < 0.01；N=3)；
Fig. 5 is expression variation (* P < 0.05, * * P < 0.01 of the eIF4E gene in patient blood；N=3).
Specific embodiment
The invention will be further described below in conjunction with the accompanying drawings.Following embodiment is only used for clearly illustrating the present invention
Technical solution, and not intended to limit the protection scope of the present invention.
Embodiment 1: the research on cellular level
The present embodiment uses RAW264.7 cell line.Recovery RAW264.7 cell is inoculated in containing 10%FBS and 1% mycillin
In sterile DMEM complete medium, it is placed in 37 DEG C, cultivates in 5%CO2 incubator.It is passed on according to cell growth state and is more renewed
Fresh DMEM complete medium.By every hole about 1X106A cell is laid in 6 orifice plates, and it is more that 200ng/ μ L rouge is separately added into after adherent
Sugared (LPS), 20ng/ μ L IL-4 (IL-4) induction extracts albumen afterwards for 24 hours and RNA is spare.
Using Western Blot(protein immunoblotting) and Real-time PCR(real-time quantitative fluorescence PCR) detection
Expression of the eIF4E on albumen and mRNA level in-site.
Western Blot:RAW264.7 extracts albumen, Coomassie brilliant blue reagent after LPS and IL-4 is induced for 24 hours respectively
Box surveys total protein concentration, calculates sample concentration (10 μ g/ μ L).80V voltage, 100g/LSDS-PAGE gel electrophoresis 2h；100V turns
Film 120min；50g/L skimmed milk power room temperature closes 1h；PBST is washed 5min/ times, and totally 3 times；Addition eIF4E antibody (1:500),
Internal reference β-actin (1:5000) primary antibody, 4 DEG C of overnight incubations；PBST is washed 5min/ times, and totally 3 times；It is incubated at room temperature secondary antibody (1:5000)
1-2h；PBST is washed 5min/ times, and totally 3 times；It sets in extensive chemical luminescence reagent ECL substrate and chemically reacts；Darkroom exposure shows special
Protein signal.Imaging system carries out image procossing, records result.Purpose band and internal reference (β-actin) band extinction area product
The expression quantity of the ratio Analysis destination protein divided.
Real-time PCR: cell sample and tissue sample total serum IgE are extracted with Trizol method, uses Reverse Transcriptase kit
Then reverse transcription carries out qPCR with premix at cDNA, calculate relative quantification using Ct value method (△ △ Ct) is compared, and by phase
GAPDH(internal reference is standardized as to expression).Experiment carries out (n=3 time independent experiment) in independent biology repeats；Each
Sample is analyzed in triplicate with technology.
The primer that PCR amplification is designed according to the gene coded sequence of eIF4E and GAPDH, by the limited public affairs of Shanghai bioengineering
Department's synthesis.Specific primer sequence is as follows:
eIF4E:
Upstream primer: 5 '-AGGACGGTGGCTGATCACA-3 ',
Downstream primer: 5 '-TCTCTAGCCAGAAGCGATCGA-3 '.
GAPDH:
Upstream primer: 5 '-CAACTTTGGCATTGTGGAAGG-3 ',
Downstream primer: 5 '-ACACATTGGGGGTAGGAACAC-3 '.
As a result, it has been found that: Fig. 1 and Fig. 2 is that protein immunoblot and fluorescent PCR detect RAW264.7 cell and lured by LPS and IL-4
Lead after being polarized to M1 and M2 type eIF4E albumen and mrna expression amount in cell.EIF4E albumen and mRNA in M1 type macrophage
Expression is higher than M2 type macrophage.Since M1 type macrophage is exempted from by proinflammatory cytokine, active oxygen, the acceleration of active nitrogen product
Recruitment and Vascular remodeling under epidemic disease endothelial.M2 type macrophage passes through anti-inflammatory cytokines transforming growth factor-β
(transforming growth factor- β, TGF-β), IL-10 play the role of antiatherosclerosis and stablize patch.Sentence
Disconnected eIF4E promotes the formation of M1 type macrophage, plays pro-inflammatory effect.
Embodiment 2: the research in animal level
ApoE-/-Mouse is that the model mouse of atherosclerosis symptoms of atherosclerosis occurs after high fat diet 12 weeks.Such as Fig. 3
With shown in Fig. 4, eIF4E is detected in ApoE using protein immunoblot and real-time fluorescence quantitative PCR-/-And Normal group
Albumen and mRNA differential expression in C57BL/6J mouse abdominal aorta.The result shows that ApoE-/-EIF4E egg in mouse abdominal aorta
White expression is higher than Normal group C57BL/6J(P< 0.05).Its mRNA expression also above control group (P< 0.05).
And take 12 weeks ApoE of high fat diet-/-Rat heart detects eIF4E albumen using immunofluorescence technique and mRNA exists
Expression in tissue: eIF4E is in ApoE for histogenic immunity fluorescence detection-/-In mouse aorta sinus portion with M1 type macrophage
Common location table in (CD86:M1 type macrophage marker) and M2 type macrophage (CD206:M2 type macrophage marker)
Up to situation.The result shows that the common location expression of the common location ratio eIF4E and CD206 of eIF4E and CD86 is more.
It is obtained from result above, eIF4E expresses significant raising in progression of atherosclerosis, promotes Atherosclerosis
The formation of change.
Embodiment 3: atherosclerotic's blood test results
As shown in figure 5, this experiment has randomly selected 16 patients after atherogenesis early stage arrives patient's aggravation
Blood sample, while the blood sample for having extracted 8 normal persons has detected eIF4E base by fluorescent quantitative PCR technique as negative control
The expression of cause changes, and eIF4E gene expresses raising with the exacerbation of patient's state of an illness as the result is shown.Therefore eIF4E gene can be made
It is applied for one molecular marked compound of early detection Atheromatosis people.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the technical principles of the invention, several improvement and deformations can also be made, these improvement and deformations
Also it should be regarded as protection scope of the present invention.
Sequence table
<110>Xuzhou medical university
<120>mankind's early atherosclerosis detection molecules marker and its application
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<141> 2019-08-12
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aggttaggac ttcctccgaa gatagtgatt ggttatcagt cccacgcaga cacagctaca 780
aagagcggct ccaccactaa aaataggttt gttgtttaag aagacacctt ctgagtattc 840
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ataaattgac taggtagtat cacatgtaat tacactgatg tggctatctt tttaataaag 1980
ttaaggcaca gttgctcagt cctaggttga gtgatggact ttgactatgt tacagttgat 2040
gaggattggg gttttggtgc atcaccattc ggtaggaaca gcggctagaa actgattgtt 2100
gggtttaaga tgtttttact taatggccag aaaattagcg taaggaaagt atatagagaa 2160
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Claims (6)
1. mankind's early atherosclerosis diagnoses molecular marked compound, characterized in that the molecular marked compound is eIF4E, core
Nucleotide sequence is as shown in SEQ ID No.1.
2. the primer pair for detecting molecular marked compound described in claim 1, which is characterized in that the nucleotide of the primer pair
Sequence are as follows:
Upstream primer: 5 '-AGGACGGTGGCTGATCACA-3 ', as shown in SEQ ID NO.2；
Downstream primer: 5 '-TCTCTAGCCAGAAGCGATCGA-3 ', as shown in SEQ ID NO.3.
3. the kit for detecting molecular marked compound described in claim 1, which is characterized in that comprising described in claim 2
Primer pair.
4. the method for detecting molecular marked compound described in claim 1, which is characterized in that by protein immunoblotting and in real time
Quantitative fluorescence PCR detects RAW264.7 cell by lipopolysaccharides and IL-4 induced polarization into eIF4E egg in cell after M1 and M2 type
White and mrna expression amount.
5. according to the method described in claim 4, it is characterized in that, the real-time quantitative fluorescence PCR method particularly includes: use
Trizol method extracts cell sample and tissue sample total serum IgE and then uses premix using Reverse Transcriptase kit reverse transcription at cDNA
QPCR is carried out, calculates relative quantification using Ct value method is compared, and relative expression is standardized as internal reference.
6. application of the molecular marked compound described in claim 1 in early detection human atherosclerosis.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114152752A (en) * | 2021-08-12 | 2022-03-08 | 南京市浦口医院 | Application of CFIm25 in detecting or treating atherosclerosis |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN109055544A (en) * | 2018-09-28 | 2018-12-21 | 新乡医学院 | Atherosclerosis molecular marker and its application |
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