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CN107022636A - A kind of aneurysmal molecular marker of diagnosis and treatment abdomen - Google Patents

A kind of aneurysmal molecular marker of diagnosis and treatment abdomen Download PDF

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Publication number
CN107022636A
CN107022636A CN201710407607.5A CN201710407607A CN107022636A CN 107022636 A CN107022636 A CN 107022636A CN 201710407607 A CN201710407607 A CN 201710407607A CN 107022636 A CN107022636 A CN 107022636A
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sync
gene
albumen
product
instrument
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李曙光
孙锦云
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Qingdao Yangshen Biomedical Co Ltd
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Beijing Medintell Bioinformatic Technology Co Ltd
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Abstract

The invention discloses a kind of gene marker, the gene marker is SYNC.SYNC can be used for the risk for judging whether abdominal aneurvsm occurs or anticipation abdominal aneurvsm occurs.In addition, SYNC can be also used for preparing the medicine for the treatment of abdominal aneurvsm.The present invention provides new diagnostic method for the clinical abdominal aneurvsm of diagnosis on a molecular scale, while providing new drug target for the gene therapy of abdominal aneurvsm.

Description

A kind of aneurysmal molecular marker of diagnosis and treatment abdomen
Technical field
The present invention relates to diagnosing tumor, therapy field, more particularly it relates to detect that SYNC is abnormal for means Diagnosing tumor method；And the tumor therapeutic agent of activation SYNC genes or protein.
Background technology
Abdominal aneurvsm (abdominal aortic aneutysm, AAA) refers to the lesion or damage due to abdominal aorta wall Lose, cause the limitation of abdominal aorta to expand, bulging, using pulsation lump as the disease of cardinal symptom.Generally by abdominal aorta Section arterial wall three-decker continuation is expanded at the arteria renalis more than 1.5 times of abdominal aorta diameter and is defined as AAA, is that one kind is normal The high case fatality rate retrograde arterial disease seen.At present, it is not fully understood for the specific pathogenesis of abdominal aneurvsm, it is existing Report shows that its morbidity and the factor such as inherent cause, inflammation, proteasome degradation, Vascular Smooth Muscle Cell Apoptosis are closely related, its morbidity With other tumours because of differences such as chemical radiation, gene mutations.People's abdominal aorta smooth muscle cell (HVSMCs) is used as abdominal aorta The main composition of middle film, plays an important role to the integrality for maintaining arterial wall structure and function.In recent years, some are reported The reduction for showing VSMC quantity in abdominal aortic aneurysm tissue is a key factor in its forming process.How VSMC is effectively suppressed Apoptosis be expected to for delay AAA formation, development new treatment thoughts are provided.
The content of the invention
An object of the present invention is that provide one kind diagnoses abdomen master by detecting SYNC genes or protein expression difference Aneurysmal method.
The second object of the present invention is that provide one kind treats abdominal aorta by activating SYNC genes or SYNC albumen The method of knurl.
The third object of the present invention is to provide a kind of method for being used to screen the medicine for the treatment of abdominal aneurvsm.
The fourth object of the present invention is to provide a kind of medicine for being used to treat abdominal aneurvsm.
To achieve these goals, present invention employs following technical scheme：
The invention provides application of the detection SYNC product in Diagnosis of Abdominal Aortic Aneurysm instrument is prepared.
Further, the product of the detection SYNC includes the product of detection SYNC gene expression amounts.
Further, the product of the detection SYNC includes that the product of SYNC gene mRNAs can be quantified, and/or can determine Measure the product of SYNC albumen.
The product of the quantitative SYNC gene mRNAs of the present invention can be based on playing its work(using the known method of nucleic acid molecules Energy：Such as PCR, such as Southern hybridization, Northern hybridization, dot blot, FISH (FISH), DNA microarray, ASO Method, high-flux sequence platform etc..It can qualitatively, quantitatively or semi-quantitatively implement analysis using the product.
Nucleic acid included in the said goods can be obtained by chemical synthesis, or be contained by being prepared from biomaterial Expect the gene of nucleic acid, then expand it to obtain using the primer for expecting nucleic acid designed for amplification.
Further, the PCR method is known method, for example, ARMS (Amplification Refractory Abruptly-changing system is not answered in Mutation System, amplification) method, RT-PCR (reverse transcriptase-PCR) method, nesting PCR methods etc..Amplification Nucleic acid can be by using dot blotting hybridization method, Surface Plasmon Resonance (SPR methods), PCR-RFLP methods, original position RT-PCR Method, PCR-SSO (sequence specific oligonucleotide) method, PCR-SSP methods, AMPFLP (amplifiable fragment length polymorphism) method, MVR-PCR methods and PCR-SSCP (single-strand conformation polymorphism) methods are detected.
The product that SYNC gene mRNAs can be quantified includes the specific amplified SYNC genes used in real-time quantitative PCR Primer, the primer sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.
The primer that product includes can by being prepared by chemical synthesis, by using those skilled in the art will know that Method with reference to Given information to be suitably designed, and prepared by chemical synthesis.
Nucleic acid recited above may also include probe, and the probe can be prepared by chemical synthesis, by using this The method that art personnel know appropriately is designed with reference to Given information, and is prepared by chemical synthesis, or can be led to Cross and prepared from biomaterial containing the gene for expecting nucleotide sequence, and expanded using the primer that nucleotide sequence is expected designed for amplification Increase it to prepare.
The product of the quantitative SYNC albumen of the present invention can be based on playing its function using the known method of antibody：For example, ELISA, radioimmunoassay, immunohistochemical method, western blot etc. can be included.
The product of the quantitative SYNC albumen of the present invention includes the antibody or its fragment of specific binding SYNC albumen.It can make With the antibody or its fragment of any structure and size, immunoglobulin class, origin etc., as long as it combines target protein.This The antibody or its fragment that the detection product of invention includes can be monoclonals or polyclonal.Antibody fragment refers to reservation antibody Antibody a part of (Partial Fragment) to the binding activity of antigen or the peptide containing an antibody part.Antibody fragment can include F (ab′)2, Fab ', Fab, scFv (scFv), the Fv (dsFv) of disulphide bonding or its polymer, dimerization V areas it is (dual anti- Body) or peptide containing CDR.The product of the quantitative SYNC albumen of the present invention can include encoding antibody or Encoding Antibody Fragment The nucleic acid of the separation of amino acid sequence, the carrier comprising the nucleic acid, and carry the cell of the carrier.
Antibody can be by obtaining well known to a person skilled in the art method.Retain target all or in part for example, preparing The mammalian cell expression vector that the polypeptide of protein or integration encode their polynucleotides is used as antigen.Exempted from using antigen After epidemic disease animal, from by immune animal adaptive immune cell and merging myeloma cell to obtain hybridoma.Then from hybridization Knurl culture collects antibody.The antibody of acquisition can finally be implemented by using SYNC albumen for being used as antigen or part thereof Antigentic specificity purifies to obtain the monoclonal antibody for SYNC albumen.Polyclonal antibody can be prepared as follows：With with above Identical antigen-immunized animal, collects blood sample from by immune animal, serum is isolated from blood, then using upper State antigen and antigentic specificity purifying is implemented to serum.The antibody or the antibody by using acquisition that can be obtained by using ferment treatment Sequence information obtain antibody fragment.
The combination of label and antibody or its fragment can be implemented by method as commonly known in the art.For example, can With following fluorescent marker protein or peptide：Clean protein or peptide with phosphate buffer, addition DMSO, buffer, etc. standard Standby dyestuff, then mixed solution, is placed 10 minutes then at room temperature.In addition, the labelling kit of commercialization can be used in mark, it is all Such as biotin labeling reagent box, such as biotin labeling reagent box-NH2, biotin labeling reagent box-SH (DojindoLaboratories)；Alkali phosphatase enzyme mark kit such as alkali phosphatase enzyme mark kit-NH2, alkaline phosphorus Sour enzyme labelling kit-SH (Dojindo Laboratories)；Peroxidase labelling kit such as peroxidase mark Remember kit-NH2, peroxidase labelling kit-NH2 (Dojindo Laboratories)；Phycobniliprotein labelled reagent Box such as phycobniliprotein labelling kit-NH2, phycobniliprotein labelling kit-SH, B- phycoerythrin labelling kit-NH2, B- phycoerythrin labelling kits-SH, R-PE labelling kit-NH2, R-PE labelling kit SH (DojindoLaboratories)；Fluorescent labeling reagent box such as fluorescein labelling kit-NH2, HiLyte Fluor (TM) 555 labelling kit-NH2, the labelling kit-NH2 of HiLyte Fluor (TM) 647 (Dojindo Laboratories)；And DyLight 547 and DyLight647 (Techno Chemical Corp.), Zenon (TM), Alexa Fluor (TM) antibody Labelling kit, Qdot (TM) antibody labeling kit (Invitrogen Corporation) and EZ- label protein marks Remember kit (Funakoshi Corporation).For correct labeling, suitable instrument can be used to detect by mark Antibody or its fragment.
As the sample of the detection product according to the present invention, the tissue sample for example obtained from biopsy subject can be used Or fluid.Sample is not particularly limited, as long as it is suitable to the measure of the present invention；For example, it can include tissue, blood, blood plasma, Serum, lymph, urine, serous cavity liquid, spinal fluid, synovia, aqueous humor, tear, saliva or its fraction or treated material Material.
In specific embodiments of the present invention, tissue of the sample from subject.
Further, the product of the quantitative SYNC genes or SYNC albumen can be detection SYNC genes or SYNC albumen Reagent, it can also be kit, chip, test paper etc. comprising the reagent or be measured using the high pass of the reagent Sequence platform.
Present invention also offers a kind of instrument for diagnosing abdominal aneurvsm, the instrument can detect SYNC gene expressions Amount.
Further, the instrument includes that the reagent of SYNC gene mRNAs can be quantified, and/or can quantify SYNC eggs White reagent.
Further, the reagent that can quantify SYNC gene mRNAs is the specific amplified used in real-time quantitative PCR The primer of SYNC genes, the primer sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.
Further, the instrument of the diagnosis abdominal aneurvsm includes but is not limited to chip, kit, test paper or high flux Microarray dataset；High-flux sequence platform is a kind of instrument of special diagnosis abdominal aneurvsm, with high throughput sequencing technologies Development, will turn into the structure of the gene expression profile of a people and very easily work.By contrasting Disease and normal person The gene expression profile of group, the exception for easily analyzing which gene is related to disease.Therefore, SYNC is known in high-flux sequence The exception of the gene purposes for falling within SYNC related to abdominal aneurvsm, equally within protection scope of the present invention.
The number for the amino acid that the detection product of the present invention, the anti-SYNC antibody or its fragment that use in diagnostic tool are recognized Mesh is not particularly limited, as long as antibody can combine SYNC.
Present invention also offers a kind of method for diagnosing abdominal aneurvsm, methods described comprises the following steps：
(1) sample of subject is obtained；
(2) expression of SYNC genes or albumen in Samples subjects is detected；
(3) the SYNC genes measured or the expression of albumen are associated with the whether ill of subject.
(4) compared with the control, the expression of SYNC genes or albumen is reduced, then the subject is judged with abdomen actively Arteries and veins knurl is judged as recurrence or abdominal aorta with risk or abdominal aortic aneurysms with abdominal aneurvsm Knurl patient is judged as prognosis mala.
Present invention also offers a kind for the treatment of method of abdominal aneurvsm, methods described includes activation SYNC genes or SYNC Albumen.
Further, methods described includes promoting the expression of SYNC genes, or promotes expression or the enhancing SYNC of SYNC albumen The activity of albumen.
, can be by after testing drug be added to cancer cell present invention also offers a kind of screening technique of tumour medicine Or some period after testing drug is applied to tumor model animal measures the expression of SYNC genes or SYNC albumen Improve the effect of tumor prognosis to determine tumour medicine.More specifically, when SYNC genes or the expression of SYNC albumen When being raised after addition or administration testing drug or when recovering normal level, the medicine may be selected and is used as improvement tumor prognosis Medicine.
Present invention also offers a kind of medicine for treating abdominal aneurvsm, the medicine includes SYNC activator.
The SYNC of invention activator is unrestricted, as long as the activator can promote or strengthen SYNC or be related to SYNC The expression of the material of upstream or downstream pathway or activity, and for the treatment effective medicine of tumour.
Present invention also offers application of the above-mentioned activator in the medicine for preparing treatment abdominal aneurvsm.
Further, the activator include SYNC genes, SYNC albumen, promoted type miRNA, promoted type transcriptional control because Son or promoted type targeting micromolecular compound.
On the one hand the activator of the present invention can be used for the missing or deficiency for supplementing endogenic SYNC albumen, by improving The expression of SYNC albumen, so as to treat the abdominal aneurvsm caused by SYNC hypoproteinosis.On the other hand it can be used for enhancing The activity of SYNC albumen, so as to treat abdominal aneurvsm.
The anti-depressant medications of the present invention can be used by any of mode compounding pharmaceutical composition in this area.This Kind of composition includes active component, add one or more pharmaceutically acceptable carriers, diluent, filler, bonding agent and its Its excipient, this depends on administering mode and designed dosage form.The known inert nothing for the treatment of of this area branch art personnel Machine or organic carrier include but is not limited to lactose, cornstarch or derivatives thereof, talcum, vegetable oil, wax, fat, polyhydroxy Based compound such as polyethylene glycol, water, sucrose, ethanol, glycerine, such, various preservatives, lubricant, dispersant, flavoring Agent.Moisturizing is cut to pieces, antioxidant, sweetener, colouring agent, stabilizer, salt, buffer solution is such can also be added thereto, these things Matter be used to help as needed formula stability or be favorably improved activity it biological effectiveness or in oral situation Lower to produce acceptable mouthfeel or smell, the preparation that can be used in such a composition can be the shape of its original chemical in itself Formula, or the form of its pharmaceutically acceptable salt is optionally used, anti-depressant medications of the invention can be administered alone, or with each Combination medicine-feeding is planted, and combining form is administered together with other healing potions.The composition so prepared may be selected as needed Any appropriate mode well known by persons skilled in the art is administered anti-depressant medications.During using pharmaceutical composition, be by The inhibitor of the invention of safe and effective amount is applied to people, wherein the micro- g kg body of oral safe and effective amount typically at least about 100 Weight.Certainly, specific dosage is also contemplated that the factors such as method of administration, patient health situation, and these are all skilled practitioners technical ability scopes Within.
The medicine of the present invention can be prepared into various formulations as needed.Including but not limited to, percutaneous, mucous membrane, nose, buccal, Tablet, solution, granule, patch, paste, capsule, aerosol or suppository sublingual or orally use.
The route of administration of the medicine of the present invention is unrestricted, as long as it can play desired therapeutic effect or preventive effect i.e. Can, including but not limited to intravenous, intraperitoneal, intraocular, intra-arterial, intrapulmonary, orally, and in vesicle, intramuscular, tracheal strips, subcutaneously , local by pleura by skin, suction, by mucous membrane, skin, stomach is intra-articular, intra-ventricle, rectum, vagina, In skull, in urethra, in liver, in knurl.In some cases, can systematically it be administered.It is to be partly administered in some cases.
The dosage of the medicine of the present invention is unrestricted, can as long as obtaining desired therapeutic effect or preventive effect To carry out appropriate determination according to symptom, sex, age etc..The medicine of the present invention or the dosage of prophylactic agent can be with use examples Determined such as to the therapeutic effect or preventive effect of disease as index.
In the context of the present invention, " diagnosis abdominal aneurvsm " includes judging whether subject suffers from abdominal aorta Knurl, judge subject whether there is with abdominal aneurvsm risk, judge abdominal aortic aneurysms whether recurred with turn Move, judge prognosis situation reactive or that judge abdominal aortic aneurysms of the abdominal aortic aneurysms to drug therapy.
" treatment " used herein is covered treatment-related in such as mankind of the mammal with relevant disease or illness Disease or morbid state, and including：
(1) prevention disease or morbid state occur in mammal, especially when the mammal is susceptible in the disease Diseased state, but when being not yet diagnosed with this morbid state；
(2) suppress disease or morbid state, that is, prevent it from occurring；Or
(3) disease or morbid state are alleviated, even if disease or morbid state disappear.
Term " treatment " is usually directed to the treatment mankind or animal (for example, being applied by animal doctor), wherein can reach some pre- The therapeutic effect of phase, for example, suppressing the development (including reduce development speed, stop development) of illness, improving illness and healing Illness.Also include the treatment as precautionary measures (such as preventing).Pair illness is not developed into also but develop into illness danger The purposes of the patient of danger, is also included within term " treatment ".
The advantages of the present invention：
The present invention's is found that a kind of molecular marker for diagnosing abdominal aneurvsm, can be in abdomen using the molecular marker The early stage that aortic aneurysm occurs can be used as judging the survival rate there is provided patient.
The medicine of the activator for including SYNC genes or albumen of the present invention can be used as controlling for new abdominal aneurvsm Treat medicine.
Brief description of the drawings
Fig. 1 displays detect difference table of the SYNC genes in abdominal aortic aneurysm tissue and normal control tissue using QPCR Reach；
Fig. 2 displays are using Western blot experiment detection SYNC albumen in abdominal aortic aneurysm tissue and normal control tissue In differential expression；
Fig. 3 displays utilize Western blot experiment detection SYNC gene expression inhibition rates.
Specific embodiment
The present invention is further detailed explanation with reference to the accompanying drawings and examples.Following examples are merely to illustrate this Invention rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in embodiment, generally according to conventional strip Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) described in condition, or according to the condition proposed by manufacturer.
The genetic chip of embodiment 1 screens difference expression gene
1st, tissue is obtained and handled：Collect Normal aorta control tissue under 2 parts of AAA arterial walls middle level living specimen and kidney 2 parts, it is sterile, without RNase under the conditions of, cut aorta wall at about 4cm below the flat arteria renalis, sterile saline is washed Bloodstain, intra-arterial outer membrane is removed by taken tissue, rapidly (<5min) freeze standby in liquid nitrogen.
2nd, RNA is extracted, cDNA is synthesized
The artery tumor tissue of Liquid nitrogen storage is individually placed in ceramic mortar crushed into powder under liquid nitrogen cryogenics environment, added Trizol reagents are homogenized, taken after centrifugation supernatant extracted twice through 1: 1 acid phenol-chloroform after again through sodium acetate and 5: 1 acidic phenols- Chloroform, isometric isopropanol precipitating dissolves precipitation after centrifugation with Milli-Q water, and UV detector determines OD values (A260/A280), denaturing formaldehyde gel electrophoresis detection 28S, 18S rRNA bands.
3rd, chip is handled
HGEC40s types chip of expression spectrum is won star gene chip Co., Ltd by Shanghai and made.With carrier two ends universal primer To 4 096 full-length gene row Polymerize chain reaction (PCR) amplifications, product length is 1 000~3000bp, using agarose electricity PCR primer is examined in swimming, and the amplified production after concentration is dissolved in 3 × SSC sampling liquids as target gene uses Cartesian The point sample instruments of Pixsys 7500 press 0.28mm spacing point samples on 18mm × 18mm slides.Slide is done through hydration, room temperature after point sample Dry, UV crosslinkings, then 10min is handled with 0.2%SDS, water and 0.2% sodium borohydride solution, dry standby.
4th, prepared by probe
Oligolex mRNA Midi Kits (Qiagen companies, the U.S.) purify mRNA, are taken according to the concentration of RNA solution suitable Total serum IgE is measured, is purified with Oligod T affinity columns, obtains pure mRNA.Reference literature [SchenaM, Shalon D, Davis RW, et al.Quantitative monitoring of gene expression patterns with a complementary DNA microarray.Science,1995,270:467-470] method reverse transcription synthesis and purifying cDNA probes, Cy3- DUTP marks normal structure mRNA, and AAA tissue mRNAs are marked with Cy5-dUTP.By Cy3-dUTP and Cy5-dUTP after ethanol precipitation The probe mixed dissolution of mark is in 20 μ l 5 × SSC+0.2%SDS hybridization solutions.
5th, hybridize and wash：Hybridization cabin is placed in after HGEC40s chip of expression spectrum and hybridization probe are done into 95 DEG C of denaturation respectively It is interior, mixed probe is added, is subject to hybridization sealed compartment closed, bubble is not left.In 60 DEG C of hybridization 16h in constant humidity hybridization case.
6th, the detection of differential gene and molecular biology information analysis：Core is scanned with GenePix 4000B Fluorescence Scanners After piece, the intensity and ratio of 2 kinds of fluorescence signals of GenePix Pro3.0 software analysis.Cy3 and Cy5 is carried out with 40 house-keeping genes Equilibrium.First the prospect value of all data is subtracted each other with background value, the intensity level of Cy3, Cy5 mark is drawn, is n's according to sum The Cy5/Cy3 of effective gene natural logrithm value lnRi=lnCy5/Cy3, with effective gene of the Ri values between 0.1~10.0 As homogenization foundation, calculate these gene Cs y5/Cy3 natural logrithms average value and uniform coefficient.By all data item Cy3 mark intensities are multiplied by homogenization coefficient, draw the Cy3 values after adjustment.The intensity level that all Cy3 values are less than 200 is taken with 200 For bioinformatic analysis result：With the intensity and ratio of GenePix Pro 3.0 software analysis, 2 kinds of fluorescence signals.
7th, bioinformatic analysis result
With the intensity and ratio of GenePix Pro 3.0 software analysis, 2 kinds of fluorescence signals.As a result difference expression gene is shown For 432, wherein up-regulated gene is 278, and down-regulated gene is 154.
Checking of the difference expression gene of embodiment 2 in large sample
The expression Select gene chip in abdominal aortic aneurysm tissue is selected to point out the SYNC of differential expression to enter for goal in research Row reversely checking.
1st, tissue is obtained and handled：Method according to embodiment 1 is collected under 30 parts of AAA arterial walls middle level living specimen and kidney 40 parts of Normal aorta control tissue.
2nd, RNA is extracted
RNA extractions are carried out according to the method for embodiment 1.
3rd, reverse transcription
Reverse transcription synthesis cDNA is carried out to l μ g total serum IgEs with RT Buffer.Using 25 μ l reaction systems, each sample Take 1 μ g total serum IgEs as template ribonucleic acid, following components is separately added into PCR pipe：DEPC water, 5 × RT Buffer, 10mmol/L dNTP, 0.1mmol/l DTT, 30 μm of mol/l Oligo dT, 200U/ μ l M-MLV, template ribonucleic acid.42 DEG C of incubations 1h, 72 DEG C of 10min, of short duration centrifugation.
4、QPCR
Using 25 μ l reaction systems, each sample sets 3 parallel pipes.Prepare following reaction system：SYBR Green gather The μ l of polymerase chain reaction system 12.5, the μ l of forward primer (5 μM) 1, reverse primer (5 μM) 1 μ l, template cDNA 2.0 μ l, no enzyme The μ l of water 8.5；The forward primer sequence for expanding SYNC genes is 5 '-GAAGCCTTGAACCCAGAA-3 ' (SEQ ID NO.1), instead It is 5 '-GTCCTCCAGGTAGAGAATG-3 ' (SEQ ID NO.2) to primer sequence；Expand the forward primer sequence of GAPDH genes It is classified as 5 '-AAAGGGTCATCATCTCTG-3 ' (SEQ ID NO.3), reverse primer sequences are 5 '- GCTGTTGTCATACTTCTC-3 ' (SEQ ID NO.4), operations are carried out on ice.Amplification program is：95 DEG C of 5min, (95 DEG C of 5s, 60 DEG C of 60s) * 40 circulations.It is fixed in real time in Light Cycler fluorescence using SYBR Green as fluorescent marker The enterprising performing PCR reaction of PCR instrument is measured, purpose band is determined by melt curve analysis analysis and electrophoresis, Δ Δ CT methods carry out relative quantification.
5th, Western blot are detected
Mechanical homogenisation after tissue is shredded, 4 DEG C of 12 000r min centrifuges 10min.Total protein is surveyed in Coomassie light blue R250 dyeing Concentration, same level is transferred to by each group protein concentration.10%SDS- polyacrylamide gels are prepared, add the μ of protein sample 100 per hole Albumen is gone to by nitrocellulose filter by half dry type electrotransfer instrument (Bio-Rad companies of the U.S.) after g, electrophoresis.Ponceau S contaminates Color determines transferring film situation and labelled protein Marker positions.5% 4 DEG C of refrigerator overnights of skimmed milk power TBS buffer blinds；Primary antibody 1: 1 000 dilutions, shake TBS after 2h and wash film 3 times at room temperature；Peroxidase labelling goat anti-rabbit igg-HRP is added by 1: 1 000 60min, TBS are washed after 3 times and developed photographic film after addition ECL 2-3min, scotography 2min.Labworks image acquisition and analysis software shooting point Analysis.
The gray value of protein band is analyzed using Image J softwares, using β-actin as internal reference, by SYNC albumen The gray value of band is normalized.Result data is represented in the way of mean+SD, is used SPSS13.0 statistical softwares carry out statistical analysis, and difference between the two is examined using t, it is believed that work as P<There is system when 0.05 Meter learns meaning.
6th, result
(1) QPCR results
As shown in figure 1, compared with normal control tissue, SYNC gene mRNA levels significantly drop in abdominal aortic aneurysm tissue Low, difference has statistical significance (P<0.05).
(2) Western blot results
As shown in Fig. 2 compared with normal control tissue, SYNC protein levels are significantly reduced in abdominal aortic aneurysm tissue, poor It is different that there is statistical significance (P<0.05).
Embodiment 3 is overexpressed SYNC gene expressions
1st, SYNC gene recombination plasmids are built
(1) coded sequence of SYNC genes is expanded；
(2) amplimer is designed；
(3) the SYNC genes after amplification are connected in expression vector pcDNA3.0, build pcDNA3.0-SYNC restructuring tables Up to carrier.
2nd, the culture of human aortic smooth muscle cell
T/G HA-VSMC cells (abbreviation HVSMC cells) are added with the DMEM high glucose mediums containing 10% hyclone (FBS) Penicillin 100units/ml, the μ g/ml of streptomysin 100, put 37 DEG C, 5%CO2Incubator in cultivate, change 1 culture every 24h Liquid, 48h is passed on 1 time.Take the logarithm growth period cell carry out subsequent experimental.
3rd, cell transfecting
Liposome Lipofectamine2000 is used for transfection reagent.2 groups of experiment point：Negative control group (transfection pcDNA3.0)；Experimental group (transfection pcDNA3.0-SYNC).Take the logarithm the HVSMC cells in growth period, be inoculated in 6 hole cells Culture plate.Tissue Culture Plate coverage rate is about 70%-80% after 24h.Rotaring transfecting mode is with reference to Lipofectamine2000 specifications Carry out.
4th, Western blot experiments detection pcDNA3.0-SYNC overexpression situation
Step be the same as Example 2.
5th, result
As shown in figure 3, compared with negative control group (transfection pcDNA3.0), experimental group (transfection pcDNA3.0-SYNC) is thin SYNC expressing quantities are significantly raised in born of the same parents, and difference has statistical significance (P<0.05).
Influence of the expression of embodiment 4SYNC genes to human aortic smooth muscle cell's apoptosis
Using Annexin V-PE/7-AAD apoptosis detection kit (be purchased from Beijing Ke Yue bio tech ltd, KTK102-020) influence of the detection SYNC gene expressions to Vascular Smooth Muscle Cell Apoptosis.
1st, cell culture and transfection
Carried out according to the method for embodiment 3.
2nd, apoptosis-inducing and detection
After cell transfecting 24 hours, each group cell uses the DMEM medium cultures of serum-free instead respectively, with apoptosis-induced.48 Hour after collected by trypsinisation cell, washed with PBS 2 times, according to explanation analysis add binding buffer (200 μ l), Annexin V-PE (10 μ l) and 7-AAD (5 μ l) lucifuge are incubated 15min, are eventually adding 300 μ l ending buffer, upper machine Detect the change of Apoptosis.
3rd, result
As a result show, each group cell non-serum culture medium starvation is stimulated after 48h, and flow cytomery apoptosis rate shows It is (18.561 ± 1.254) % to show pcDNA3.0-SYNC transfection groups apoptosis rate；And negative control group apoptosis rate is (31.318 ± 1.425) %；PcDNA3.0-SYNC transfection groups apoptosis rate is substantially reduced compared with negative control group, and difference has system Meter learns meaning (P<0.05), the above results show, promote SYNC gene expressions to suppress withering for human aortic smooth muscle cell Die.Because the apoptosis increase of human aortic smooth muscle cell result in the generation of abdominal aneurvsm, therefore promote SYNC gene tables Up to can realize treatment abdominal aneurvsm purpose.
The explanation of above-described embodiment is only intended to understand the method and its core concept of the present invention.It should be pointed out that for this For the those of ordinary skill in field, under the premise without departing from the principles of the invention, some improve can also be carried out to the present invention And modification, these are improved and modification will be also fallen into the protection domain of the claims in the present invention.
SEQUENCE LISTING
<110>Beijing Yang Shen biology information technologies Co., Ltd
<120>A kind of aneurysmal molecular marker of diagnosis and treatment abdomen
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213>Artificial sequence
<400> 1
gaagccttga acccagaa 18
<210> 2
<211> 19
<212> DNA
<213>Artificial sequence
<400> 2
gtcctccagg tagagaatg 19
<210> 3
<211> 18
<212> DNA
<213>Artificial sequence
<400> 3
aaagggtcat catctctg 18
<210> 4
<211> 18
<212> DNA
<213>Artificial sequence
<400> 4
gctgttgtca tacttctc 18

Claims (10)

1. detect application of the SYNC product in diagnosis abdominal aneurvsm instrument is prepared.
2. application according to claim 1, it is characterised in that the product of the detection SYNC includes detection SYNC gene tables Up to the product of amount.
3. application according to claim 1 or 2, it is characterised in that the product of the detection SYNC includes quantifying The product of SYNC gene mRNAs, and/or the product of SYNC albumen can be quantified.
4. application according to claim 3, it is characterised in that the reagent that can quantify SYNC gene mRNAs includes real When quantitative PCR in the primer of specific amplified SYNC genes that uses, the primer sequence such as SEQ ID NO.1 and SEQ ID Shown in NO.2；The reagent that SYNC albumen can be quantified includes the antibody of specific binding SYNC albumen.
5. a kind of instrument for diagnosing abdominal aneurvsm, it is characterised in that the instrument includes that SYNC gene expression amounts can be detected Instrument.
6. instrument according to claim 5, it is characterised in that the instrument includes that SYNC gene mRNAs can be quantified Reagent, and/or the reagent of SYNC albumen can be quantified.
7. instrument according to claim 6, it is characterised in that the reagent that can quantify SYNC gene mRNAs is real-time The primer of the specific amplified SYNC genes used in quantitative PCR, the primer sequence such as SEQ ID NO.1 and SEQ ID NO.2 It is shown.
8. a kind of medicine for treating abdominal aneurvsm, it is characterised in that the medicine includes SYNC activator.
9. medicine according to claim 8, it is characterised in that the activator can promote or strengthen SYNC or be related to The expression of the material of SYNC upstreams or downstream pathway or activity.
10. application of the activator in the medicine for preparing treatment abdominal aneurvsm described in claim 8 or 9.
CN201710407607.5A 2017-06-02 2017-06-02 A kind of aneurysmal molecular marker of diagnosis and treatment abdomen Withdrawn CN107022636A (en)

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Citations (2)

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CN103988080A (en) * 2011-10-11 2014-08-13 加利福尼亚大学董事会 Biomarker for abdominal aortic aneurysm
CN106222259A (en) * 2016-07-27 2016-12-14 北京泱深生物信息技术有限公司 A kind of molecular marker of diagnosis and treatment multiple myeloma

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Publication number Priority date Publication date Assignee Title
CN103988080A (en) * 2011-10-11 2014-08-13 加利福尼亚大学董事会 Biomarker for abdominal aortic aneurysm
CN106222259A (en) * 2016-07-27 2016-12-14 北京泱深生物信息技术有限公司 A kind of molecular marker of diagnosis and treatment multiple myeloma

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