CN110499365A - Application of the AGAP9 in the product of preparation diagnosis osteoarthritis - Google Patents
Application of the AGAP9 in the product of preparation diagnosis osteoarthritis Download PDFInfo
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- CN110499365A CN110499365A CN201910934540.XA CN201910934540A CN110499365A CN 110499365 A CN110499365 A CN 110499365A CN 201910934540 A CN201910934540 A CN 201910934540A CN 110499365 A CN110499365 A CN 110499365A
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- agap9
- osteoarthritis
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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
- G01N33/6893—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
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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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
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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
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/105—Osteoarthritis, e.g. cartilage alteration, hypertrophy of bone
Abstract
The invention discloses a kind of diagnostic tool of osteoarthritis, which is to realize diagnostic purpose by detection AGAP9 gene and its expression product.Research of the present invention has shown that compared with normal synovial tissue, the mRNA expression of Human Osteoarthritis AGAP9 gene significantly raises.According to the existing correlation between AGAP9 gene and osteoarthritis, the kit of diagnosis osteoarthritis can be prepared, which can clinically be widely applied.In addition, the invention discloses inhibition AGAP9 gene expressions can inhibit synovial cell proliferation, show that AGAP9 gene can be used for researching and developing the drug for the treatment of osteoarthritis, research achievement of the invention provides theories integration to develop new clinical application.
Description
Technical field
The present invention relates to medical diagnosis on disease fields, more particularly it relates to detect the abnormal disease for means of AGAP9
Diagnostic method.
Background technique
A kind of osteoarthritis (OA) chronic, the progressivity joint disease more typical as the middle-aged and the old, can behave as difference
The symptoms such as painful swelling of joints, the limitation of activity of degree are destroyed, joint synovitis disease reaction with articular cartilage and subchondral bone progressive
For major pathologic features, pathogenesis is not fully understood so far, and early diagnosis is difficult, and clinically common diagnostic means are past
Toward middle and advanced stage patient is directed to, early stage patient is as a special population, and the horizontal influence to its Health outcome of early diagnosis is more
It is direct and obvious.
Osteoarthritis is considered as a kind of chronic disease, can be kept over a long time " silencing ", its early stage of clinical diagnosis
It is rather difficult；Diagnosis is normally based on clinical symptoms and Imaging examination.However x-ray has a pitiful sensitivity and precision,
An accumulated damage is only provided, error is relatively large, cannot detect osteoarthritis in advance or monitor the progress of joint injury.CT
And similarly there are some problems in MRI.Due to the limitation of these tools, it is currently available for the osteoarthritis of assessment early stage,
Only biomarker: research concentrates on the construction package of cartilage matrix, especially II Collagen Type VI catabolite.However both at home and abroad
The results show of some scholars detects merely II Collagen Type VI catabolite, not can solve diagnosis early stage osteoarthritis still
Sensitivity and precision problem, slowly just form such common recognition in this way: simple detection H-type Collagen Degradation Products are unworkable.Closely
Year, people more paid close attention on cell factor IL-1, in particular, it is considered as the central factor and monokaryon of osteoarthritis developes
Perhaps, nucleotide polymorphism gene biological marker and by technologies such as metabolism group is conducive to detect early stage osteoarthritis.It may
Substitution method, such as molecular labeling can quantify, and reliably, sensitively detect the joint of osteoarthritis in early stage
Variation.The experiment is only easier universal clinical detection side by more mature at present there is presently no that step in the future is gone to
Method attempts its feasibility.
The early diagnosis for especially carrying out osteoarthritis at the genetic level on a molecular scale has become Bones and joints
The development trend of scorching diagnostic field, in terms of diagnosis, application No. is: 201510548635.X, 2015105495645,
2015105486241, the patent of 201510627048.X, 2015107250040,201510724747.6,2015107257552 text
Offer the gene marker for disclosing and can be used for osteoarthritis diagnosis.The application is found newly under the enlightenment of the prior art
It can be used for the biomarker of osteoarthritis diagnosis.
Summary of the invention
One of the objects of the present invention is to provide a kind of for diagnosing the gene marker of osteoarthritis.The present invention utilizes survey
Sequence and QPCR are the experiment proves that AGAP9 gene is significantly higher than Normal synovial group in the expression of Osteoarthritic Synovium tissue
It knits, therefore can be using AGAP9 gene as the gene marker of diagnosis osteoarthritis.
The second object of the present invention is to provide a kind of drug targets for treating osteoarthritis.The present invention utilizes cell in vitro
Experiments have shown that inhibiting AGAP9 gene to can inhibit synovial cell proliferation, therefore can be using AGAP9 gene as treatment osteoarthritis
Drug targets.
To achieve the goals above, the present invention adopts the following technical scheme:
According to an aspect of the present invention, the present invention provides a kind of for diagnosing the product of osteoarthritis, the product
Reagent including detecting AGAP9 gene expression.
Further, the reagent includes the reagent that can quantify AGAP9 gene mRNA, and/or can quantify AGAP9 albumen
Reagent.
Further, the product of present invention front includes but is not limited to chip, kit, test paper or high-flux sequence
Platform；High-flux sequence platform is a kind of tool of special diagnosis osteoarthritis, right with the development of high throughput sequencing technologies
The building of the gene expression profile of one people will become very easily work.By the gene for comparing Disease and normal population
Express spectra, the exception for being easy to analyze which gene are related to disease.Therefore, AGAP9 gene is known in high-flux sequence
The abnormal purposes for also belonging to AGAP9 gene related to osteoarthritis, equally within protection scope of the present invention.
Kit of the invention includes the primer of specific amplified AGAP9 gene.As a specific embodiment of the invention,
The primer sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.
Kit of the invention can also include: SYBR Green polymerase chain reaction system, for expanding AGAP9 base
The primer pair of cause and/or house-keeping gene.
Preferably, the SYBR Green polymerase chain reaction system includes PCR buffer, dNTPs, SYBR Green
Fluorescent dye.
In above-mentioned technical proposal, the prior art that the PCR buffer is known to the skilled person, it is preferable that institute
Stating PCR buffer includes: 25mM KCL, 2.5mM MgCL2, 200mM (NH4)2SO4。
The primer pair is that those skilled in the art can design according to the conventional design principle of design of primers；Preferred
In embodiment, positive the sequence 5 '-TAAGAAGAAGCAACTGTA-3 ' (SEQ ID NO.1) of AGAP9 gene is expanded, reversely
Sequence 5 '-GTGTGATATGATGAGGTA-3 ' (SEQ ID NO.2)；Preferred β-the actin of house-keeping gene, is expanding the gene just
It is 5 '-GTGGGGCGCCCCAGGCACCA-3 ' (SEQ ID NO.3), reverse primer sequences 5 '-to primer sequence
CTCCTTAATGTCACGCACGATTT-3’(SEQ ID NO.4)。
Further, kit of the invention can also include M-MLV reverse transcription system, which includes: T is repeated
Oligonucleotides Oligo (dT), reverse transcription reaction liquid, M-MLV reverse transcriptase, RNase inhibitor, dNTPs.
Preferably, reverse transcription reaction liquid includes: 250mM Tris-HCL (pH8.3), 375mM KCL, 15mM MgCL2、
50mM DTT。
RNase inhibitor commonly used in the art can be selected in RNase inhibitor, and preferably Bacillus coli expression is noncompetitive
Inhibit the recombinant protease of RNA enzyme.
Preferably, kit of the invention also includes that RNA extracts reagent, RNA extract reagent include Trizol, it is chloroform, different
Propyl alcohol, 75% ethyl alcohol.
Kit of the invention is stored in -20 DEG C, reduces multigelation to the greatest extent.
According to another aspect of the present invention, the present invention also provides the reagents of detection AGAP9 diagnoses Bones and joints in preparation
Application in scorching product.
Further, the reagent of the detection AGAP9 includes the reagent for detecting AGAP9 gene expression.
Further, the reagent of the detection AGAP9 gene expression includes the reagent that can quantify AGAP9 gene mRNA,
And/or the reagent of AGAP9 albumen can be quantified.
The reagent of quantitative AGAP9 gene mRNA includes: such as PCR, Southern hybridization, Northern hybridization, dot blot, glimmering
Light in situ hybridization (FISH), DNA microarray, ASO method, high-flux sequence platform etc. qualitatively, quantitatively or semi-quantitatively detect
Any reagent used in the method for mrna expression.
Further, the reagent of the quantitative AGAP9 gene mRNA includes primer and/or probe used in QPCR.
In specific embodiments of the present invention, the reagent of the quantitative AGAP9 gene mRNA includes used in QPCR
Primer, the primer sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.
Primer recited above can be prepared by chemical synthesis, using those skilled in the art will know that method refer to
Given information is prepared to be suitably designed by chemical synthesis.
Mentioned-above probe can be prepared by chemical synthesis, by using those skilled in the art will know that method
It appropriately designs with reference to Given information, and is prepared by chemical synthesis, or can be by containing the phase from biomaterial preparation
Hope the gene of nucleic acid sequence, and using it is expected designed for amplification nucleic acid sequence primer amplification it prepare.
The reagent of quantitative AGAP9 albumen includes such as ELISA, radioimmunoassay, immunohistochemical method, Western
Trace etc. is able to detect any reagent used in the method for protein expression level.
The reagent that the present invention can be used for quantifying AGAP9 albumen includes the antibody for being directed to AGAP9 albumen, and the antibody can be
Monoclonal antibody, polyclonal antibody, multi-specific antibody (such as bispecific antibody).
The sample of the mentioned-above product testing of the invention is not particularly limited, as long as it is suitable for survey of the invention
It is fixed；For example, it may include tissue, blood, blood plasma, serum, lymph, urine, serous cavity liquid, spinal fluid, synovia, aqueous humor,
Tear, saliva or its fraction or treated material.In specific embodiments of the present invention, the sample is from tested
The tissue of person.
The present invention also provides a kind of methods for diagnosing osteoarthritis, and described method includes following steps:
(1) sample that subject contains AGAP9 gene expression product is obtained；
(2) expression of AGAP9 gene or albumen in sample is detected；
(3) it associates whether by the expression of the AGAP9 gene or albumen that measure with subject's illness；
(4) compared with normal synovial tissue, if the expression of AGAP9 gene or albumen increases, subject's quilt
It is diagnosed as osteoarthritis, or judges that risk of the subject with osteoarthritis is high.
In the context of the present invention, " diagnosis osteoarthritis " both includes judging whether subject has suffered from Bones and joints
Inflammation also includes the risk that judges subject and whether there is with osteoarthritis.
As used herein, term " antibody ", it is intended that be specifically bound to specific antigen or with specific antigen interaction
Any antigen-binding molecule or molecular complex, it includes at least one complementary determining regions (CDR).Term " antibody " includes containing
Have 4 polypeptide chains (that is, by cystine linkage 2 weights (H) chain interconnected and 2 light (L) chain) and its polymer (such as
IgM).Each heavy chain contains heavy chain variable region and heavy chain constant region.Heavy chain constant region contain 3 domain Cs H1, CH2 and
CH3.Each light chain contains light chain variable region and constant region of light chain.Constant region of light chain contains a structural domain (CL1).VH and VL
Area can be further subdivided into hypervariable region (referred to as complementary determining region (CDR)), more conservative be known as framework region (FR) wherein being studded with
Region.Each VH and VL is made of three CDR and four FR, is arranged in the following order from aminoterminal to c-terminus: FR1,
CDR1, FR2, CDR2, FR3, CDR3 and FR4.
As used herein, term " antibody " further includes the antigen-binding fragment of entire antibody molecule.The antigen binding of antibody
Segment can use any suitable standard technique, such as proteolytic digestion or be related to encoding antibody variable and optionally constant
The recombination renovation technique of the manipulation and expression of the DNA in area, from for example complete antibody derivative molecule.Such DNA is
It is knowing and/or be easy to obtain from such as commercial source, DNA library (including such as bacteriophage-antibody library), or it can be synthesized.
The DNA can be sequenced and chemically or Protocols in Molecular Biology is manipulated, thus for example will be one or more variable
Area and/or constant region are arranged in suitable configuration, or introduce codon, generate cysteine residues, modify, add or delete ammonia
Base acid etc..
The non-limiting example of antigen-binding fragment includes: (i) Fab segment；(ii) 2 segment of F (ab')；(iii) Fd piece
Section；(iv) Fv segment；(v) scFv (scFv) molecule；(vi) dAb segment；And (vii) by analog antibody hypervariable region (such as
Isolated complementary determining region (CDR), such as CDR3 peptide) amino acid residue composition minimum recognition unit or restricted type FR3-
CDR3-FR4 peptide.Antigen-binding fragment further includes the molecule of other engineering, such as domain-specific antibody, single domain antibody, knot
Antibody, the double antibody, three antibody, four antibody, miniantibody, nano antibody (example that antibody, chimeric antibody, the CDR of structure domain missing are transplanted
Such as monovalent nano antibody, divalent nano antibody), little module immune drug (smallmodularimmunopharmaceutic
Als) (SMIP) and shark can be changed the domain IgNAR.
" monoclonal antibody " is by the B- lymphocyte of monoclonal or by will coding single antibody (or its antigen binding
Segment) antibody light chain and heavy chain variable region the antibody that is generated to cell therein or its offspring of nucleic acid transfection.Monoclonal is anti-
Body generates by methods known to those skilled in the art, such as by merging manufacture from myeloma cell and immune spleen cell
Hybrid antibody forms cell to carry out.These fused cells and their offspring are referred to as " hybridoma ".
" multi-specificity antibody " can be to the different epitopes of a target polypeptide have specificity or may containing pair
More than one target polypeptide has the antigen-binding domain of specificity.See, for example, Tutt etc., 1991, J.Immunol.147:60-
69；Kufer etc., 2004, TrendsBiotechnol.22:238-244.Antibody or its segment can functionally link (example
Such as, pass through chemical coupling, genetic fusion, noncovalent associations or other modes) to one or more other molecular entities, it is such as another
A antibody or antibody fragment, to generate the bispecific or multi-specificity antibody that have second of binding specificity.
According to a further aspect of the invention, the present invention provides AGAP9 genes in the drug for preparing treatment osteoarthritis
In application.
According to a further aspect of the invention, the present invention provides inhibit the reagent of AGAP9 gene expression to treat in preparation
Application in the drug of osteoarthritis.
Further, the drug includes that can inhibit or weaken AGAP9 or be related to the object of the upstream AGAP9 or downstream pathway
The expression of matter or active ingredient.
Inhibit or weaken AGAP9 or is related to the expression or active at subpackage of the substance of the upstream AGAP9 or downstream pathway
Include siRNA, shRNA, inhibition miRNA, antibody, other small molecule compounds.
Inhibit or weaken AGAP9 or is related to expression or the active ingredient one of the substance of the upstream AGAP9 or downstream pathway
Aspect passes through the expression for inhibiting AGAP9 albumen, thus osteoarthritis caused by treating because of AGAP9 albumen excess.It on the other hand can
With for inhibiting the activity of AGAP9 albumen, to treat osteoarthritis.
Drug of the invention can be prepared into various dosage forms 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 administration method of drug of the invention is unrestricted, as long as it can play desired therapeutic effect or preventive effect i.e.
Can, including but not limited to intravenously, in peritonaeum, intraocularly, intra-arterial, intrapulmonary is taken orally, in vesicle, intramuscular, intratracheally, subcutaneously
, local by pleura by skin, sucking, by mucous membrane, skin, stomach is intra-articular, intra-ventricle, rectum, vagina,
In skull, in urethra, in liver.In some cases, it can systematically be administered.It is locally to be administered in some cases.
The dosage of drug of the invention is unrestricted, can as long as obtaining desired therapeutic effect or preventive effect
To carry out appropriate determination according to symptom, gender, age etc..Example can be used in the dosage of therapeutic agent or prophylactic agent of the invention
Such as the therapeutic effect of disease or preventive effect are determined as index.
In the context of the present invention, " AGAP9 gene " (NC_000010.11 (47501854..47523638,
Complement)) sequence can inquire in international public GenBank GeneBank.
" treatment " used herein is covered treatment-related in such as mankind of the mammal with related disease or illness
Disease or morbid state, and include:
(1) prevent disease or morbid state occurs in mammals, especially when the mammal is susceptible in the disease
Diseased state, but when being not yet diagnosed with this morbid state；
(2) inhibit disease or morbid state, that is, prevent its generation；Or
(3) alleviate disease or morbid state, even if disease or morbid state subside.
Term " treatment " is usually directed to treatment mankind or animal (for example, being applied by animal doctor), wherein can reach certain pre-
The therapeutic effect of phase, for example, inhibiting the development (including reduce development speed, stop development) of illness, improving illness and healing
Illness.It further include the treatment as precautionary measures (such as prevention).To not yet development be illness but have development be the illness endanger
The purposes of the patient of danger, is also included in term " treatment ".
Detailed description of the invention
Fig. 1 shows the statistical chart for detecting AGAP9 gene differential expression situation in mRNA level in-site using QPCR；
Fig. 2 shows the statistical chart for detecting AGAP9 gene differential expression situation on protein level using immunoblotting；
Fig. 3 shows the statistical chart that AGAP9 gene expression cell proliferation influences.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this
It invents rather than limits the scope of the invention.Test method without specific conditions in embodiment, usually according to conventional strip
Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory
Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.
Embodiment 1 screens difference expression gene
1, it draws materials:
Synovial tissue's (OA group) of 5 OA patients is from row knee prosthesis or patient OA of villusectomy.It is used
Case meets the diagnostic criteria about OA of Altam proposition.3 normal synovial tissues (Nor group) are from trauma surgery patient
Synovial tissue of joint.Clinical sample used in this research know to patient and inform and Ethics Committee through hospital is logical
It crosses.
The clinical information of the research object of selection is as shown in table 1.
1 clinical information of table
2, the acquisition of RNA is organized
Total RNA is extracted from tissue sample, is examined using concentration and purity of the Nanodrop2000 to mentioned RNA
It surveys, agarose gel electrophoresis detects RNA integrality, and Agilent2100 measures RIN value.Single requirement for construction data base RNA total amount 5 μ g is dense
Degree >=200ng/ μ L, OD260/280 is between 1.8~2.2.
3, mRNA library construction
3 ' end of eukaryote mRNA has the structure of ployA tail, raw with the enrichment with magnetic bead eukaryon with Oligo (dT)
Object mRNA.Fragmentation buffer is added, mRNA is broken into short-movie section, using mRNA as template, with hexabasic base with power traction
Object (random hexamers) synthesizes first cDNA chain, and buffer, dNTPs, RNase H and DNA is then added
Polymerase I synthesizes Article 2 cDNA chain, after purifying by QiaQuick PCR kit and EB buffer is added to elute
It does end and repairs and connect sequence measuring joints, then carry out clip size selection with agarose gel electrophoresis, finally carry out PCR expansion
Increase, is sequenced using machine on the library built up.
4, upper machine sequencing
Machine sequencing process (mRNA) on llumina Hiseq x-ten
(1) library is enriched with, 15 cycles of PCR amplification；
(2) 2% agarose gels recycle purpose band (Certified Low Range Ultra Agarose)；
(3) TBS380 (Picogreen) is quantitative, mixes upper machine by ratio data；
(4) bridge-type PCR amplification is carried out on cBot, generates clusters；
(5) Hiseq x-ten microarray dataset carries out 2*100bp/300bp sequencing.
5, bioinformatic analysis
It is as follows that sequencing data obtains later mRNA analytic process:
(1) base of quality < 20 is fallen to the 5 ' of reads and 3 ' Duan Jinhang trim, trim with cutadapt, and deletes N
Reads (quality of sequencing data being had a look with fastQC, the data taken are completed to can skip this step when processing) greater than 10%；
(2) tophat is compared to mRNA with reference on genome.Reference genome version used in mRNA is GRCh38.p7,
Fasta and gff file download is from NCBI；
(3) expression quantity of cuffquant quantification of mrna and normalization output；
(4) cuffdiff packet compares control group with the differential expression of disease group mRNA.
6, result
Significant difference mRNA screening conditions: P<0.05, | log2FC |>1.It is obtained with the above standard screening in Normal synovial group
It knits and has differences gene 1068 between Osteoarthritic Synovium tissue, wherein expression up-regulation gene 516, expresses the base of downward
Because there is 552.
2 large sample of embodiment verifies the difference expression gene filtered out
Based on sequencing early period as a result, according to the size of P value, we select AGAP9 gene to verify.
1, sample collection
Osteoarthritic Synovium tissue and normal synovial tissue each 42 are collected according to the method for embodiment 1.
2, it is verified in mRNA level in-site
2.1 extract tissue RNA
Step is the same as embodiment 1.
2.2 reverse transcription
Reverse transcription uses Primescript 1stStrand cDNA synthesis kit kit, operating procedure are as follows
It carries out:
(1) following reaction liquid is added in microcentrifugal tube, as shown in table 2:
2 reaction liquid of table
| Reagent | Dosage |
| RNA | 2.0μg |
| dNTP | 1.0μl |
| Oligo(dT) | 2.0μl |
| Rnase free dH2O | Add to 10.0 μ l |
(2) 70 DEG C of incubation 5min, are rapidly cooled to 4 DEG C；
Following reaction reagent is added in microcentrifugal tube, reaction system is made, as shown in table 3:
The preparation of 3 reaction system of table
It gently shakes, after rapid centrifugation, 42 DEG C of reactions 1h, 70 DEG C of 10min terminate reaction, 4 DEG C of coolings, -20 DEG C of preservations.
Using SYBP Premix Ex TapTMII kit is carried out in Eppendorf Real-time PCR analyzer,
Concrete operations are as follows:
(1) following PCR reaction solution is prepared on ice, as shown in table 4:
The preparation of 4 PCR reaction solution of table
| Reagent | Dosage |
| SYBR | 10.0μl |
| Forward primer | 1.0μl |
| Reverse primer | 1.0μl |
| cDNA | 2.0μl |
| ddH2O | 6.0μl |
| Total amount | 20.0μl |
Primer sequence design is as follows:
AGAP9 gene:
5'-TAAGAAGAAGCAACTGTA-3'(SEQ ID NO.1)；
5’-GTGTGATATGATGAGGTA-3’(SEQ ID NO.2)
β-actin:
5'-GTGGGGCGCCCCAGGCACCA-3'(SEQ ID NO.3)；
5’-CTCCTTAATGTCACGCACGATTT-3’(SEQ ID NO.4)
(2) machine on executes following programs: 95 DEG C of initial denaturation 3min；95 DEG C of denaturation 15s.49 DEG C of annealing 20s, 72 DEG C of extensions
20s, totally 40 recycle.
As a result relative quantification method, formula 2 are used-△△ctIt calculates.Experiment is repeated 3 times.
△ ct=ct (A)-ct (β-actin)
△ △ ct=△ ct (experimental group)-△ ct (control group)
The results show that there is the AGAP9 gene in 42 Osteoarthritic Synovium tissues in 42 Osteoarthritic Synovium tissues
MRNA level in-site is significantly raised compared to the average value of normal synovial tissue；Statistical result is as shown in Figure 1, with normal synovial tissue phase
Than the mRNA level in-site of AGAP9 gene obviously increases in Osteoarthritic Synovium tissue, and difference has statistical significance (P < 0.05).
AGAP9 is inquired into the diagnostic value of osteoarthritis using Receiver operating curve, analyzes result such as 5 institute of table
Show.
5 ROC result of table statistics
3 AGAP9 gene pairs Osteoarthritic Synovium cell Proliferation of embodiment influences
1, AGAP9 gene expression is interfered
According to AGAP9 gene-correlation sequence, designed and synthesized by Shanghai JiMa pharmacy Technology Co., Ltd for AGAP9's
siRNA(siRNA-AGAP9).SiRNA-AGAP9 sequence is as follows: 5 '-UUACUUAGUUUACGAAAAGUATT- of positive-sense strand
3 ' (SEQ ID NO.5), antisense strand 5 '-CUUUUCGUAAACUAAGUAAAUTT-3 ' (SEQ ID NO.6).
2, cell culture
People's bone arthritis synovial cell (article No.: 408OA-05A, Cell applications, INC.) be grown in containing
In the synovial cell growth culture medium of 10%FBS and mycillin (article No.: 415-500, Cell applications, INC.),
37 DEG C are placed in, 5%CO2It is cultivated in wet incubator.
3, cell transfecting and expression detection
According to 2*105/ hole cell inoculation carries out siRNA-AGAP9 when cell density is to 75% or so in 6 orifice plates
With siRNA-NC (general negative control siRNA is purchased from Shanghai JiMa pharmacy Technology Co., Ltd) transfection.Transfection procedure is according to rouge
The specification of plastid 2000 carries out.6 orifice plates are placed on ice by transfection after 48 hours, and every hole is added RIPA lysate and (contains 1mmol/L
Protease inhibitors PMSF) 150 μ l effect 1min, extracts total protein, 4 DEG C, 12000rmin-1It is centrifuged 15min, takes supernatant,
After BCA protein reagent box protein is quantitative, then SDS-PAGE electrophoresis is carried out, transferring film, 5% rouge milk powder room temperature closes 1h, and 4 DEG C of incubations are anti-
Body antibody；After overnight, TBST is washed film 3 times, each 10min, and secondary antibody is incubated at room temperature 1h, and TBST washes film 3 times, each 10min, ECL
Chemiluminescence exposes 3-5min, development.The results show that AGAP9 gene expression can be effectively suppressed in siRNA-AGAP9, inhibiting rate is
73%, difference has statistical significance P < 0.05.
4, cell Proliferation
People's bone arthritis synovial cell is inoculated in 96 orifice plates, and 100 μ l (2000 cells) is added in every hole, next day cell patch
Cell transfecting is carried out according to above method after wall, cell culture fluid is as a control group.16 hours are added before culture terminates
1u Ci's3H collects cell B liquid scintillation instrument detection proliferation.As a result as shown in figure 3, siRNA-AGAP9 can obviously inhibit synovial cell
Proliferation, difference have statistical significance P < 0.05.
The explanation of above-described embodiment is only intended to understand method and its core concept of the invention.It should be pointed out that for this
For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention
And modification, these improvement and modification will also be fallen into the protection scope of the claims in the present invention.
Sequence table
<110>People's Hospital of Deyang City
<120>application of the AGAP9 in the product of preparation diagnosis osteoarthritis
<150> 201811260546.5
<151> 2018-10-26
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gtgtgatatg atgaggta 18
<210> 3
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gtggggcgcc ccaggcacca 20
<210> 4
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
ctccttaatg tcacgcacga ttt 23
<210> 5
<211> 23
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 5
uuacuuaguu uacgaaaagu att 23
<210> 6
<211> 23
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 6
cuuuucguaa acuaaguaaa utt 23
Claims (10)
1. a kind of for diagnosing the product of osteoarthritis, which is characterized in that the product includes the examination for detecting AGAP9 gene expression
Agent.
2. product according to claim 1, which is characterized in that the reagent includes that can quantify AGAP9 gene mRNA
Reagent, and/or the reagent of AGAP9 albumen can be quantified.
3. product according to claim 1 or 2, which is characterized in that the product includes chip, kit, test paper or height
Flux microarray dataset.
4. product according to claim 3, which is characterized in that the kit includes drawing for specific amplified AGAP9 gene
Object, the primer sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.
5. product according to claim 4, which is characterized in that the kit includes SYBR Green polymerase chain reaction
Answer system；The SYBR Green polymerase chain reaction system includes PCR buffer, dNTPs, SYBR Green fluorescence dye
Material.
6. product according to claim 5, which is characterized in that the kit includes M-MLV reverse transcription system；It is described inverse
Transcription system includes: T repeat oligonucleotides Oligo (dT), reverse transcription reaction liquid, M-MLV reverse transcriptase, RNase inhibitor,
dNTPs。
7. detecting application of the reagent of AGAP9 in the product of preparation diagnosis osteoarthritis.
8. application according to claim 7, which is characterized in that the product includes described in any one of claims 1-6
Product.
Application of the 9.AGAP9 in the drug of preparation treatment osteoarthritis.
10. inhibiting application of the reagent of AGAP9 gene expression in the drug of preparation treatment osteoarthritis.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2018112605465 | 2018-10-26 | ||
| CN201811260546.5A CN109295207A (en) | 2018-10-26 | 2018-10-26 | Application of the AGAP9 in the product of preparation diagnosis osteoarthritis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110499365A true CN110499365A (en) | 2019-11-26 |
| CN110499365B CN110499365B (en) | 2020-09-29 |
Family
ID=65157928
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811260546.5A Pending CN109295207A (en) | 2018-10-26 | 2018-10-26 | Application of the AGAP9 in the product of preparation diagnosis osteoarthritis |
| CN201910934540.XA Active CN110499365B (en) | 2018-10-26 | 2019-09-29 | Application of AGAP9 in preparation of product for diagnosing osteoarthritis |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811260546.5A Pending CN109295207A (en) | 2018-10-26 | 2018-10-26 | Application of the AGAP9 in the product of preparation diagnosis osteoarthritis |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN109295207A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105755146A (en) * | 2016-04-29 | 2016-07-13 | 北京致成生物医学科技有限公司 | Application of SERPINB3 gene in preparing osteoarthritis diagnosis preparation |
| CN105886628A (en) * | 2016-04-29 | 2016-08-24 | 肖刻 | Application of SPRR1A gene in preparation of osteoarthritis diagnosis product |
-
2018
- 2018-10-26 CN CN201811260546.5A patent/CN109295207A/en active Pending
-
2019
- 2019-09-29 CN CN201910934540.XA patent/CN110499365B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105755146A (en) * | 2016-04-29 | 2016-07-13 | 北京致成生物医学科技有限公司 | Application of SERPINB3 gene in preparing osteoarthritis diagnosis preparation |
| CN105886628A (en) * | 2016-04-29 | 2016-08-24 | 肖刻 | Application of SPRR1A gene in preparation of osteoarthritis diagnosis product |
Non-Patent Citations (3)
| Title |
|---|
| HONGWEI QIN等: ""Inhibition of the JAK/STAT Pathway Protects Against α-Synuclein-Induced Neuroinflammation and Dopaminergic Neurodegeneration"", 《 THE JOURNAL OF NEUROSCIENCE》 * |
| YUNDONG HE等: ""Androgen receptor splice variants bind to constitutively open chromatin and promote abiraterone-resistant growth of prostate cancer"", 《NUCLEIC ACIDS RESEARCH》 * |
| 宋朋飞等: ""滑膜病变在骨关节炎中的表现"", 《中国骨伤》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109295207A (en) | 2019-02-01 |
| CN110499365B (en) | 2020-09-29 |
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