CN105695621B

CN105695621B - Application of the REPS1 genes in diagnosis and treatment osteoarthritis product is prepared

Info

Publication number: CN105695621B
Application number: CN201610271798.2A
Authority: CN
Inventors: 边洋; 肖枫; 杨明; 杨承刚
Original assignee: Gu'an Bojian Biotechnology Co Ltd
Current assignee: Gu'an Bojian Biotechnology Co Ltd
Priority date: 2016-04-27
Filing date: 2016-04-27
Publication date: 2017-12-22
Anticipated expiration: 2036-04-27
Also published as: CN105695621A

Abstract

The invention discloses a kind of molecular marker REPS1 genes and its expression product for osteoarthritis early diagnosis.The present invention proves that REPS1 genes have differences expression in the synovial tissue of Human Osteoarthritis and normal person using genetic chip and Western blot methods, can be as the index of early diagnosis osteoarthritis.In addition, can be as the target of osteoarthritis treatment, for instructing the research and development of new drug the invention also discloses REPS1 genes and its expression product.

Description

Application of the REPS1 genes in diagnosis and treatment osteoarthritis product is prepared

Technical field

The present invention relates to biological technical field, more particularly to use of the REPS1 genes in the diagnosis, treatment of osteoarthritis On the way.

Background technology

Osteoarthritis is a kind of retrogression pathological changes, is due to increasing age, obesity, strain, wound, joint birth defect, pass Articular cartilage degeneration damage, joint margins and the subchondral bone reactive hyperplasia caused by factors such as deformity are saved, also known as bone closes Save disease, degenerative arthritis, senescent arthritis, hypertrophiarthritis etc..Clinical manifestation is arthralgia, the pressure slowly developed Bitterly, stiff, arthroncus, limitation of activity and joint deformity etc..

Demography trend shows, to the year two thousand twenty, the crowd influenceed by joint disease will be added to 50%, and the whole world will There are 5.7 hundred million people to be perplexed by osteoarthritis.Chinese society progresses into aging society, therefore we will face osteoarthritis The generally popular period of morbidity.It is particularly significant and urgent to the sick research.

God in articular cartilage, synovial membrane, subchondral bone, ligament or periarticular can occur for the primary exception of osteoarthritis Through musculature.The infringement of articular cartilage is that osteoarthritis most significantly changes.Articular cartilage forfeiture is the key of 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 With metalloprotease osteoarthritic joint cartilage loss in be most important reason.There is no 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. Lead.Synovial membrane plays during the occurrence and development of osteoarthritis disorders important for maintaining the normal function in joint Effect.

The content of the invention

It is an object of the invention to provide a kind of point that can be used for osteoarthritis (Osterarthritis, OA) early diagnosis Sub- mark.Compared to the diagnostic method of traditional osteoarthritis, it is timely to diagnose having for osteoarthritis using gene marker Property, specificity and sensitivity so that patient just can know disease risks in disease early stage, for risk height, take corresponding Prevention and treatment measure.

To achieve these goals, the present invention adopts the following technical scheme that：

The invention provides application of the product of detection REPS1 gene expressions in the instrument for preparing diagnosis osteoarthritis.

Further, the product of the detection REPS1 gene expressions include detection REPS1 gene mRNA levels product and/ Or the product of detection REPS1 protein levels.

Further, the product of the detection REPS1 gene expressions includes：Pass through RT-PCR, real-time quantitative PCR, immune inspection Survey, in situ hybridization or the expression of chip detection REPS1 genes and its expression product to be to diagnose the product of osteoarthritis.

Further, the product that osteoarthritis is diagnosed with RT-PCR comprises at least a pair of specific amplified REPS1 genes Primer；The primer of the product including at least a pair of specific amplified REPS1 genes that osteoarthritis is diagnosed with real-time quantitative PCR； The product that osteoarthritis is diagnosed with immune detection includes：The antibody combined with REPS1 protein-specifics；It is described miscellaneous with original position Handing over the product of diagnosis osteoarthritis includes：With the probe of the nucleic acid array hybridizing of REPS1 genes；It is described to diagnose Bones and joints with chip Scorching product includes：Protein chip and genetic chip；Wherein, protein chip includes the antibody combined with REPS1 protein-specifics, Genetic chip includes the probe with the nucleic acid array hybridizing of REPS1 genes.

A pair of specific amplified REPS1 genes that the product with real-time quantitative PCR diagnosis osteoarthritis comprises at least Primer is as shown in SEQ ID NO.3 and SEQ ID NO.4.

The product of the detection REPS1 gene expressions can be the reagent of detection REPS1 gene expressions, can also include The kit of the reagent, chip, test paper etc. or the high-flux sequence platform using the reagent.

The instrument of the diagnosis osteoarthritis includes but is not limited to chip, kit, test paper or high-flux sequence platform； High-flux sequence platform is a kind of instrument of special diagnosis osteoarthritis, with the development of high throughput sequencing technologies, to one The structure of the gene expression profile of people, which will turn into, very easily to work.By the gene expression for contrasting Disease and normal population Spectrum, the exception for easily analyzing which gene are related to disease.Therefore, the exception of REPS1 genes is known in high-flux sequence The purposes for falling within REPS1 genes related to osteoarthritis, equally within protection scope of the present invention.

Present invention also offers a kind of instrument for diagnosing osteoarthritis, the instrument includes detection REPS1 gene expressions Reagent；The reagent includes the primer and/or probe, the antibody for detecting REPS1 albumen of detection REPS1 gene mRNAs.

The instrument includes but is 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 being used to detect being directed to for REPS1 gene transcription levels The oligonucleotide probe of REPS1 genes；The protein-chip includes solid phase carrier and is fixed on the REPS1 eggs of solid phase carrier White specific antibody；The genetic chip can be used for multiple genes of the detection including REPS1 genes (for example, being closed with bone The scorching related multiple genes of section) expression.The protein-chip can be used for detection multiple including REPS1 albumen The expression of protein (such as multiple protein related to osteoarthritis).By by multiple with osteoarthritis mark Detect simultaneously, be greatly improved the accuracy rate of osteoarthritis diagnosis.

Wherein, the kit includes gene detecting kit and protein immunization detection kit；The genetic test examination Agent box includes being used for the reagent for detecting REPS1 gene transcription levels；The protein immunization detection kit includes REPS1 albumen Specific antibody.Further, the reagent is including the use of RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or chip Reagent needed for during method detection REPS1 gene expression doses.Preference, the reagent are included for REPS1 genes Primer and/or probe.Easily designed according to the nucleotide sequence information of REPS1 genes and can be used for detecting REPS1 gene tables Up to horizontal primer and probe.

The test paper includes the reagent of detection REPS1 gene expressions.

The high-flux sequence platform includes the reagent of detection REPS1 gene expressions.

Probe with the nucleic acid array hybridizing of REPS1 genes can be DNA, RNA, DNA-RNA chimera, PNA or other Derivative.The length of the probe does not limit, 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 gene.Because 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, most long not surpass typically 30 base-pairs are crossed, complementary length is optimal with 15-25 base-pair with purpose nucleotide sequence.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 REPS1 albumen includes monoclonal antibody, polyclonal antibody.The REPS1 eggs White specific antibody include complete antibody molecule, antibody any fragment or modification (for example, chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc..As long as the fragment can retain the binding ability with REPS1 albumen.For protein level Antibody preparation when well known to a person skilled in the art and the present invention can prepare the antibody using any method.

In specific embodiments of the present invention, the primers of the detection REPS1 gene mRNAs include SEQ ID NO.3 and Primer pair shown in SEQ ID NO.4.

Present invention also offers the accelerator of REPS1 genes and/or its expression product to prepare the medicine for the treatment of osteoarthritis Application in thing.

The accelerator includes promoting the reagent of REPS1 gene expressions and promotes the reagent of REPS1 gene expression products；Institute State and promote the reagent of REPS1 gene expressions to include promoting the reagent of genetic transcription, promote the reagent of gene translation, promote REPS1 The reagent of protein content；The reagent of the promotion REPS1 gene expression products includes promoting REPS1 gene expression product stability Reagent, promote the reagent of REPS1 gene expression products activity, promote the reagent of REPS1 gene expression product functions.

Specifically, the reagent of the promotion REPS1 gene expressions includes：Reagent containing REPS1 genes, carry REPS1 Reagent that the carrier or host cell of gene are formed, the reagent containing REPS1 protein.

On the one hand the accelerator of the present invention can be used for the missing or deficiency for supplementing endogenic REPS1 albumen, by carrying The expression of high REPS1 albumen, so as to treat the osteoarthritis caused by REPS1 hypoproteinosis.On the other hand can be used for promoting The activity or function of REPS1 albumen, so as to treat osteoarthritis.

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 and eukaryotic.Conventional prokaryotic host cell Example includes Escherichia coli, hay bacillus etc..Conventional eukaryotic host cell includes yeast cells, insect cell and mammal Cell.It is preferred that the host cell is eukaryotic, such as Chinese hamster ovary celI, COS cells.

Present invention also offers a kind of pharmaceutical composition for being used to treat osteoarthritis, described pharmaceutical composition is included above Described REPS1 genes and/or the accelerator of its expression product.

Further, medicine of the invention also includes pharmaceutically acceptable carrier, carrier, and this kind of carrier is included (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 acid carbonate；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 medicine of the present invention imports tissue either cell can be divided into external or internal mode.Vitro formats Including the medicine containing REPS1 genes or the medicine containing REPS1 protein are imported in cell, then by cell transplantation or return It is defeated to arrive in vivo.Internal mode is included directly by the medicine containing REPS1 genes or the infusion of medicine body containing REPS1 protein In inner tissue.

The medicine of the present invention can also be with the drug combination of other treatment osteoarthritis, and multi-medicament is used in combination can be significantly Mention the success rate for the treatment of.

In the context of the present invention, " REPS1 genes " includes any function of REPS1 genes and REPS1 genes etc. The polynucleotides of jljl.REPS1 genes include and REPS1 genes in the public GenBank GeneBank in the current world (NC_000006.12) DNA sequence dna has more than 70% homology, and encodes identical function protein DNA sequence；

Preferably, the coded sequence of REPS1 genes includes following any DNA molecular：

(1) DNA sequence dna in sequence table shown in SEQ ID NO.1；

(2) under strict conditions with 1) the DNA sequence dna hybridization that limits and coding identical function protein DNA sequence；

(3) DNA sequence dna limited with (1) or (2) has 70%, preferably, more than 90% homology, and encodes identical work( Can protein DNA molecule.

In specific embodiments of the present invention, the coded sequence of the REPS1 genes is shown in SEQ ID NO.1 DNA sequence dna.

In the context of the present invention, REPS1 gene expression products include the part of REPS1 albumen and REPS1 albumen Peptide.The partial peptide of the REPS1 albumen contains the functional domain related to osteoarthritis.

" REPS1 albumen " includes any functional equivalent of REPS1 albumen and REPS1 albumen.The functional equivalent Including REPS1 albumen conservative variation protein or its active fragment, or its reactive derivative, allelic variant, natural mutation Body, induced mutants, can be with the protein coded by the DNA of REPS1 DNA hybridization under high or low stringent condition.

Preferably, REPS1 albumen is the protein for having following amino acid sequences：

(1) protein being made up of the amino acid sequence in sequence table shown in SEQ ID NO.2；

(2) amino acid sequence shown in SEQ ID NO.2 by the substitution of one or several amino acid residues and/or is lacked Lose and/or addition and with the amino acid sequence shown in SEQ ID NO.2 have identical function as the ammonia shown in SEQ ID NO.2 Protein derived from base acid sequence.The number of the amino acid of substitution, missing or addition is usually 1-50, preferably 1-30 It is individual, more preferably 1-20, most preferably 1-10.

(3) there is at least 80% homology (also known as sequence identity) with the amino acid sequence shown in SEQ ID NO.2, It is highly preferred that the homology with the amino acid sequence at least about 90% to 95% shown in SEQ ID NO.2, be often 96%, 97%, 98%th, the polypeptide that the amino acid sequence of 99% homology is formed.

In specific embodiments of the present invention, the REPS1 albumen is that have the amino acid sequence shown in SEQ ID NO.2 The protein of row.

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 that changes single amino acids or small percentage or indivedual additions to amino acid sequence, Missing, insertion, replacement are conservative modifications, and the change of wherein protein produces 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 REPS1 albumen Albumen.Do not limited for the peptide or protein with REPS1 protein fusions, as long as the fusion protein of gained retains REPS1 eggs White biological activity.

The REPS1 albumen of the present invention also includes the non-conservative modification to the amino acid sequence shown in SEQ ID NO.2, as long as Protein by modification remains able to the biological activity for retaining REPS1 albumen.It is mutated in such modifying protein Amino acid number be typically 10 or less, such as 6 or less, such as 3 or less.

In the context of the present invention, " diagnosis osteoarthritis " had both included judging whether subject suffers from Bones and joints Inflammation, also include judging that subject whether there is the risk with osteoarthritis.

In the context of the present invention, " treatment osteoarthritis " divides from the state change of disease, can include disease Alleviate, the complete healing of disease.

The advantages of the present invention：

Present invention firstly discovers that REPS1 gene expressions are related to osteoarthritis, by detecting in subject synovial tissue REPS1 expression, it can be determined that whether subject, which suffers from osteoarthritis or judge that subject whether there is, suffers from osteoarthritis Risk, so as to instruct clinician to provide prevention scheme or therapeutic scheme to subject.

Present invention finds a kind of new molecular marked compound-REPS1 genes, compared to traditional detection means, gene diagnosis More in time, it is more special, sensitiveer, the early diagnosis of osteoarthritis can be realized, so as to reduce the death rate of osteoarthritis.

Brief description of the drawings

Fig. 1, which is shown, utilizes expression of the genechip detection REPS1 genes in synovial tissue；

Fig. 2 shows the expression in synovial cell using Western blot detection REPS1 albumen；

Fig. 3 shows the overexpression situation for detecting REPS1 genes on transcriptional level using QPCR；

Fig. 4 shows the overexpression situation using Western blo detection REPS1 albumen.

Specific embodiment

The present invention is further detailed explanation with reference to the accompanying drawings and examples.Following examples are merely to illustrate this Invention rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in embodiment, generally according to conventional strip Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or according to the condition proposed by manufacturer.

The differential expression of REPS1 genes in the OA synovial tissues of embodiment 1 and normal synovial tissue

The synovial tissue of 50 OA patients comes from patient OA of row knee prosthesis or villusectomy.Case symbol used Close the diagnostic criteria on OA that Altam is proposed.30 normal synovial tissues come from trauma surgery patient synovial tissue of joint. Take supernatant after OA patient's synovial fluid (SF) high speed centrifugation, -80 DEG C of storages are stand-by.Clinical sample used in this research, to patient Know and inform and the Ethics Committee through hospital passes through.

1st, RNA extraction is organized

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, it is ground to pestle powdered；

2) sample is transferred in a 2mL without RNase centrifuge tube；

3) 300uL Lysis solution are added, is placed in homogenizer, is fully ground 1-5min；

4) 12000g, 4 DEG C, 10min is centrifuged, transfer supernatant is into new 1.5mL centrifuge tube；

5) 600ul RNase-Free Water are added, are mixed with whirlpool device；

6) 20ul Proteinase Ks are added, in 55 DEG C of warm bath 15min, is constantly vortexed and mixes；

7) 14000g, room temperature, 1min is centrifuged, makes pellet cell debris take supernatant to be transferred to other one in centrifugation bottom of the tube In the individual centrifuge tube without RNase 1.5mL；

8) 450ul 95% ethanol is added, is vortexed and mixes；

RNA is adsorbed：

9) lysates of the 650ul containing ethanol is taken to be added in centrifugal column, 14000g centrifugations 1min；

10) lower floor is abandoned, resets collecting pipe on post；

11) capacity according to lysate, repeat 9)~10) step；

12) 400ulWash solution, 14000g centrifugations 2min is added；

13) lower floor is abandoned, post is placed on a new collecting pipe；

DNase processing：

14) 100ulEnzyme Incubation Buffer and 15ulDNase I, 14000g centrifugation 1min are added；

15) solution in collecting pipe is moved into post again；

16) room temperature places 15min；

RNA is washed：

17) 400ulWash solution, 14000g centrifugation 1min are added, lower floor is abandoned, resets collecting pipe on post；

18) 400ulWash solution, 14000g centrifugation 2min are added, abandon collecting pipe；

RNA is eluted：

19) pillar is put into 1.7mL Elution pipes；

20) 30ul Elution Buffer are added；

21) 200g centrifuges 2min, solution is fully combined with post, and then 14000g centrifuges 1min.

2nd, the quality analysis (NanoDrop1000 spectrophotometers) of RNA sample

NanoDrop1000 spectrophotometers detect RNA sample：OD260/OD280 is 1.8-2.2.

3rd, gene chip hybridization and scanning

After the linearized amplification of total serum IgE, cy3-UTP is marked, and the cRNAs after fluorescence labeling uses RNEASY Mini Kit Purifying, fragmentation processing is carried out with Amhion RNA Fragmentation Reagents to the cRNAs marked.Using U.S. People's full genome chip of expression spectrum (4x 44K genes) of Agilent companies of state, 65 DEG C of hybridization 17h in chip hybridization stove, then Elution, dyeing, finally with Agilent DNA MicroarrayScanner scanner scannings.

4th, chip data processing and analysis

Chip after hybridization imports data to analysis software, for two groups of ratios after chip scanner reads data point Natural logrithm absolute value be more than 2.0 or the gene less than 0.5 as difference expression gene.

5th, statistical procedures

Data analysis is carried out using the statistical softwares of SPSS 13.0, group difference compares using one-way analysis of variance method, P< 0.05 difference has significant.

6th, result

As a result (as shown in Figure 1) is shown, compared with normal synovial tissue, REPS1 bases in Human Osteoarthritis synovial tissue The mRNA level in-site of cause significantly reduces, and difference has statistical significance (P<0.05).

The differential expression of REPS1 albumen in the OA synovial tissues of embodiment 2 and normal synovial tissue

1st, step

1.1 synovial tissue's cell injuring models

After synovial tissue's PBS of sterile acquisition, about 1mmx1mmx1mm is cut into repeatedly with aseptic operation scissors Tissue block, after adding clostridiopetidase A II 37 DEG C of (0.5mg/ml), digestion 2h, filtered through 200 mesh gauzes, will after supernatant is removed in centrifugation Cell is resuspended in DMEM nutrient solutions, is placed in 37 DEG C, 5%CO₂Cultivated in cell culture incubator.When cell grow up to fusiformis and in flakes after, Carry out Secondary Culture.After cell reached for 3 generation, mouse anti human CD3, CD14, CD19 and PE mark of FITC marks are separately added into The mouse anti human CD11b of note is marked, flow cytomery identification.Above-mentioned 4 kinds of marks are that negative cell is into fibre Dimension sample synovial cell (Fibroblast-like Synoviocytes, FLS) is used for this research.

The extraction of 1.2 total protein of cell

The phase well-grown fiber-like synovial cell that takes the logarithm carries out the extraction of total protein of cell, entirely thin according to EpiQuik The specification of born of the same parents' extracts kit carries out the operation of protein extraction.

1.3Western blot are detected

The protein quantification of extraction is subjected to SDS-PAGE electrophoresis, carry out afterwards transferring film, closing, primary antibody is incubated, secondary antibody is incubated, Colour developing.

2nd, statistical procedures

The gray value of protein band is analyzed using Image J softwares, using β-actin as internal reference, by REPS1 albumen The gray value of band is normalized.Result data is represented in a manner of mean+SD, is used SPSS13.0 statistical softwares carry out statistical analysis, and difference between the two is examined using t, it is believed that works as P<There is system when 0.05 Meter learns meaning.

3rd, result

As a result as shown in Fig. 2 compared with normal synovial tissue, the expression water of REPS1 albumen in Osteoarthritic Synovium tissue Flat to significantly reduce, difference has statistical significance (P<0.05).

The REPS1 gene expression plasmids of embodiment 3 are built

1st, the structure of REPS1 expression vectors

Amplimer is designed according to the coded sequence (as shown in SEQ ID NO.1) of REPS1 genes, design of primers is according to normal Rule method is carried out.From cDNA library (clontech companies, the article No. into Human fetal spleen：638831) the REPS1 bases of amplification total length in The coded sequence of cause, above-mentioned coded sequence is inserted into eukaryotic expression vector pcDNA3.1, the restructuring for connecting acquisition carries Body pcDNA3.1-REPS1 is used for subsequent experimental.

2nd, transfect

OA fibroblast-like synoviocytes are divided into two groups, respectively control group (transfection pcDNA3.1 empty carriers) and REPS1 Overexpression group (transfection pcDNA3.1-REPS1).The transfection of carrier is carried out using liposome 2000, specific transfection method is according to saying The instruction of bright book is carried out.PcDNA3.1 empty carriers and pcDNA3.1-REPS1 working concentration are 0.5 μ g/ml.

3rd, QPCR is detected

3.1 extraction cell total rnas are operated using conventional method.

3.2 reverse transcription

Reverse transcription synthesis cDNA is carried out to l μ g total serum IgEs with RT Buffer.Using 25 μ l reaction systems, each sample 1 μ g total serum IgEs are taken to be separately added into following components in PCR pipe as template ribonucleic acid：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.

3.3 QPCR

Using 25 μ l reaction systems, each sample sets 3 parallel pipes, all amplified reactions in triplicate more than to protect Demonstrate,prove the reliability of result.Prepare following reaction system：The μ l of SYBR Green PCRs system 12.5, forward primer (5 μM/μ l) 1 μ l, reverse primer (5 μM/μ l) 1 μ l, template cDNA 2.0 μ l, no μ l of enzyme water 8.5；Expand the forward direction of REPS1 genes Primer sequence is 5 '-GCATCACAACCTTCTATT-3 ' (SEQ ID NO.3), reverse primer sequences 5 '- GCAATTCGCTATTCAATC-3’(SEQ ID NO.4)；Expand the forward primer sequences of GAPDH genes for 5 '- TTTAACTCTGGTAAAGTGGATAT-3 ' (SEQ ID NO.5), reverse primer sequences 5 '- GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO.6), operations are carried out on ice.Amplification program is：95℃ 5min, (95 DEG C of 10s, 60 DEG C of 60s) * 45 circulations.Using SYBR Green as fluorescent marker, in Light Cycler fluorescence The enterprising performing PCR reaction of real-time PCR, determines purpose band, Δ Δ CT methods carry out phase by melt curve analysis analysis and electrophoresis To quantitative.

3.4 statistical method

Experiment is all completed according to being repeated 3 times, and result data is represented in a manner of mean+SD, Statistical analysis is carried out using SPSS13.0 statistical softwares, the difference between two groups is examined using t, it is believed that works as P<Have when 0.05 It is statistically significant.

4th, Western is detected

Specific steps are the same as embodiment 2.

2nd, result

As shown in figure 3, compared with transfecting the cell of pcDNA3.1 empty carriers, in the cell for transfecting pcDNA3.1-REPS1 REPS1 mRNA level in-site significantly raises, and difference has statistical significance (P<0.05)；It is as shown in figure 4, empty with transfection pcDNA3.1 The cell of carrier is compared, and the protein level for transfecting REPS1 in pcDNA3.1-REPS1 cell significantly raises, and difference has statistics Learn meaning (P<0.05).

OA fibroblast-like synoviocyte proliferation experiments under the incentive condition of embodiment 4

1st, OA fibroblast-like synoviocytes proliferation experiment under incentive condition

1.1 step

OA fibroblast-like synoviocytes are inoculated in 96 porocyte culture plates, per hole 2x10³The μ l of cells/ holes/200,37 DEG C, 5%CO₂After incubator is incubated 24 hours, add (1 in synovial fluid：5 dilutions), continue to be incubated 24 hours, afterwards respectively in institute 10 μ l CK-8 solution are added in the culture hole that need to be detected per hole, continue to be incubated 1h in cell culture incubator, are determined each at 450nm Hole absorbance (OD values).

1.2 statistical method

Experiment is all completed according to being repeated 3 times, and result data is represented in a manner of mean+SD, Statistical analysis is carried out using SPSS13.0 statistical softwares, difference between the two is examined using t, it is believed that works as P<Have when 0.05 It is statistically significant.

1.3 result

As a result as shown in table 1, compared with being not added with the control group of synovial fluid stimulation, the FLS propagation for adding synovial fluid to stimulate is obvious Accelerate, difference has statistical significance (P<0.05).

The FLS cell OD values of table 1

Experimental group	OD values (optical density)
		It is not added with synovial fluid	0.1598±0.008
Add synovial fluid	0.2762±0.007

The influence that the REPS1 gene overexpressions of embodiment 5 are bred to OA fibroblast-like synoviocytes under incentive condition

1st, step

With 2 × 10⁵/ ml density is inoculated in 96 porocyte culture plates, after cell attachment, is entered according to the method for embodiment 3 Row cell transfecting, each experimental group design three wells, per the μ l of hole 100, are positioned over 37 DEG C, 5%CO₂It is incubated, transfects in incubator After 24h, add (1 in synovial fluid：5 dilutions), continue to be incubated 24 hours, add respectively in the culture hole of required detection per hole afterwards Enter 10 μ l CK-8 solution, continue to be incubated 1h in cell culture incubator, determine each hole absorbance (OD values) at 450nm.

2nd, statistical method

Experiment is all completed according to being repeated 3 times, and result data is represented in a manner of mean+SD, Statistical analysis is carried out using SPSS13.0 statistical softwares, the difference between REPS1 gene overexpressions group and control group uses t Examine, it is believed that work as P<There is statistical significance when 0.05.

3rd, result

As a result as shown in table 2, compared with transfecting the cell of pcDNA3.1 empty carriers, transfection pcDNA3.1-REPS1 is inhibited Cell under synovial fluid stimulates is bred, and difference has statistical significance (P<0.05).

The FLS cell OD values of table 2

Experimental group	OD values (optical density)
		pcDNA3.1	0.2695±0.004
pcDNA3.1-REPS1	0.1715±0.003

The influence that the REPS1 gene overexpressions of embodiment 6 are bred to OA fibroblast-like synoviocytes

1st, step

With 2 × 10⁵/ ml density is inoculated in 96 porocyte culture plates, after cell attachment, is entered according to the method for embodiment 3 Row cell transfecting, each experimental group design three wells, per the μ l of hole 100, are positioned over 37 DEG C, 5%CO₂It is incubated, transfects in incubator Add 10 μ l CK-8 solution after 48h per hole in the culture hole of required detection respectively, continue to be incubated in cell culture incubator 1h, determine each hole absorbance (OD values) at 450nm.

2nd, statistical method

3rd, result

As a result as shown in table 3, compared with transfecting the cell of pcDNA3.1 empty carriers, transfection pcDNA3.1-REPS1 cells increase Grow and substantially slow down, difference has statistical significance (P<0.05).

The FLS cell OD values of table 3

Experimental group	OD values (optical density)
		pcDNA3.1	0.1549±0.005
pcDNA3.1-REPS1	0.0842±0.001

The explanation of above-described embodiment is only intended to understand the method and its core concept of the present invention.It should be pointed out that for this For the those of ordinary skill in field, under the premise without departing from the principles of the invention, some improvement can also be carried out to the present invention And modification, these are improved and modification will be also fallen into the protection domain of the claims in the present invention.

Claims

1. detect application of the product of REPS1 gene expressions in the instrument for preparing diagnosis osteoarthritis.

2. application according to claim 1, it is characterised in that the product includes：Pass through reverse transcription PCR, real-time quantitative PCR, immune detection, in situ hybridization chip or high-flux sequence detection of platform REPS1 gene expressions are to diagnose the production of osteoarthritis Product.

3. application according to claim 2, it is characterised in that the product that osteoarthritis is diagnosed with RT-PCR at least wraps Include the primer of a pair of specific amplified REPS1 genes；The product that osteoarthritis is diagnosed with real-time quantitative PCR comprises at least a pair The primer of specific amplified REPS1 genes；The product that osteoarthritis is diagnosed with immune detection includes：It is special with REPS1 albumen Property combine antibody；The product that osteoarthritis is diagnosed with situ hybridization includes：With the nucleic acid array hybridizing of REPS1 genes Probe；The product that osteoarthritis is diagnosed with chip includes：Protein chip and genetic chip；Wherein, protein chip include with The antibody that REPS1 protein-specifics combine, genetic chip include the probe with the nucleic acid array hybridizing of REPS1 genes.

4. application according to claim 3, it is characterised in that the product that osteoarthritis is diagnosed with real-time quantitative PCR The primer of a pair of the specific amplified REPS1 genes comprised at least is as shown in SEQ ID NO.3 and SEQ ID NO.4.

5. a kind of instrument for diagnosing osteoarthritis, it is characterised in that the instrument includes the reagent of detection REPS1 gene expressions； The reagent includes the primer and/or probe, the antibody for detecting REPS1 albumen of detection REPS1 gene mRNAs.

6. instrument according to claim 5, it is characterised in that the primer of the detection REPS1 gene mRNAs includes SEQ Primer pair shown in ID NO.3 and SEQ ID NO.4.

Application of the accelerator of 7.REPS1 genes and/or its expression product in the medicine for preparing treatment osteoarthritis.

8. application according to claim 7, it is characterised in that the accelerator includes promoting the examination of REPS1 gene expressions Agent, the reagent for promoting REPS1 gene expression product stability, the reagent for promoting REPS1 gene expression products activity, promotion The reagent of REPS1 gene expression product functions.

9. application according to claim 8, it is characterised in that promote the reagent of REPS1 gene expressions to include containing REPS1 Reagent, the reagent containing REPS1 protein that the reagent of gene, the carrier for carrying REPS1 genes or host cell are formed.

10. a kind of pharmaceutical composition for being used to treat osteoarthritis, it is characterised in that described pharmaceutical composition includes appointing in 7-9 Accelerator described in one.