CN108611409A - Biomarker for diagnosis and treatment rheumatoid arthritis and osteoarthritis - Google Patents
Biomarker for diagnosis and treatment rheumatoid arthritis and osteoarthritis Download PDFInfo
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Abstract
The invention discloses application of the RBBP6 genes in rheumatoid arthritis and/or osteoarthritis diagnosis, RBBP6 genes are in low expression in rheumatoid arthritis and/or Human Osteoarthritis tissue, expression by detecting RBBP6 genes can diagnose whether patient suffers from rheumatoid arthritis and/or osteoarthritis, and the present invention substantially increases the sensibility and specificity of rheumatoid arthritis and/or osteoarthritis diagnosis.In addition, the application the invention also discloses RBBP6 genes in rheumatoid arthritis and/or osteoarthritis treatment, the achievement in research of the invention provides new candidate to develop novel rheumatoid arthritis and/or osteoarthritis treatment drug.
Description
Technical field
The present invention relates to biotechnologies, more particularly to RBBP6 genes in rheumatoid arthritis and osteoarthritis
Diagnosis, the purposes in treatment.
Background technology
Rheumatoid arthritis is a kind of autoimmune disease, and characterized by multi-joint is involved, being in progress, it is broken joint occur
Bad and deformity, often involves periphery joint.The cause of disease is unclear, and organ involvement such as interstitial lung lesion and Sjogren syndrome be also very outside joint
It is common.Appropriate early treatment can improve joint symptoms and function, reduce the death rate, reduce complication.
One system review in 2010 is the study found that the estimation of the rheumatoid arthritis incidence in North America and Northern Europe exists
0.5%~1.1%.The incidence of developing country is relatively lower (0.1%~0.5%).Rheumatoid arthritis women more often
See (men and women's incidence 1：3).Any age can fall ill, and it is referred to as 55.6 years old that one, which is looked back the middle position age of onset of cohort study,；English
Newly diagnosis rheumatoid arthritis case load is about 20,000 every year for state.Thus it calculates, every general practitioner can see and treat patients 1 for every 2 years
Neopathy patient.
Research from UK ＆ EURO, the U.S. finds omni-doctor, and there are Diagnosis of Rheumatoid Arthritis delays.State of Britain
2009 annual report of the National Audit Office of family, is diagnosed as before rheumatoid arthritis, and patient goes medical average out to 4 at its omni-doctor to take second place
More, having 18% patient that need to even go to a doctor 8 times can just make a definite diagnosis.In primary medical care, the diagnosis of early stage rheumatoid arthritis and its
His skeletal muscle problem is equally difficult：Clinical symptoms performance is not easy to recognize, and inflammatory markers such as erythrocyte sedimentation rate and c reactive protein are again without spy
Sign property meaning, and specificity marker feminine gender such as rheumatoid factor (seronegativity occurs in 31% patient) and cyclic citrullinated peptid
(anti-CCP, 33% patients serum are negative) and common seronegativity shows.
It is disabled caused by rheumatoid arthritis that 28% patient can be made to lose the job in 1 year ability.It is opened from symptom
Begin, " window phase " in 3 months starts to treat, can delay progression of disease.And delay treatment then increases iconography joint and breaks
Bad and lethal risk.It is more easy to single therapy occur to fail and lead to drug resistance moreover, starting treatment again after 3~6 months.
Rheumatoid arthritis is exactly a kind of rheumatoid arthritis inside joint disease, it may appear that the one of painful swelling of joints
A little symptoms.But the symptom of also many diseases and rheumatoid arthritis is very similar, is particularly easy to that people is allowed to generate
It misleads, such as osteoarthritis.
Osteoarthritis is that punching is degenerative arthritis either osteoproliferation, has influenced a kind of disease of human synovial,
Chief reason is exactly that the cartilage of articular surface caused by joint is born a heavy burden for a long time receives the bone tissue of damage surrounding
Hyperplasia, when Osteoarthritis appears on finger, it is possible to rheumatoid arthritis can be misdiagnosed as.
So it can be urgently in the early stage effectively method of difference diagnostics classes rheumatic arthritis and osteoarthritis to find a kind of
Problem to be solved ensures that patient is correctly treated in order to avoid because of sing misdiagnosis and mistreatment delay treatment.
In recent years, it studies in molecular level and is constantly gushed for diagnosing the marker of rheumatoid arthritis or osteoarthritis
It is existing, the patent of application number as listed below：201310596649.X 201580058686.2,201380044584.6,
201310483384.2 200610070393.9,201510725004.0,201510627048.X, 201510724747.6,
201510548635.X 201510548624.1,201510549564.5,201510725755.2.Yet there are no it is any about
The molecular marker of difference diagnosis rheumatoid arthritis and osteoarthritis, applicant has carried out in this context originally to grind
Study carefully.
Invention content
The technical issues of in order to solve in the prior art, can be used for rheumatoid the purpose of the present invention is to provide one kind and closes
Section inflammation and/or the molecular marker of osteoarthritis early diagnosis.Rheumatoid arthritis and bone are distinguished using gene marker
Arthritis has promptness, specificity and sensitivity, to make patient that can be made a definite diagnosis in disease early stage, improves cure rate.
According to an aspect of the present invention, the present invention provides the products of detection RBBP6 gene expressions to prepare diagnostics classes
Application in the tool of rheumatic arthritis and/or osteoarthritis.
Further, it is described detection RBBP6 gene expressions product include detect RBBP6 gene mRNA levels product and/
Or the product of detection RBBP6 protein levels.
Further, the product of the detection RBBP6 gene expressions includes：Pass through RT-PCR, real-time quantitative PCR, immune inspection
Survey, in situ hybridization or chip detection RBBP6 genes and its expression product expression with diagnose rheumatoid arthritis and/or
The product of osteoarthritis.
Further, the product that rheumatoid arthritis and/or osteoarthritis are diagnosed with RT-PCR includes at least a pair
The primer of specific amplified RBBP6 genes；The production that rheumatoid arthritis and/or osteoarthritis are diagnosed with real-time quantitative PCR
Product include at least the primer of a pair of of specific amplified RBBP6 genes；It is described to diagnose rheumatoid arthritis and/or bone with immune detection
Arthritic product includes：The antibody combined with RBBP6 protein-specifics；It is described to diagnose rheumatoid arthritis in situ hybridization
And/or the product of osteoarthritis includes：With the probe of the nucleic acid array hybridizing of RBBP6 genes；It is described to diagnose rheumatoid with chip
The product of property arthritis and/or osteoarthritis includes：Protein chip and genetic chip；Wherein, protein chip includes and RBBP6 eggs
The antibody specifically bound in vain, genetic chip include the probe with the nucleic acid array hybridizing of RBBP6 genes.
A pair that the product of the real-time quantitative PCR diagnosis rheumatoid arthritis and/or osteoarthritis includes at least is special
The primer of different amplification RBBP6 genes is as shown in SEQ ID NO.1 and SEQ ID NO.2.
The product of the detection RBBP6 gene expressions can be the reagent for detecting RBBP6 gene expressions, can also be to include
Kit, chip, test paper of the reagent etc. can also be the high-flux sequence platform using the reagent.
The tool of the diagnosis rheumatoid arthritis and/or osteoarthritis includes but not limited to chip, kit, examination
Paper or high-flux sequence platform；High-flux sequence platform is a kind of special diagnosis rheumatoid arthritis and/or osteoarthritis
Tool very easily work will be become to the structure of the gene expression profile of a people with the development of high throughput sequencing technologies
Make.By comparing the gene expression profile of Disease and normal population, the exception for being easy to analyze which gene is related to disease.
Therefore, know that the exception of RBBP6 genes is related to rheumatoid arthritis and/or osteoarthritis in high-flux sequence to also belong to
The purposes of RBBP6 genes, equally within protection scope of the present invention.
The present invention also provides the tool of a kind of diagnosis rheumatoid arthritis and/or osteoarthritis, the tool includes
Detect the reagent of RBBP6 gene expressions；The reagent includes primer and/or probe, the detection for detecting RBBP6 gene mRNAs
The antibody of RBBP6 albumen.
The tool includes but not limited to chip, kit, test paper or high-flux sequence platform.
Wherein, the chip includes genetic chip, protein-chip；The genetic chip includes solid phase carrier and fixation
In the oligonucleotide probe of solid phase carrier, the oligonucleotide probe includes for detecting being directed to for RBBP6 gene transcription levels
The oligonucleotide probe of RBBP6 genes；The protein-chip includes solid phase carrier and is fixed on the RBBP6 eggs of solid phase carrier
White specific antibody；The genetic chip can be used for detecting multiple genes including RBBP6 genes (for example, being closed with bone
Section is scorching and/or the relevant multiple genes of rheumatoid arthritis) expression.The protein-chip can be used for detecting
The table of multiple protein (such as with the relevant multiple protein of osteoarthritis or rheumatoid arthritis) including RBBP6 albumen
Up to level.By being detected multiple simultaneously with marker that is distinguishing rheumatoid arthritis and osteoarthritis, it is greatly improved not
The arthritic accuracy rate of diagnosis of same type.
Wherein, the kit includes gene detecting kit and protein immunization detection kit；The genetic test examination
Agent box includes the reagent for detecting RBBP6 gene transcription levels；The protein immunization detection kit includes RBBP6 albumen
Specific antibody.Further, the reagent includes using RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or chip
Method detects reagent needed for RBBP6 gene expression dose processes.Preference, the reagent include being directed to RBBP6 genes
Primer and/or probe.It is easy to design according to the nucleotide sequence information of RBBP6 genes and can be used for detecting RBBP6 gene tables
Up to horizontal primer and probe.
The test paper includes the reagent for detecting RBBP6 gene expressions.
The high-flux sequence platform includes the reagent for detecting RBBP6 gene expressions.
Probe with the nucleic acid array hybridizing of RBBP6 genes can be DNA, RNA, DNA-RNA chimera, PNA or other
Derivative.There is no limit for the length of the probe, as long as completing specific hybrid, being specifically bound with purpose nucleotide sequence,
Any length can.The length of the probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the probe
Length can be grown to 60,80,100,150,300 base-pairs or longer or even whole genes.Since different probe lengths is to miscellaneous
Efficiency, signal specificity is handed over to have different influences, the length of the probe is typically at least 14 base-pairs, and longest does not surpass generally
30 base-pairs are crossed, it is best with 15-25 base-pair with the length of purpose nucleotide sequence complementation.The probe self-complementary sequence
Row are most preferably less than 4 base-pairs, in order to avoid influence hybridization efficiency.
Further, the specific antibody of the RBBP6 albumen includes monoclonal antibody, polyclonal antibody.The RBBP6 eggs
White specific antibody include complete antibody molecule, antibody any segment or modification (for example, chimeric antibody, scFv,
Fab, F (ab ') 2, Fv etc..As long as the segment can retain the binding ability with RBBP6 albumen.For protein level
Antibody preparation when well known to a person skilled in the art and the present invention may use any method to prepare the antibody.
In specific embodiments of the present invention, it is described detection RBBP6 gene mRNAs primer include SEQID NO.1 and
Primer pair shown in SEQ ID NO.2.
The present invention also provides RBBP6 genes and/or its expression product to prepare treatment rheumatoid arthritis and/or bone
Application in arthritic drug.
Further, the drug includes RBBP6 genes or the accelerating agent of its expression product.The accelerating agent includes promoting
The ingredient of RBBP6 gene expressions, promotes RBBP6 gene expression products to live at the ingredient for promoting RBBP6 gene expression product stability
The ingredient of property.
Further, described that the ingredient of RBBP6 gene expressions is promoted to include the reagent for promoting genetic transcription, promote gene translation
Reagent, promote RBBP6 protein contents reagent.
Specifically, the ingredient of the promotion RBBP6 gene expressions includes：Reagent containing RBBP6 genes carries RBBP6
The carrier or host cell of gene are formed by reagent, the reagent containing RBBP6 protein.
On the one hand the accelerating agent of the present invention can be used for supplementing the missing or deficiency of endogenic RBBP6 albumen, by carrying
The expression of high RBBP6 albumen, to treatment rheumatoid arthritis and/or osteoarthritis caused by RBBP6 hypoproteinosis.Separately
On the one hand the activity or function that can be used for promoting RBBP6 albumen, to treat rheumatoid arthritis and/or Bones and joints
It is scorching.
The carrier of the present invention for carrying gene is various carriers known in the art, such as commercially available carrier including plasmid,
Clay, bacteriophage, virus etc..
In the present invention, term " host cell " includes prokaryotic cell and eukaryocyte.Common prokaryotic host cell
Example includes Escherichia coli, hay bacillus etc..Common eukaryotic host cell includes yeast cells, insect cell and mammal
Cell.Preferably, the host cell is eukaryocyte, such as Chinese hamster ovary celI, COS cells.
According to a further aspect of the invention, the present invention also provides one kind for treat rheumatoid arthritis and/or
The drug of osteoarthritis, the drug include RBBP6 genes recited above and/or the accelerating agent of its expression product.
Further, drug of the invention further includes pharmaceutically acceptable carrier, carrier, and this kind of carrier includes (but not
It is limited to)：Diluent, excipient such as water etc., filler such as starch, sucrose etc.；Adhesive such as cellulose derivative, alginates, bright
Glue and polyvinylpyrrolidone；Wetting agent such as glycerine；Disintegrant such as agar, calcium carbonate and sodium bicarbonate；Sorbefacient quaternary ammonium
Compound；Surfactant such as hexadecanol；Absorption carrier such as kaolin and soap clay；Lubricant such as talcum powder, calcium stearate
With magnesium, polyethylene glycol etc..
The mode that the drug of the present invention imports tissue either cell can be divided into external or internal mode.Vitro formats
Including importing the drug containing RBBP6 genes or the drug containing RBBP6 protein in cell, then by cell transplantation or return
It is defeated to arrive in vivo.Internal mode includes directly by the drug containing RBBP6 genes or the infusion of medicine body containing RBBP6 protein
In inner tissue.
The drug of the present invention can also be with the drug combination of other treatment rheumatoid arthritis or osteoarthritis, a variety of drugs
The success rate for the treatment of can be mentioned significantly by being used in combination.
In the context of the present invention, " RBBP6 genes " includes any function etc. of RBBP6 genes and RBBP6 genes
The polynucleotides of jljl.RBBP6 genes (NC_000016.10 (24539587..24572863)) can be in international public nucleic acid sequence
Correlated series are inquired in column database GeneBank.
In the context of the present invention, RBBP6 gene expression products include the mRNA of RBBP6, RBBP6 albumen.RBBP6 eggs
Include RBBP6 holoproteins or part thereof peptide in vain.The partial peptide contains and rheumatoid arthritis or the relevant work(of osteoarthritis
It can domain.
" RBBP6 albumen " includes any functional equivalent of RBBP6 albumen and RBBP6 albumen.The functional equivalent
Including RBBP6 albumen conservative variation protein or its active fragment or its reactive derivative, allelic variant, natural mutation
Body, induced mutants, can be with the encoded protein of DNA of the DNA hybridization of RBBP6 under high or low stringent condition.
It is known that, conventionally, the modification of one or more amino acid does not interfere with the function of protein in a protein.
Those skilled in the art can approve the amino acid for changing single amino acids or small percentage or to amino acid sequence it is individual add,
Missing, insertion, replacement are conservative modifications, and the change of wherein protein generates the protein with identity function.Function phase is provided
As the Conservative substitution tables of amino acid be well known in the art.
Example by the protein for adding an amino acid or more amino acid modification is the fusion of RBBP6 albumen
Albumen.For the peptide or protein with RBBP6 protein fusions, there is no limit as long as the fusion protein of gained retains RBBP6 eggs
White biological activity.
In the context of the present invention, " diagnosis rheumatoid arthritis and/or osteoarthritis " had both included judging subject
Rheumatoid arthritis or osteoarthritis are whether suffered from, have also included judging that subject whether there is to suffer from rheumatoid joint
Scorching or osteoarthritis risk.
In the context of the present invention, " treatment " divides from the state change of disease, may include alleviation, the disease of disease
It is complete healing, in addition, further including the prevention of disease.
The advantages of the present invention：
Present invention firstly discovers that RBBP6 gene expressions are related to rheumatoid arthritis or osteoarthritis, pass through detection
The expression of RBBP6 in subject synovial tissue, it can be determined that subject whether with rheumatoid arthritis or osteoarthritis or
Person judges that subject whether there is the risk with rheumatoid arthritis or osteoarthritis, to instruct clinician to tested
Person provides prevention scheme or therapeutic scheme.
Present invention finds a kind of molecular marker that can have not only diagnosed rheumatoid arthritis but also osteoarthritis can be diagnosed,
The molecular marker can not only distinguish normal person and arthritic, can with effective district classify rheumatoid arthritis patients and
Human Osteoarthritis.
Present invention finds a kind of drugs that can have not only treated rheumatoid arthritis but also can treat osteoarthritis.One medicine is more
With expansion drug indication reduces financial burden.
Description of the drawings
Fig. 1 shows the statistical chart for detecting RBBP6 gene differential expression situations on transcriptional level using QPCR；
Fig. 2 shows the statistical chart for detecting RBBP6 gene differential expression situations on protein level using Western blot；
Fig. 3 shows the statistical chart for the overexpression situation for detecting RBBP6 genes on transcriptional level using QPCR；Wherein, A：
Osteoarthritis fibroblast-like synoviocyte；B：Rheumatoid arthritis fibroblast-like synoviocyte；
Fig. 4 shows the statistical chart for the overexpression situation for detecting RBBP6 albumen on protein level using Western blot；
A：Osteoarthritis fibroblast-like synoviocyte；B：Rheumatoid arthritis fibroblast-like synoviocyte.
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:ColdSpring HarborLaboratory
Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.
Embodiment 1 is screened in Osteoarthritic Synovium tissue, rheumatoid arthritis synovial tissue and normal synovial tissue
Difference expression gene
The synovial tissue of 5 rheumatoid arthritis patients comes from hospital orthopedics row knee prosthesis or villusectomy
Rheumatoid arthritis patients, the diagnosis of all cases meet the rheumatoid arthritis classification of American Rheumatism Association's revision in 1987
Standard, wherein women 3, male 2, average age (55 ± 8) year, average course of disease (12 ± 7) year.5 Human Osteoarthritis
Synovial tissue comes from hospital orthopedics row knee prosthesis or the Human Osteoarthritis of villusectomy, and case used meets
The diagnostic criteria about OA that Altam is proposed, wherein women 2, male 3, average age (63 ± 5) year, average course of disease (12
± 8) year.6 normal synovial tissues come from trauma surgery patient synovial tissue of joint.Clinical sample used in this research,
Patient know and informs and passes through through Ethics Committee of the court.
1, the extraction (utilizing Norgen RNA extracts kits) of tissue RNA
1) in the clear area of less RNase interference, in vitro synovial tissue's sample is weighed about using the mortar containing appropriate liquid nitrogen
20mg is ground to powdered with pestle；
2) sample is transferred in the centrifuge tube of a 2mL without RNA enzyme；
3) 300 μ L Lysis solution are added, is placed in homogenizer, is fully ground 1-5min；
4) 12000g 4 DEG C, centrifuges 10min, shifts in supernatant to the centrifuge tube of new 1.5mL；
5) 600 μ l RNase-Free Water are added, with vortex device mixing；
6) 20 μ l Proteinase Ks are added, in 55 DEG C of warm bath 15min, be constantly vortexed mixing；
7) 14000g, room temperature centrifuge 1min, make pellet cell debris in centrifugation bottom of the tube, supernatant is taken to be transferred to other one
In a centrifuge tube without RNA enzyme 1.5mL；
8) 95% ethyl alcohol of 450 μ l, vortex mixing is added；
RNA is adsorbed：
9) 650 lysates of the μ l containing ethyl alcohol are taken to be added in centrifugal column, 14000g centrifuges 1min；
10) lower layer is abandoned, resetting collecting pipe is on column；
11) according to the capacity of lysate, repeat 9)~10) step；
12) 400 μ l Wash solution, 14000g are added and centrifuge 2min；
13) lower layer is abandoned, column is placed on a new collecting pipe；
DNase processing：
14) 100 μ l Enzyme Incubation Buffer are added and 15 μ l DNase I, 14000g centrifuge 1min；
15) solution in collecting pipe is moved into column again；
16) it is placed at room temperature for 15min；
RNA is washed：
17) 400 μ l Wash solution, 14000g are added and centrifuge 1min, abandon lower layer, resetting collecting pipe is on column；
18) 400 μ l Wash solution, 14000g centrifugation 2min are added, abandon collecting pipe；
RNA is eluted：
19) pillar is put into 1.7mL Elution pipes；
20) 30 μ l Elution Buffer are added；
21) 200g centrifuges 2min, and solution is made fully to be combined with column, and then 14000g centrifuges 1min.
2, the quality analysis of RNA sample
The concentration and purity of carried RNA are detected using Nanodrop2000, it is complete that agarose gel electrophoresis detects RNA
Whole property, Agilent2100 measure RIN values.Single requirement for construction data base RNA total amounts 5 μ g, concentration >=200ng/ μ L, OD260/280 between
Between 1.8~2.2.
3, fragmentation RNA
Illumina platforms are sequenced for short sequence fragment, and mRNA average lengths may reach several kb, it is therefore desirable to
It is interrupted at random.It, can be by RNA random fractures at the small fragment of 200bp or so using metal ion.
4, reversion synthesis cDNA
Under the action of reverse transcriptase, using random primer, one chain cDNA of synthesis is inverted by template of mRNA, carries out two chains
When synthesis, dTTP is replaced with dUTP in dNTPs reagents, it includes A/U/C/G to make base in the second chains of cDNA.
5, adaptor is connected
The cDNA structures of double-strand are cohesive end, and End Repair Mix are added and are mended into flat end, then at 3 ' ends
End adds an A base, the connector for connecting Y-shaped.
6, bis- chains of UNG enzymic digestions cDNA
Before PCR amplification, the second chains of cDNA are digested with UNG enzymes, to make only to include the first chains of cDNA in library.
7, machine is sequenced on Illumina x-ten
Illumina x-ten microarray datasets carry out 2*150bp sequencings.
8, bioinformatic analysis
It is as follows that sequencing data obtains later rawdata analytic processes：
(1) trim is carried out to the 5 ' of reads and 3 ' sections with cutadapt, trim falls quality<20 base, and delete N
Reads more than 10%；
(2) tophat is compared onto reference gene group.Reference gene group version used be GRCh38.p7, fasta and
Gff file downloads are from NCBI；
(3) expression quantity of cuffquant quantification of mrna and normalization output；
(4) compare differential expression of the control group with disease group mRNA with DEGseq packets under R environment.Significant difference mRNA sieves
Select condition：p-value<0.05.
9, result
It is obtained between normal person and Human Osteoarthritis with the above standard screening, normal person and rheumatoid arthritis are suffered from
There is the gene totally 367 of differential expression between person, between patient with rheumatoid arthritis and Human Osteoarthritis.
The large sample of 2 difference expression gene of embodiment is verified
It is based on high-flux sequence early period that as a result, according to the size of P value, we select RBBP6 genes to verify,
It is expressed in rheumatoid arthritis and Human Osteoarthritis and lowers, and compared with patient with rheumatoid arthritis, in bone
Lower modulation bigger in arthritic.
One, the differential expression of RBBP6 genes is detected on transcriptional level
1,100, rheumatoid arthritis synovial tissue, Osteoarthritic Synovium tissue are collected in the way of embodiment 1
100, normal synovial tissue 100.
2, the extraction and quality testing of RNA are carried out by way of example
3, reverse transcription
Reverse transcription is carried out to l μ g total serum IgEs with RT Buffer and synthesizes cDNA.Using 25 μ l reaction systems, each sample
It takes 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 amplifications are examined
Using 25 μ l reaction systems, 3 parallel pipes are arranged in each sample, and all amplified reactions are repeated three times above to protect
Demonstrate,prove the reliability of result.Prepare following reaction system：12.5 μ l of SYBR Green PCRs system, forward primer (5
μM/μ l) 1 μ l, reverse primer (5 μM/μ l) 1 μ l, template cDNA 2.0 μ l, 8.5 μ l of no enzyme water；Expand the forward direction of RBBP6 genes
Sequence 5 '-AGCCATCTTCTTCCTCAG-3 ' (SEQ ID NO.1), reverse sequence 5 '-CACAACAGCATCAGTCATAA-3 '
(SEQ ID NO.2)；The preferred GAPDH of house-keeping gene, the forward primer sequence for expanding the gene are 5 '-
ATGTTCCAATATGATTCCA-3 ' (SEQ ID NO.3), reverse primer sequences 5 '-GATTTCCATTGATGACAAG-3 '
(SEQ ID NO.4).Operations are carried out on ice.Amplification program is：95 DEG C of 5min, (95 DEG C of 10s, 60 DEG C of 60s) * 45
Cycle.With SYBR
Green carries out PCR reactions on Light Cycler fluorescence real-time quantitative PCR instrument, passes through as fluorescent marker
Melt curve analysis is analyzed and electrophoresis determines that purpose band, Δ Δ CT methods carry out relative quantification, and the results are shown in Figure 1, with Normal synovial
Tissue is compared, and RBBP6 gene mRNA levels are significantly lowered in rheumatoid arthritis synovial tissue；It is slided with rheumatoid arthritis
Membrane tissue is compared, and RBBP6 gene mRNA levels are significantly lowered in Osteoarthritic Synovium tissue, and difference all has statistical significance (*
P<0.05).Using the upper bound of 95% credibility interval of normal synovial tissue mean value as the cut-off values of diagnostic test, at this
It is normally 94% with the sensitivity of osteoarthritis with RBBP6 diagnosis, specificity 90%, the results are shown in Table 1 under part；With
It is 91% that RBBP6, which diagnoses normal person and the sensitivity of patient with rheumatoid arthritis, and specificity 92%, the results are shown in Table 2.
Using the upper bound of 95% credibility interval of Osteoarthritic Synovium tissue mean value as the cut-off values of diagnostic test, with this condition,
The sensitivity that osteoarthritis and rheumatoid arthritis are diagnosed with RBBP6 is 90%, and specificity 93%, the results are shown in Table 3.
1 normal person of table distinguishes with Human Osteoarthritis
2 normal person of table distinguishes with patient with rheumatoid arthritis
3 Human Osteoarthritis of table is distinguished with patient with rheumatoid arthritis
Two, the differential expression of RBBP6 genes is detected on protein level
1, histone extracts
(1) 50mg tissues are first weighed, are fully ground with liquid nitrogen；
(2) 500 μ l hybrid workings liquid (RIPA lysates are added in every part of sample：PMSF=100:1) fully shaking；
(3) centrifuge 13000rpm/min centrifuge 10 minutes, after take supernatant to be sub-packed in -80 DEG C of preservations.
2, Western blot are detected
The protein quantification of extraction is subjected to SDS-PAGE electrophoresis, carry out later transferring film, closing, primary antibody is incubated, secondary antibody is incubated,
Colour developing.
3, statistical procedures
The gray value of protein band is analyzed using Image J softwares, using β-actin as internal reference, by RBBP6 albumen
The gray value of band is normalized.Result data is indicated in a manner of mean+SD, is used
SPSS13.0 statistical softwares are next for statistical analysis, and difference between the two is examined using t, it is believed that works as P<There is system when 0.05
Meter learns meaning.
4, result
The results are shown in Figure 2, and compared with normal synovial tissue, RBBP6 protein contents are notable in Osteoarthritic Synovium tissue
Decline；Compared with rheumatoid arthritis synovial tissue, RBBP6 albumen is remarkably decreased in Osteoarthritic Synovium tissue, and difference is equal
With statistical significance (* P<0.05).
3 RBBP6 gene expression plasmids of embodiment are built
1, the structure of RBBP6 expression vectors
Amplimer is designed according to the coded sequence (as shown in SEQ ID NO.5) of RBBP6 genes.From at Human fetal spleen
CDNA library (clontech companies, article No.：638831) coded sequence of the RBBP6 genes of amplification overall length, above-mentioned cDNA sequences in
Row are inserted into after restriction enzyme BamHI and XhoI double digestion through the true of restriction enzyme BamHI and XhoI double digestion
In nucleus expression vector pcDNA3.1, the recombinant vector pcDNA3.1-RBBP6 for connecting acquisition is used for subsequent experimental.
2, synovial tissue's cell injuring model
After the synovial tissue of the rheumatoid arthritis of sterile acquisition and osteoarthritis is cleaned with PBS, aseptic operation is used
Scissors cuts into the tissue block of about 1mm x 1mm x 1mm repeatedly, after 37 DEG C of clostridiopetidase A II (0.5mg/ml), digestion 2h is added,
It is filtered through 200 mesh gauzes, after supernatant is removed in centrifugation, cell is resuspended in DMEM culture solutions, is placed in 37 DEG C, 5%CO2Cell incubator
Interior culture.When cell grow up to fusiformis and in flakes after, carry out secondary culture.After cell reached for 3 generation, it is separately added into FITC labels
The mouse anti human CD11b of mouse anti human CD3, CD14, CD19 and PE label be marked, flow cytomery identification.On
It is that negative cell is used for fibroblast-like synoviocyte (Fibroblast-like Synoviocytes, FLS) to state 4 kinds of labels
In this research.
3, it transfects
The fibroblast-like synoviocyte of preparation is divided into two groups, respectively control group (transfection pcDNA3.1 empty carriers) and
RBBP6 overexpressions group (transfection pcDNA3.1-RBBP6).The transfection of carrier is carried out using liposome 2000, specific transfection method is pressed
The instruction of book carries out as directed.The working concentration of pcDNA3.1 empty carriers and pcDNA3.1-RBBP6 are 0.5 μ g/ml.
4, QPCR is detected
4.1 extraction cell total rnas are operated using conventional method.
4.2 reverse transcriptions and QPCR steps are the same as embodiment 2.
4.3 result
As shown in figure 3, compared with the cell of transfection pcDNA3.1 empty carriers, in the cell for transfecting pcDNA3.1-RBBP6
The mRNA level in-site of RBBP6 significantly raises, and difference has statistical significance (P<0.05)
5, Western is detected
The extraction of 5.1 total protein of cell
The well-grown fibroblast-like synoviocyte of logarithmic phase is taken to carry out the extraction of total protein of cell, it is complete according to EpiQuik
The specification of cell extraction kit carries out the operation of protein extraction.
5.2Western blot detections
Step is the same as embodiment 2.
5.3 result
As shown in figure 4, compared with the cell of transfection pcDNA3.1 empty carriers, in the cell for transfecting pcDNA3.1-RBBP6
The protein level of RBBP6 significantly raises, and difference has statistical significance (P<0.05).
The influence that 4 RBBP6 gene overexpressions of embodiment are proliferated fibroblast-like synoviocyte
1, step
With 2 × 105/ ml density is inoculated in 96 porocyte culture plates, after cell is adherent, according to embodiment 3 method into
Row cell transfecting, each experimental group design three wells, per 100 μ l of hole, are positioned over 37 DEG C, 5%CO2It is incubated, transfects in incubator
10 μ l CK-8 solution are added after 48h per hole in the culture hole of required detection respectively, continue to be incubated in cell incubator
1h measures each hole absorbance value (OD values) at 450nm.
2, statistical method
Experiment is all completed according to being repeated 3 times, and result data is indicated in a manner of mean+SD,
Using SPSS13.0 statistical softwares come for statistical analysis, the difference between RBBP6 gene overexpressions group and control group uses t
It examines, it is believed that work as P<There is statistical significance when 0.05.
3, result
As a result such as table 4 and the display of table 5 pcDNA3.1-RBBP6 is transfected compared with the cell of transfection pcDNA3.1 empty carriers
Cell Proliferation obviously slows down, and difference has statistical significance (P<0.05).
4 osteoarthritis fibroblast-like synoviocyte of table measures OD values
| Experimental group | OD values (optical density) |
| pcDNA3.1 | 0.1554±0.006 |
| pcDNA3.1-RBBP6 | 0.0513±0.003 |
5 rheumatoid arthritis synovial raji cell assay Raji OD values of table
| Experimental group | OD values (optical density) |
| pcDNA3.1 | 0.1584±0.005 |
| pcDNA3.1-RBBP6 | 0.0499±0.004 |
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, 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 domain of the claims in the present invention.
Sequence table
<110>Beijing Yang Shen biology information technologies Co., Ltd
<120>Biomarker for diagnosis and treatment rheumatoid arthritis and osteoarthritis
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
agccatcttc ttcctcag 18
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
cacaacagca tcagtcataa 20
<210> 3
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
atgttccaat atgattcca 19
<210> 4
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
gatttccatt gatgacaag 19
<210> 5
<211> 5379
<212> DNA
<213>People source (Homo sapiens)
<400> 5
atgtcctgtg tgcattataa attttcctct aaactcaact atgataccgt cacctttgat 60
gggctccaca tctccctctg cgacttaaag aagcagatta tggggagaga gaagctgaaa 120
gctgccgact gcgacctgca gatcaccaat gcgcagacga aagaagaata tactgatgat 180
aatgctctga ttcctaagaa ttcttctgta attgttagaa gaattcctat tggaggtgtt 240
aaatctacaa gcaagacata tgttataagt cgaactgaac cagcgatggc aactacaaaa 300
gcaattgatg actcttccgc gtctatttct ctggcccagc ttacaaagac tgccaatctg 360
gctgaagcca atgcttctga agaagataaa attaaagcaa tgatgtcgca atctggccat 420
gaatacgacc caatcaatta catgaagaaa cctctaggtc caccacctcc atcttacacg 480
tgtttccgtt gtggtaaacc tggacattat attaagaatt gcccaacaaa tggggataaa 540
aactttgaat ctggtcctag gattaaaaag agcactggaa ttcccagaag tttcatgatg 600
gaagtgaaag atcctaatat gaaaggtgca atgcttacca acactggaaa atatgcaata 660
ccaactatag atgcagaagc atatgcaatt gggaagaaag agaaacctcc cttcttacca 720
gaggagccat cttcttcctc agaagaagat gatcctatcc cagatgaatt gttgtgtctc 780
atctgcaagg atattatgac tgatgctgtt gtgattccct gctgtggaaa cagttactgt 840
gatgaatgta taagaacagc actcctggaa tcagatgagc acacatgtcc gacgtgtcat 900
caaaatgatg tttctcctga tgctttaatt gccaataaat ttttacgaca ggctgtaaat 960
aacttcaaaa atgaaactgg ctatacaaaa agactacgaa aacagttacc tcctccacca 1020
cccccaatac cacctccgag accactgatt cagaggaacc tacaacctct gatgagatct 1080
ccgatatcaa gacaacaaga tcctcttatg attccagtga catcttcatc aactcaccca 1140
gctccgtcta tatcttcatt aacttctaat cagtcttcct tggcccctcc tgtgtctgga 1200
aatccgtctt ctgctccagc tcctgtacct gatataactg caacagtatc catatcagtt 1260
cattcagaaa aatcagatgg accttttcgg gattctgata ataaaatatt gccagctgca 1320
gctcttgcat cagagcactc aaagggaacc tcctcaattg caattaccgc tcttatggaa 1380
gagaagggtt accaggtgcc tgttcttgga accccatctt tgcttggaca gtcattattg 1440
catggacagt tgatccccac aactggtcca gtaagaataa atactgctcg tccaggtggt 1500
ggtcgaccag gctgggaaca ttccaacaaa cttggctatc tggtttctcc accacaacaa 1560
attagaagag gggagaggag ctgctacaga agtataaacc gtgggcgaca ccacagcgaa 1620
agatcacaga ggactcaagg cccgtcacta ccagcaactc cagtctttgt acctgttcca 1680
ccacctcctt tgtatccgcc tcctccccat acacttcctc tccctccggg tgttcctcct 1740
ccacagtttt ctcctcagtt tcctcctggc cagccaccac ccgctgggta tagtgtccct 1800
cctccagggt ttcctccagc tcctgccaat ttatcaacac cttgggtatc atcaggagtg 1860
cagacagctc attcaaatac catcccaaca acacaagcac cacctttgtc cagggaagaa 1920
ttctatagag agcagcgacg actaaaagaa gaggaaaaga aaaagtccaa gctagatgag 1980
tttacaaatg attttgctaa ggaattgatg gaatacaaaa agattcaaaa ggagcgtagg 2040
cgctcatttt ccaggtctaa atctccctat agtggttctt cgtattcaag aagttcatat 2100
acttattcta aatcaagatc tggttcaaca cgttcacgct cttattctcg atcattcagc 2160
cgctcacatt ctcgttccta ttcacggtca cctccatacc ccagaagagg cagaggcaag 2220
agccgcaatt accgttcacg gtctagatct catggatatc atcgatctag gtcaaggtca 2280
cccccttaca gacgctatca ttcacgatca agatctcctc aagcgtttag gggacagtct 2340
cctaataaac gtaatgtacc tcaaggggaa acagaacgtg aatattttaa tagatacaga 2400
gaagttccac caccatatga catgaaagca tattatggga gaagtgttga ctttagagac 2460
ccatttgaaa aagaacgcta ccgagaatgg gagagaaaat atagagagtg gtatgaaaaa 2520
tattataaag gttatgctgc tggagcacag cctagaccct cagcaaatag agagaacttt 2580
tctccagaga gatttttgcc acttaacatc aggaattctc ccttcacaag aggccgcaga 2640
gaagactatg ttggtgggca aagtcataga agtcgaaaca taggtagcaa ctatccagaa 2700
aagctttcag caagagatgg tcacaatcag aaggataata caaagtcaaa agagaaggag 2760
agtgaaaacg ctccaggaga tggtaaagga aataagcata agaaacacag aaaaagaaga 2820
aaaggggagg aaagtgaggg ttttctgaac ccagagttat tagagacttc taggaaatca 2880
agagaaccta caggtgttga agaaaataaa acagactcat tgtttgttct cccaagtaga 2940
gatgatgcca cacctgttag agatgaacca atggatgcag aatcaatcac ttttaaatca 3000
gtgtctgaaa aagacaagag agaaagggat aaaccaaaag caaagggtga taaaaccaaa 3060
cggaagaatg atggatctgc tgtgtccaaa aaagaaaata ttgtaaaacc tgctaaagga 3120
ccccaagaaa aagtagatgg agaacgtgag agatctcctc gatctgaacc tccaattaaa 3180
aaagccaaag aggagactcc gaagactgac aatactaaat catcatcttc ctctcagaag 3240
gatgaaaaaa tcactggaac ccccagaaaa gctcactcta aatcagcaaa agaacaccaa 3300
gaaacaaaac cagtcaaaga ggaaaaagtg aagaaggact attccaaaga tgtcaaatca 3360
gaaaagctaa caactaagga agaaaaggcc aagaagccta atgagaaaaa caaaccactt 3420
gataataagg gagaaaaaag aaaaagaaaa actgaagaaa aaggcgtaga taaagatttt 3480
gagtcttctt caatgaaaat ctcgaaacta gaagtgactg aaatagtgaa accatcacca 3540
aagcgcaaaa tggaacctga tactgaaaaa atggatagga cccctgaaaa ggacaaaatt 3600
tctttaagtg cgccagccaa aaaaatcaaa ctcaacagag aaactgggaa gaaaattgga 3660
agtacagaaa atatatcaaa cacaaaagaa ccctctgaaa aattggagtc aacatctagc 3720
aaagttaaac aagaaaaagt caaaggaaag gtcagacgaa aagtgactgg aactgaagga 3780
tccagctcaa ctctggtgga ttacaccagt acgagctcaa ctggaggcag tcctgtgcgg 3840
aaatctgaag aaaaaacaga tacaaagcga actgtgatta aaacgatgga agaatataat 3900
aatgacaata ccgcgccagc tgaagatgtt atcattatga ttcaggttcc tcaatccaaa 3960
tgggataaag atgactttga atctgaagaa gaagatgtta aatccacaca gcctatatca 4020
agtgtaggaa aacctgctag tgttataaaa aatgttagta caaagccatc aaatatagtc 4080
aagtatcctg agaaagaaag tgagccatcc gagaaaattc agaaattcac caaggacgtg 4140
agccatgaaa tcatacaaca tgaggttaaa agttcaaaaa actctgcatc tagtgaaaaa 4200
gggaaaacca aagatcgaga ttattcagtg ttggaaaagg agaaccctga aaagaggaag 4260
aacagcactc agccagagaa agagagtaat ttggaccgtc tgaatgaaca aggaaatttt 4320
aaaagtctgt ctcaatcttc caaagaggct agaacgtcag ataaacatga ttccactcgt 4380
gcttcctcaa ataaagactt cactcccaat agagacaaaa aaactgacta tgacaccaga 4440
gagtattcaa gttccaaacg tagagatgaa aagaatgaat taacaagacg aaaagactct 4500
ccttctcgga ataaagattc tgcatctgga cagaaaaata aaccaaggga agagagagat 4560
ttgcctaaaa aaggaacagg agattccaaa aaaagtaatt ctagtccctc aagagacaga 4620
aaacctcatg atcacaaagc cacttatgat actaaacggc caaatgaaga gacaaaatct 4680
gtagataaaa atccttgtaa ggatcgtgag aagcatgtat tagaagcaag gaacaataaa 4740
gagtcaagtg gcaataaact actttatata cttaacccac cagagacaca ggttgaaaaa 4800
gagcaaatta ctgggcaaat tgacaagagt actgtcaagc ctaaacccca gttaagtcat 4860
tcctctagac tttcctctga cttaactaga gaaactgatg aagctgcttt tgaaccagac 4920
tataatgaaa gtgacagtga aagtaatgtt tctgtaaaag aagaggaatc ttcaggaaac 4980
atttctaagg acctgaaaga taaaatagtg gagaaagcaa aagagagcct ggacacagca 5040
gcagttgtcc aggtgggcat aagcaggaat cagagccaca gcagccccag cgtcagcccc 5100
agcagaagcc acagtccttc tggaagccag acccgaagcc acagtagcag tgccagctca 5160
gcagaaagtc aggacagcaa gaagaagaag aaaaagaagg aaaagaaaaa acacaagaaa 5220
cataaaaagc ataagaagca taagaaacat gcaggcactg aagtggaatt ggaaaaaagc 5280
caaaaacaca aacacaagaa aaagaagtca aagaagaaca aagataaaga gaaggagaag 5340
gagaaagatg accaaaaagt gaaatctgtc actgtgtaa 5379
Claims (10)
1. detecting the product of RBBP6 gene expressions in preparing the tool of diagnosis rheumatoid arthritis and/or osteoarthritis
Using.
2. application according to claim 1, which is characterized in that the product includes：By RT-PCR, real-time quantitative PCR,
Immune detection, in situ hybridization chip or high-flux sequence detection of platform RBBP6 gene expressions with diagnose rheumatoid arthritis and/
Or the product of osteoarthritis.
3. application according to claim 2, which is characterized in that it is described with RT-PCR diagnose rheumatoid arthritis and/or
The product of osteoarthritis includes at least the primer of a pair of of specific amplified RBBP6 genes；It is described to diagnose rheumatoid with real-time quantitative PCR
Property arthritis and/or the product of osteoarthritis include at least the primers of a pair of of specific amplified RBBP6 genes；It is described to use immune detection
Diagnosis rheumatoid arthritis and/or the product of osteoarthritis include：The antibody combined with RBBP6 protein-specifics；The use
In situ hybridization diagnoses rheumatoid arthritis and/or the product of osteoarthritis includes：With the nucleic acid array hybridizing of RBBP6 genes
Probe；The product that rheumatoid arthritis and/or osteoarthritis are diagnosed with chip includes：Protein chip and genetic chip；
Wherein, protein chip includes the antibody combined with RBBP6 protein-specifics, and genetic chip includes the nucleic acid sequence with RBBP6 genes
Arrange the probe of hybridization.
4. application according to claim 3, which is characterized in that described to diagnose rheumatoid arthritis with real-time quantitative PCR
And/or primer such as SEQ ID NO.1 and the SEQ ID of a pair of of specific amplified RBBP6 genes that the product of osteoarthritis includes at least
Shown in NO.2.
5. the tool of a kind of diagnosis rheumatoid arthritis and/or osteoarthritis, which is characterized in that the tool includes detection
The reagent of RBBP6 gene expressions；The reagent includes the primer and/or probe, detection RBBP6 eggs for detecting RBBP6 gene mRNAs
White antibody.
6. tool according to claim 5, which is characterized in that the primer of the detection RBBP6 gene mRNAs includes SEQ
Primer pair shown in ID NO.1 and SEQ ID NO.2.
7.RBBP6 genes and/or its expression product are in preparing the drug for the treatment of rheumatoid arthritis and/or osteoarthritis
Using.
8. application according to claim 7, which is characterized in that the drug include promote RBBP6 gene expressions ingredient,
Promote the ingredient of RBBP6 gene expression product stability, promote the active ingredient of RBBP6 gene expression products.
9. application according to claim 8, which is characterized in that it includes containing RBBP6 to promote the ingredient of RBBP6 gene expressions
The reagent of gene, the carrier for carrying RBBP6 genes or host cell are formed by reagent, the reagent containing RBBP6 protein.
10. the drug described in a kind of any one of claim 7-9.
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| CN111549113A (en) * | 2020-04-17 | 2020-08-18 | 中山大学 | Rheumatoid arthritis diagnostic marker and application thereof |
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