CN109295208A - Application of the PI15 as osteoarthritis marker - Google Patents
Application of the PI15 as osteoarthritis marker Download PDFInfo
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- CN109295208A CN109295208A CN201811260578.5A CN201811260578A CN109295208A CN 109295208 A CN109295208 A CN 109295208A CN 201811260578 A CN201811260578 A CN 201811260578A CN 109295208 A CN109295208 A CN 109295208A
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- gene
- osteoarthritis
- albumen
- expression
- nucleic acid
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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
- C12Q2600/118—Prognosis of disease development
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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
Application the invention discloses PI15 as osteoarthritis marker.PI15 can be used for judging whether subject has the risk for suffering from osteoarthritis or judge whether subject suffers from osteoarthritis.In addition, PI15 can be also used for the drug of preparation treatment osteoarthritis.The present invention provides new diagnostic method for the clinical osteoarthritis of diagnosis on a molecular scale, while new drug target is provided for the gene therapy of osteoarthritis.
Description
Technical field
The present invention relates to diagnosis, treatment, prediction prognosis fields, more particularly it relates to be to detect PI15 extremely
The diagnosis of means, prediction method of prognosis.
Background technique
Osteoarthritis (Osterarthritis, OA) is also referred to as degenerative arthritis, be in the elderly and overweight people most
Universal disease.OA is the disease in joint, but be different from rheumatoid arthritis (RA), this disease be not it is systematic, lead to
Often only influence one or several joints.This disease leads to total destruction of articular cartilage, the hardening and spur shape of lower section bone
At, cause locomitivity lose and pain.Final result often needs total joint replacement.
Demography trend shows that the year two thousand twenty, the crowd influenced by joint disease will be added to 50%, and the whole world will
There is puzzlement of 5.7 hundred million people by osteoarthritis.Chinese society progresses into aging society, therefore we will face osteoarthritis
The generally popular period of morbidity.It is particularly significant to the research of the disease and urgent.
Mind in articular cartilage, synovial membrane, subchondral bone, ligament or periarticular can occur for the primary exception of osteoarthritis
Through musculature.The damage of articular cartilage is that osteoarthritis most significantly changes.Articular cartilage forfeiture is the key that osteoarthritis
Lesion.With the decline of proteoglycan content, articular cartilage is gradually slowly degraded.Due to the proteoglycans of Human Osteoarthritis,
Collagen and hyaluronic acid synthesis rate increase, so that the catabolic activity of tissue is especially high.Generally, it is considered that protein resolvase
It is most important reason in osteoarthritic joint cartilage loss with metalloprotease.Without blood vessel in articular cartilage tissue
And nerve, the reaction to wound, inflammation are situated between by such as cell factor of the protein in articular cartilage, synovial tissue or joint fluid etc.
It leads.Synovial membrane plays during the occurrence and development of osteoarthritis disorders important for maintaining the normal function in joint
Effect.
The inspection of iconography is clinically based primarily upon for the diagnosis of osteoarthritis at present.Between x-ray performance predominantly joint
Gap is narrow, and subchondral bony sclerosis and cystis degeneration, joint margins spur are formed, and articular surface withers, deforms and subluxation of joint etc..
MRI can show the lesion of the joint structures such as early stage cartilage, meniscus, be conducive to early diagnose.CT is used for the diagnosis of discopathy, excellent
In x-ray.Joint space narrows, Subchondral bone sclerosis, and marginality spur spinal joint is linked to be bone bridge.Also visible bone cyst and abnormal
Shape.As found, these variations can be used as the foundation of diagnosis and the degree of estimation joint injury.OA is generally no or seldom in early stage
There is symptom, only in the activity of inflammation secondary, pain or influence joint, patient just looks for a doctor.At this point, joint injury occurs
Long.Because method of the exploitation for the diagnosis of early stage osteoarthritis is a problem to be solved.
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
Bone is diagnosed by detection PI15 gene or protein expression difference one of the objects of the present invention is to provide one kind to close
Save scorching method.
The second object of the present invention is that providing one kind predicts that bone is closed by detection PI15 gene or protein expression difference
The method for saving scorching prognosis.
The third object of the present invention is to provide one kind by activation PI15 gene or PI15 albumen to treat osteoarthritis
Method.
The fourth object of the present invention is to provide a kind of for treating the drug of osteoarthritis.
To achieve the goals above, present invention employs following technical solutions:
The present invention provides use of the product of detection PI15 gene or PI15 albumen in preparation osteoarthritis diagnostic tool
On the way.
The present invention also provides the products of detection PI15 gene or PI15 albumen to predict osteoarthritis prognostic tool in preparation
In purposes.
Further, the product of the detection PI15 gene or PI15 albumen includes the table for detecting PI15 gene or PI15 albumen
Up to horizontal product.The product includes the nucleic acid that can combine PI15 gene or the substance (example that can combine PI15 albumen
Such as antibody).The nucleic acid is able to detect the expression of PI15 gene；The substance is able to detect the expression water of PI15 albumen
It is flat.
The product of detection PI15 gene of the invention can play its function based on the known method of nucleic acid molecules is used: such as
PCR, such as Southern hybridization, Northern hybridization, dot blot, fluorescence in situ hybridization (FISH), DNA microarray, ASO method, height
Flux microarray dataset etc..It can qualitatively, quantitatively or semi-quantitatively implement analysis using the product.
Include that nucleic acid in the said goods can be obtained by chemical synthesis, or by containing from biomaterial preparation
It is expected that the gene of nucleic acid, then using primer amplification designed for amplification expectation nucleic acid, it is obtained.
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 method etc..Amplification
Nucleic acid can be by using dot blotting hybridization method, Surface Plasmon Resonance (SPR method), PCR-RFLP method, original position RT-PCR
Method, PCR-SSO (sequence specific oligonucleotide) method, PCR-SSP method, AMPFLP (amplifiable fragment length polymorphism) method,
MVR-PCR method and PCR-SSCP (single-strand conformation polymorphism) method detect.
Nucleic acid recited above includes the primer for expanding PI15 gene, and the primer for including in product can be by passing through chemistry
Synthesis to prepare, by using those skilled in the art will know that method be suitably designed with reference to Given information, and passing through
Synthesis is learned to prepare.
In specific embodiments of the present invention, the nucleic acid is amplimer used in QPCR experiment, the primer
Sequence such as SEQ ID NO.1 (positive sequence) and SEQ ID NO.2 (reverse sequence) shown in.
Nucleic acid recited above may also include probe, and the probe can be prepared by chemical synthesis, by using this
The method that field technical staff knows appropriately is designed with reference to Given information, and is prepared by chemical synthesis, or can lead to
The gene for containing desired nucleic acid sequence from biomaterial preparation is crossed, and is expanded using the primer designed for amplification expectation nucleic acid sequence
Increase it to prepare.
The product of detection PI15 albumen of the invention can play its function based on the known method of antibody is used: for example,
It may include ELISA, radioimmunoassay, immunohistochemical method, western blot etc..
The product of detection PI15 albumen of the invention includes the antibody or its segment for specifically binding PI15 albumen.It can make
With the antibody or its segment of any structure and size, immunoglobulin class, origin etc., as long as it combines target protein.This
The antibody or its segment for including in the testing product of invention can be monoclonal or polyclonal.Antibody fragment refers to reservation antibody
Peptide to the active antibody of the combination of antigen a part of (Partial Fragment) or containing antibody a part.Antibody fragment may include F
(ab′)2, Fab ', Fab, scFv (scFv), the Fv (dsFv) of disulphide bonding or its polymer, the area dimerization V it is (dual anti-
Body) or peptide containing CDR.The product of detection PI15 albumen of the invention may include encoding antibody or Encoding Antibody Fragment
The isolated nucleic acid of amino acid sequence, the carrier comprising the nucleic acid, and carry the cell of the carrier.
Antibody can be obtained by the way that well known to a person skilled in the art methods.For example, preparation retains target all or in part
The mammalian cell expression vector of their polynucleotides of the polypeptide or integration coding of protein is as antigen.Exempted from using antigen
After epidemic disease animal, from the immune animal adaptive immune cell of process and myeloma cell is merged to obtain hybridoma.Then from hybridization
Tumor culture collects antibody.It can finally be implemented by using antibody of the PI15 albumen for being used as antigen or part thereof to acquisition
Antigentic specificity purifies to obtain the monoclonal antibody for PI15 albumen.Polyclonal antibody can be prepared as follows: with it is above
Identical antigen-immunized animal collects blood sample from by immune animal, serum is isolated from blood, then using upper
It states antigen and antigentic specificity purifying is implemented to serum.It can be by the antibody that is obtained with enzymatic treatment or by using the antibody of acquisition
Sequence information obtain antibody fragment.
The combination of marker and antibody or its segment 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. standards
Standby dyestuff, then mixed solution, then at being placed at room temperature for 10 minutes.In addition, the labelling kit of commercialization can be used in label, 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 kit-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-NH2,647 labelling kit-NH2 of HiLyte Fluor (TM) (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- marker protein mark
Remember kit (Funakoshi Corporation).For correct labeling, suitable instrument can be used to detect by label
Antibody or its segment.
As the sample according to testing product of the 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 for measurement of the invention；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
Material.
In specific embodiments of the present invention, tissue of the sample from subject.
In the present invention, " prognosis " refers to the process of patient by after surgical procedure or result.In the present specification, in advance
Can be afterwards by 1 after surgical procedure, 2,3,4,5,6,7,8,9,10,15,20 years or more long when life state.Prognosis can be with
It is predicted by checking biomarker, that is, PI15 albumen or encoding the gene of PI15 albumen.Prognosis prediction can be performed such that
According to biomarker with or without or being raised and lowered, determine that the prognosis of patient is good or bad, or determination is good
The probability of good prognosis or poor prognosis.
In the present invention, " prognosis bona " refers to by after surgical procedure, patient's long-term (such as 3,5,6,7,8,9,
10,15,20 years or longer) there is no critical condition.Alternatively, good prognosis can mean in such survival interior for a long time, without transfer, nothing
Recurrence or nothing are sent out again.
In the present invention, " prognosis mala " refers to patient by short-term after surgical procedure (such as 1,2,3,4,5 year
Or it is shorter) in occur pathological condition.
Prediction prognosis is referred to the process of prediction status of patient or as a result, is not meant to be predicted with 100% accuracy
The process or result of status of patient.Prediction prognosis refers to whether a possibility that determining certain processes or result increases, and simultaneously unexpectedly
Taste by determining a possibility that certain processes or result occurs or not compared with certain processes or result.Such as this
For invention, in the present invention in the horizontal patient reduced of PI15 gene or PI15 albumen, with the patient's phase for not showing this feature
Than more likely observing particular procedure or result.
Further, the product of the detection PI15 gene or PI15 albumen can be detection PI15 gene or PI15 albumen
Reagent is also possible to include kit, chip, test paper of the reagent etc., is also possible to measure using the high pass of the reagent
Sequence platform.
The present invention also provides a kind of tool for diagnosing osteoarthritis, the tool is able to detect PI15 gene or PI15 egg
White expression.The tool includes the nucleic acid that can combine PI15 gene or the substance (example that can combine PI15 albumen
Such as antibody).The nucleic acid is able to detect the expression of PI15 gene；The substance is able to detect the expression water of PI15 albumen
It is flat.
Further, the property of the nucleic acid and the substance is the same as noted earlier.
Further, the tool of the diagnosis osteoarthritis includes but is not limited to that chip, kit, test paper or high pass measure
Sequence platform；High-flux sequence platform is a kind of tool of special diagnosis osteoarthritis, with the development of high throughput sequencing technologies,
Very easily work will be become to the building of the gene expression profile of a people.By the base for comparing Disease and normal population
Because of express spectra, the exception for being easy to analyze which gene is related to disease.Therefore, PI15 gene is known in high-flux sequence
The abnormal purposes for also belonging to PI15 gene related to osteoarthritis, equally within protection scope of the present invention.
The present invention also provides a kind of tool for predicting osteoarthritis prognosis, the prediction osteoarthritis prognostic tool includes
The nucleic acid of PI15 gene can be combined or the substance (such as antibody) of PI15 albumen can be combined.The nucleic acid is able to detect
The mRNA level in-site of PI15 gene；The substance is able to detect the expression of PI15 albumen.
Further, the property of the nucleic acid and the substance is the same as noted earlier.
Further, the tool of the prediction osteoarthritis prognosis includes but is not limited to chip, kit, test paper or high pass
Measure microarray dataset；High-flux sequence platform is a kind of tool of special diagnosis osteoarthritis, with high throughput sequencing technologies
Development will become very easily work to the building of the gene expression profile of a people.Pass through comparison Disease and normal person
The gene expression profile of group, the exception for being easy to analyze which gene are related to disease.Therefore, PI15 is known in high-flux sequence
The exception of the gene purposes for also belonging to PI15 gene related to osteoarthritis, equally within protection scope of the present invention.
The number for the amino acid that anti-PI15 antibody used in testing product of the invention, diagnostic tool or its segment are identified
Mesh is not particularly limited, as long as antibody can combine PI15.
The present invention also provides a kind of diagnosis osteoarthritis or the methods for predicting osteoarthritis prognosis, and the method includes such as
Lower step:
(1) sample of subject is obtained；
(2) expression of PI15 gene or albumen in Samples subjects is detected；
(3) it associates whether by the expression of the PI15 gene or albumen that measure with the illness of subject.
(4) compared with the control, the expression of PI15 gene or albumen reduces, then the subject is diagnosed as Bones and joints
Inflammation or the subject are confirmed as prognosis mala.
The present invention also provides a kind for the treatment of methods of osteoarthritis, and the method includes activation PI15 gene or PI15 eggs
It is white.
Further, the method includes promoting the expression of PI15 gene, or expression or the enhancing PI15 of promotion PI15 albumen
The activity of albumen.
The present invention also provides a kind of drugs of activator containing PI15 gene or PI15 albumen.
The present invention also provides application of the above-mentioned activator in the drug of preparation treatment osteoarthritis.
The activator of PI15 gene or PI15 albumen of the invention is unrestricted, as long as can promote or enhance PI15
Or it is related to the expression or activity of the substance of the upstream PI15 or downstream pathway, and for treating the effective drug of osteoarthritis.
Further, the activator include PI15 gene, PI15 albumen, promoted type miRNA, promoted type transcriptional control because
Son or promoted type target small molecule compound.
The activator further includes carrier or host cell comprising carrying PI15 gene.
On the one hand activator of the invention can be used for supplementing the missing or deficiency of endogenic PI15 albumen, pass through raising
The expression of PI15 albumen, thus osteoarthritis caused by treating because of PI15 hypoproteinosis.On the other hand can be used for enhancing PI15
The activity of 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, " 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.
" 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 ".
The advantages of the present invention:
Of the invention has found a kind of molecular marker for diagnosing osteoarthritis, can be closed in bone using the molecular marker
Saving the scorching early stage occurred can be used as judging, provide the survival rate of patient.
In addition, the present invention is capable of providing significant information to determine treatment side for patient by the prognosis of prediction patient
Case strategy.
Of the invention includes that the therapeutic agent of the activator of PI15 gene or albumen can be used as the treatment of new osteoarthritis
Drug.
Detailed description of the invention
Fig. 1 shows the statistical chart for detecting PI15 gene differential expression situation in mRNA level in-site using QPCR.
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) trim is carried out to the 5 ' of reads and 3 ' sections with cutadapt, trim falls the base of quality < 20, 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 PI15 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
| Reagent | Dosage |
| 5x1st Strand Synthesis Buffer | 4.0μl |
| PrimeScript RTase | 1.0μl |
| RNase Inhibitor | 1.0μl |
| Rnase free dH2O | 4.0μl |
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:
PI15 gene:
5'-GGACCTTCTTACTTACTG-3'(SEQ ID NO.1)；
5’-TTCACTTCATCATACCAT-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.49.9 DEG C of annealing 20s, 72 DEG C are prolonged
20s is stretched, totally 43 circulations.
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 PI15 gene mRNA in 40 Osteoarthritic Synovium tissues in 42 Osteoarthritic Synovium tissues
Level is remarkably decreased compared to the average value of normal synovial tissue；Statistical result is as shown in Figure 1, compared with normal synovial tissue, bone
The mRNA level in-site of PI15 gene is decreased obviously in arthritis synovial tissue, and difference has 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
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<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
Claims (10)
1. detecting the product of PI15 gene or PI15 albumen in preparation diagnosis osteoarthritis or the tool of prediction osteoarthritis prognosis
In application.
2. application according to claim 1, which is characterized in that the detection PI15 gene or the product of PI15 albumen include
Detect the product of the expression of PI15 gene or PI15 albumen.
3. application according to claim 1 or 2, which is characterized in that the product includes can be in conjunction with the core of PI15 gene
Acid can be in conjunction with the substance of PI15 albumen；The nucleic acid is able to detect the expression of PI15 gene；The substance can
Detect the expression of PI15 albumen.
4. application according to claim 3, which is characterized in that the nucleic acid is specifically expanded used in real-time quantitative PCR
Increase the primer of PI15 gene as shown in SEQ ID NO.1 and SEQ ID NO.2.
5. a kind of diagnosis osteoarthritis or the tool for predicting osteoarthritis prognosis, which is characterized in that the tool includes that can examine
Survey the tool of the expression of PI15 gene or PI15 albumen.
6. tool according to claim 5, which is characterized in that the tool include can in conjunction with PI15 gene nucleic acid or
Person can be in conjunction with the substance of PI15 albumen；The nucleic acid is able to detect the expression of PI15 gene；The substance is able to detect
The expression of PI15 albumen.
7. tool according to claim 6, which is characterized in that the nucleic acid is specifically expanded used in real-time quantitative PCR
Increase the primer of PI15 gene as shown in SEQ ID NO.1 and SEQ ID NO.2.
8. a kind of drug for treating osteoarthritis, which is characterized in that the drug includes the activation of PI15 gene or PI15 albumen
Agent.
9. drug according to claim 8, which is characterized in that the activator can promote or enhance PI15 or be related to
The expression or activity of the substance of the upstream PI15 or downstream pathway.
10. application of the activator described in claim 8 or 9 in the drug of preparation treatment osteoarthritis.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811260578.5A CN109295208A (en) | 2018-10-26 | 2018-10-26 | Application of the PI15 as osteoarthritis marker |
| CN201910934566.4A CN110499366B (en) | 2018-10-26 | 2019-09-29 | Application of PI15 as osteoarthritis marker |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811260578.5A CN109295208A (en) | 2018-10-26 | 2018-10-26 | Application of the PI15 as osteoarthritis marker |
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| CN109295208A true CN109295208A (en) | 2019-02-01 |
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| CN201811260578.5A Pending CN109295208A (en) | 2018-10-26 | 2018-10-26 | Application of the PI15 as osteoarthritis marker |
| CN201910934566.4A Active CN110499366B (en) | 2018-10-26 | 2019-09-29 | Application of PI15 as osteoarthritis marker |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201910934566.4A Active CN110499366B (en) | 2018-10-26 | 2019-09-29 | Application of PI15 as osteoarthritis marker |
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| CN110499366A (en) | 2019-11-26 |
| CN110499366B (en) | 2020-10-27 |
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