CN105349644B

CN105349644B - Application of the TSPAN15 in preparation Osteoarthritis diagnostic preparation

Info

Publication number: CN105349644B
Application number: CN201510752721.2A
Authority: CN
Inventors: 林进; 吴志宏; 范彧; 叶伟亮; 杨跃梅; 陈俊; 康艳娜
Original assignee: Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Current assignee: Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Priority date: 2015-11-06
Filing date: 2015-11-06
Publication date: 2019-01-18
Anticipated expiration: 2035-11-06
Also published as: CN105349644A

Abstract

The present invention relates to application of the TSPAN15 in preparation Osteoarthritis diagnostic preparation.Inventor has found and verifies that TSPAN15 is high in Osteoarthritis tissue to express by large sample, further in cartilage cell the experimental results showed that, the inhibitor of TSPAN15 can be used for preparing treatment medicine for treating arthritis.The present invention provides new treatment medicine for treating arthritis research direction for clinic, has important application value.

Description

Application of the TSPAN15 in preparation Osteoarthritis diagnostic preparation

Technical field

The present invention relates to biomedicine fields, and in particular to TSPAN15 answering in preparation Osteoarthritis diagnostic preparation With.

Background technique

Osteoarthritis (osteoarthritis, OA) be it is a kind of with advancing age and what incidence obviously increased move back Row arthritis disease and the elderly's arthralgia and the main reason for disabling.According to the statistics of the World Health Organization, kneecap Property arthritis accounts for the 4th in women illness rate, and with advancing age, illness rate also obviously rises, the people greater than 65 years old 50% or more the clinical manifestation for having an Osteoarthritis in group.The treatment method of Osteoarthritis is mainly adopted in early days at present With non-operative treatment, the degeneration of cartilage is relieved pain, protects and delayed；Advanced stage cartilage destruction is serious, strips off or episome, spur Hyperplasia etc., this stage will use operative treatment, such as joint debridement, osteotomy or fusion, artificial joint replacement etc., and Very long conservative therapy and expensive surgery cost chronic disease this for Osteoarthritis are not suitable for especially.Therefore, OA is specified Pathogenesis, look for prevention OA, early intervention and the method for cartilage degeneration process delayed just to become medically urgently to be resolved Problem.

Inventor carries out high-flux sequence to 7 Osteoarthritis synovial tissues and 4 check samples, in conjunction with biological information Method carries out genescreen, and highly expressed candidate gene TSPAN15 is picked out in the apparent gene of differential expression, existing to grind There is no TSPAN15 and the relevant reports of Osteoarthritis in studying carefully, and further, inventor has carried out molecular cytobiology method Verifying, it was confirmed that TSPAN15 high expression in Osteoarthritis tissue, TSPAN15 inhibitor can be used for preparing treatment bone and close Save scorching drug.The present invention provides a kind of therapy targets of new Osteoarthritis, have important clinical value.

Summary of the invention

The purpose of the present invention is to provide a kind of Osteoarthritis diagnostic preparation, the Osteoarthritis diagnostic preparation detection The expression product of TSPAN15 gene and/or TSPAN15 gene.

Further, the Osteoarthritis diagnostic preparation uses PCR kit for fluorescence quantitative, genetic chip, immunization method Detect the expression of TSPAN15 gene in Osteoarthritis tissue, it is preferred that contain one in the PCR kit for fluorescence quantitative To the primer of specific amplification TSPAN15 gene；It include the nucleic acid array hybridizing with TSPAN15 gene in the genetic chip Probe, it is furthermore preferred that the upstream primer containing specific detection TSPAN15 gene and downstream in PCR kit for fluorescence quantitative Primer, upstream primer sequence are SEQ ID NO.9, and downstream primer sequence is SEQ ID NO.10.

Further, the diagnostic preparation of the Osteoarthritis is using in immunization method detection Osteoarthritis synovial tissue The expression product of TSPAN15 gene, it is preferred that the immunization method is that ELISA is detected and/or colloidal gold detects.

Further, the ELISA method of the detection TSPAN15 expression product is to use ELISA detection kit.The reagent Commercially available TSPAN15 monoclonal antibody can be used in antibody in box.Further, the kit includes: coating TSPAN15 mono- The solid phase carrier of clonal antibody, enzyme labelled antibody, the substrate of enzyme, protein standard substance, negative controls, dilution, cleaning solution, enzyme are anti- Answer terminate liquid etc..

Further, the colloidal gold method of the detection TSPAN15 albumen is using detection kit, and the antibody can be used Commercially available TSPAN15 monoclonal antibody.Further, the gold-immunochromatographyreagent reagent for assay box using colloidal gold immunochromatographimethod technology or Colloidal gold percolation.Further, detection zone (T) specking on the gold-immunochromatographyreagent reagent for assay box nitrocellulose filter has anti- TSPAN15 monoclonal antibody, quality control region (C) specking have Immunoglobulin IgG.

The purpose of the present invention is to provide above-mentioned Osteoarthritis diagnostic preparations in preparation Osteoarthritis diagnostic tool Application.

The purpose of the present invention is to provide a kind of preparation for treating Osteoarthritis, contain inhibition in the preparation The reagent or compound of transcription or the expression of TSPAN15 gene.

Further, the suppressor containing activation TSPAN15, activation inhibit in the preparation of the treatment Osteoarthritis The albumen of TSPAN15 expression, the siRNA for importing inhibition TSPAN15 transcription or expression, activation promote TSPAN15mRNA to degrade MicroRNA, the molecule for promoting TSPAN15 protein degradation, the expression for inhibiting the factor for promoting TSPAN15 to express and albumen are imported.

Preferably, the preparation of the treatment Osteoarthritis contains one group or several groups of siRNA:SEQ ID in following NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6.It is furthermore preferred that It is SEQ ID NO.5 and SEQ ID NO.6 that the preparation of the treatment Osteoarthritis, which contains siRNA sequence,.

The purpose of the present invention is to provide the preparations of above-mentioned treatment Osteoarthritis in preparation Osteoarthritis therapeutic agent Or the application in reagent.

The purpose of the present invention is to provide a kind of promotion chondrocyte proliferation preparation, the promotion chondrocyte proliferation preparation In the compound containing the expression product for inhibiting TSPAN15 gene and/or TSPAN15 gene.

Further, the suppressor promoted containing activation TSPAN15 in chondrocyte proliferation preparation, activation inhibit The albumen of TSPAN15 expression, the siRNA for importing inhibition TSPAN15 transcription or expression, activation promote TSPAN15mRNA to degrade MicroRNA, the molecule for promoting TSPAN15 protein degradation, the expression for inhibiting the factor for promoting TSPAN15 to express and albumen are imported.

Preferably, described one group or several groups of siRNA:SEQ ID that promotes chondrocyte proliferation preparation to contain in following NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6.It is furthermore preferred that It is SEQ ID NO.5 and SEQ ID NO.6 that the preparation of the treatment Osteoarthritis, which contains siRNA sequence,.

The purpose of the present invention is to provide above-mentioned promotion chondrocyte proliferation preparations to promote chondrocyte proliferation work in preparation Application in tool.

To achieve the above object, the present invention passes through high-flux sequence combination bioinformatics method first and screens candidate base Because of TSPAN15 gene, and then pass through the molecular cytobiology method validation relationship of TSPAN15 gene and Osteoarthritis: The high expression in synovial tissue of TSPAN15 gene, has good correlation with Osteoarthritis, can be used for preparing bone joint Scorching auxiliary diagnosis preparation has important clinical value.

The expression of the known suppressor of those skilled in the art and its expression product usually can be using one of following methods It is and/or several: by the albumen of the inhibition of gene expression of the suppressor of activating genes of interest, activating genes of interest, using RNA Perturbation technique inhibits destination gene expression, activation that the microRNA of target gene mRNA degradation, importing is promoted to promote target gene It encodes the molecule of protein degradation, inhibit to promote the factor of destination gene expression and the expression of albumen.

RNA interference (RNAi) refers to that exogenous and endogenous double-stranded RNA induces the mRNA of homologous target gene in vivo Selective degradation, the phenomenon that leading to posttranscriptional gene silencing, be it is a kind of efficiently, specifically blocked using small double-stranded RNA it is internal The expression of certain specific gene, promotes mRNA to degrade, and cells show is made to go out the technology of specific gene missing phenotype.SiRNA design After the completion can be using direct synthesis technique or building SiRNA expression vector, the siRNA prepared can be coprecipitated by calcium phosphate The Mechanical Methods, cationic-liposome such as shallow lake method, electroporation, DEAE- glucan and polybrene method, microinjection or particle gun The approach such as reagent method transfect cell.

Fluorescence quantitative PCR method is that PCR product is marked by the probe of the specificity of fluorescent dye or fluorescent marker Tracking, real-time online monitoring reaction process can analyze product in conjunction with corresponding software, calculate sample to be tested template Initial concentration.The appearance of quantitative fluorescent PCR greatly simplifies the process of quantitative detection, and is truly realized absolute quantitation. The appearance of a variety of detection systems keeps the selectivity of experiment stronger.Automatic operation improves work efficiency, rapid reaction, repetition The good, high sensitivity of property, high specificity, result are clear.

Genetic chip is also known as DNA microarray (DNAmicroarray), can be divided into three kinds of main Types: 1) be fixed on poly- The nucleic acid probe or cDNA segment on object substrate (nylon membrane, nitrocellulose membrane etc.) surface are closed, the target of isotope labelling is usually used Gene is hybrid with it, and is detected by radiography technology.2) DNA probe array on a glass is fixed with point sample method, Hybridized by the target gene with fluorescent marker and is detected.3) oligonucleotide probe directly synthesized on the hard surfaces such as glass Array hybridizes with the target gene of fluorescent marker and is detected.Genetic chip is as a kind of advanced, extensive, high-throughput detection Technology, applied to the diagnosis of disease, advantage has the following aspects: first is that the sensitivity and accuracy of height；Second is that quickly It is easy；Third is that a variety of diseases can be detected simultaneously.

Known antigen or antibody are adsorbed on surface of solid phase carriers by enzyme-linked immunosorbent assay (ELISA), make enzyme mark The technology that the antigen-antibody reaction of note is carried out in solid phase surface.The technology can be used for detecting macromolecular antigen and specific antibody Deng having many advantages, such as that quick, sensitive, easy, carrier is easy to standardize.ELISA detection kit is according to testing goal and operation Step can be divided into indirect method, double-antibody method, competition law, double site one-step method, prize law survey IgM antibody, using Avidin and The ELISA of biotin.Horseradish peroxidase (HRP) or alkaline phosphatase may be selected in chromogenic substrate in ELISA detection kit Enzyme (AP).

Common immune colloid gold detection technique: (1) immune colloid gold light microscopic decoration method cell suspension smear or tissue are cut Piece can be dyed with the antibody of colloid gold label, can also be enhanced with silver-colored developer solution and be marked on the basis of colloid gold label, So that the silver atoms being reduced is deposited on marked gold particle surface, the sensibility of colloid gold label can be remarkably reinforced.(2) it is immunized Colloidal gold staining method for electron microscopy with the antibody of colloid gold label or antiantibody and negative staining Virus Sample or can be organized in conjunction with ultra-thin section, Then negative staining is carried out.It can be used for observation and the viral diagnosis of morphology of virus.(3) dot immunogold filtration assay application miillpore filter is made Antigen or antibody point are first added sample to be examined on film by carrier after closing, corresponding with the antibody test of colloid gold label after washing Antigen or antibody.(4) antigen of specificity or antibody are fixed on film by colloidal gold immunity chromatography with ribbon, colloidal gold Labelled reagent (antibody or monoclonal antibody) is adsorbed on bonding pad, when sample to be examined is added in the sample pad of test strips one end Afterwards, it moves forward, reacts to each other after dissolving the colloid gold label reagent on bonding pad through capillary action, it is fixed when being moved to When the region of antigen or antibody, the conjugate of object and gold marked reagent to be checked occurs specific binding therewith again and is trapped, and assembles It is taken in detection, colour developing result can be observed by the naked eye.The method has developed into diagnosis test paper, and use is very convenient.

The purpose of the present invention is to provide a kind of gene detecting kit for detecting Osteoarthritis, the kit detection Gene TSPAN15, using special upstream primer and downstream primer, upstream primer sequence is SEQ ID NO.9, downstream primer sequence It is classified as SEQ ID NO.10.

Further, which is suitable for presently, there are all types fluorescence quantitative gene extender in the market, clever Sensitivity is high, it is quantitative quick and precisely, stability it is good, have a good application prospect.

Further, above-mentioned PCR kit for fluorescence quantitative component includes: specific primer, internal control primer, quantitative fluorescent PCR Reaction solution.The internal reference is GAPDH.

The kit also includes RNA extraction agent.

It is an object of the present invention to provide a kind of Osteoarthritis protein detection kit, the detection kit detection TSPAN15 albumen.Further, the kit further includes other detection reagents.

It is an object of the present invention to provide it is a kind of detect Osteoarthritis genetic chip, the genetic chip include with The probe of the nucleic acid array hybridizing of TSPAN15 gene.

Detailed description of the invention

Each group TSPAN15mRNA relative expression quantity after Fig. 1 siRNA interference

Influence of Fig. 2 siRNA to chondrocyte proliferation

Specific embodiment

Present invention will be further explained below with reference to specific examples, for explaining only the invention, and should not be understood as to this The limitation of invention.It will be understood by those skilled in the art that: without departing from the principle and spirit of the present invention may be used To carry out a variety of change, modification, replacement and modification to these embodiments, the scope of the present invention is limited by claim and its equivalent It is fixed.In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition or according to item proposed by manufacturer Part examinations.

The collection of 1 case of embodiment

Take go to a doctor osteoarthritis patient to during in December, 2014 in hospital orthopedics in October, 2012, case group is collected altogether 7, the other diseases patient that control is hospitalized from same time orthopaedics collects totally 4.Obtain the synovial membrane group of all research objects Sample is knitted, -80 DEG C of low temperature refrigerators of number postposition save.Case group meets knee joint OA diagnostic criteria, and it is artificial to carry out knee joint Joint replacement patient；Control group is the patient that meniscus injury and cruciate ligament carry out arthrocsopic surgery treatment.

2 high-flux sequence of embodiment and analysis

RNA extraction is carried out to tissue, agarose gel electrophoresis after RNA is extracted can be extracted from electrophoresis result with preliminary judgement RNA sample it is up-to-standard whether, if can be used for further transcriptome analysis.And then it is divided by NanoDrop1000 Photometer detects the extraction situation of RNA sample, the sample requirement of RNA-seq sequencing: OD260/OD280 1.8-2.2.

Microarray dataset is the 2500 high-flux sequence platform of HiSeq of Illumina company, carries out high-throughput transcript profile depth Sequencing, we use Fast-QC (http://www.bioinformatics.babraham.ac.uk/projects/ after sequencing Fastqc/) software carries out total evaluation, the quality Distribution value including base, the position point of mass value to the quality of sequencing data Cloth, G/C content, PCR duplication content, the frequency etc. of kmer.In differential genes expression analysis, according to obtaining FPKM value, using internationally recognized algorithm EBSeq carry out differential screening.Wherein, when screening, LOG2FC>1 or<-1, FDR< 0.05.In order to better understand the function of difference expression gene, we have carried out Gene Onlogy and letter to difference expression gene Number path analysis, and functional annotation and protein interaction network analysis are carried out to difference expression gene, in view of above data Analysis as a result, we have screened difference expression gene TSPAN15 in conjunction with document.

3 osteoarthritis patient of embodiment and control synovial tissue TSPAN15 expression conditions

One, material and method

1, material

39 osteoarthritis patient synovial tissues and 8 control synovial tissues are chosen, it is grouped and is numbered.Disease Example group meets knee joint OA diagnostic criteria, carries out knee joint artificial joint replacement patient；Control group be meniscus injury and The patient of cruciate ligament progress arthrocsopic surgery treatment.

2, method

The extraction of synovial tissue's total serum IgE of 2.1 osteoarthritis patients and control

UsingReagent (invitrogen, article No. 15596-018) carries out sample rna extraction, experimental implementation It is carried out by product description.

RNA uses Nanodrop2000 ultraviolet specrophotometer measurement RNA purity and concentration after extracting, freeze in -70 DEG C. RNA quality judging standard: the OD260/OD280 value of RNA sample is between 1.7-2.2；Total serum IgE electrophorogram have clearly 28S, 18S band；70 DEG C of water-baths keep the temperature 1 hour after electrophorogram and the map no significant difference before water-bath heat preservation.

2.2 reverse transcriptions synthesize cDNA

UsingIII Reverse Transcriptase (invitrogen, article No. 18080-044) into Row cDNA reverse transcription, experimental implementation are carried out by product description.It is spare that -20 DEG C of refrigerators are put in the cDNA preservation of acquisition.

2.3 Real-Time PCR

2.3.1 instrument and analysis method

With 7500 type fluorescence quantitative PCR instrument of ABI, the relative quantitative assay of data is carried out using 2- △ △ CT method.

2.3.2 design of primers

Using online primer-design software, gene order is referring to NCBI:NM_012339.3 (TSPAN15), interior participation in the election GAPDH is synthesized by invitrogen company after design of primers.Specific primer sequence is as follows:

1 primer sequence of table

Operating process is as follows:

2 RealTime reaction system of table

Component	Additional amount
		2×mix	10μl
Upstream primer (10uM)	0.5μl
		Downstream primer (10uM)	0.5μl
Template	2μl
		Sterile purified water is added	To 25 μ l

(1) reaction system: Power is usedGreen PCR Master Mix (invitrogen, article No. 4367659) it is expanded, experimental implementation is carried out by product description.

Amplification program are as follows: 95 ° of 5min, (95 DEG C of 15sec, 60 DEG C of 45sec) × 35 circulations.

(2) primer screening

After each sample cDNA is mixed, 5 times of gradient dilutions are carried out as template, sample respectively takes 2 μ l to make template after dilution, It is expanded respectively with target gene primer and reference gene primer, while in 60-95 DEG C of progress melt curve analysis analysis, according to expansion Increasing Efficiency height and the unimodal principle of solubility curve carry out primer screening.

(3) sample RealTimePCR is detected

2 μ l will be taken to make template after cDNA10 times of each sample dilution, respectively with target gene primer and reference gene primer into Row amplification.Simultaneously in 60-95 DEG C of progress solubility curve analysis.

Two, experimental result

Real-time quantitative PCR amplification curve inflection point understands that amplification curve entirety collimation is good, shows the amplification effect of each reaction tube Rate is close, and the limit is flat and present without raising up, and exponent phase slope is larger, illustrates that amplification efficiency is higher；Sample amplified production is molten Solution curve be all it is unimodal, illustrate that amplified production only has one, be specific amplification；According to the relative quantification formula of qRT-PCR: 2- Δ Ct × 100% compares expression of the TSPAN15 gene in Osteoarthritis tissue and control tissue.As the result is shown: QRT-PCR stable amplification result, wherein expression of the TSPAN15 gene in Osteoarthritis tissue is the close of control tissue 3 times, result above demonstrates the confluence analysis TSPAN15 gene of high-throughput transcript profile expression data in osteoarthritis patient Highly expressed result.

The separation and culture of 4 cartilage cell of embodiment

One, experimental method

(1) it will be immediately placed in PBS sterile petri dish, remove after sterile knee cartilage sample that traumatic amputation is removed All soft tissues around articular cartilage obtain kneed full-thickness cartilage piece；

(2) cartilage is trimmed to by about 3mm × lmm × lmm size tissue block with aseptic operation knife, shreds cartilaginous tissue, After the PBS buffer solution of these cartilage fragments dual anti-liquid containing Benzylpenicillin sodium salt/gentamicin is rinsed for several times later, it is put into and fills In the sterile petri dish of 12 culture medium of DMEM/F；

(3) the cartilage fragment of these trimmings is moved into centrifuge tube, is centrifuged about 5 points under low temperature with 1000 revs/min of speed Clock；

(4) 0.25% trypsase of 5ml is added after discarding supernatant, digests 30-50 on 37 DEG C of warm blenders Minute, then gently piping and druming discards supernatant liquid, and D-Hanks liquid rinses 3-5 times；

(5) be added 5ml 0.15% II Collagenase Type, be put into constant temperature and shake in case, swayed at 37 DEG C digestion 6 hours, every 3 Cell of a hour receipts；

(6) 200 mesh net filtration postdigestive cell suspensions discard again after low-temperature centrifugation 5 minutes (about 2800 revs/min) Then supernatant washs 3 times in 12 culture solution of DMEM/F, the DMEM/F12 culture medium for containing 10% fetal calf serum is added, with suction Pipe is gently blown and beaten, and so that cell suspension is evenly distributed, to obtain the cell suspension containing cartilage cell；

(7) by cell with 1 × 10⁸/ L is inoculated in 25CM²In culture bottle (5ml/ bottles), it is placed in 37 DEG C of constant temperature, 5%CO₂Saturation Culture in humidified incubator, the frequency for replacing culture solution is every two days 1 time, is observed and is shone using inverted phase contrast microscope Phase starts to pass on as long as cartilage cell converges after in blocks and adherent rate reaches 85%-90%；

(8) before passage starts, culture solution is first cleaned, is then rinsed cell 2-3 times with HANKS liquid；

(9) 0.05% trypsase 1ml is added in culture bottle, is exhausted after bottom of bottle is uniformly distributed after it, so 0.05% tryptic digestive juice is added again afterwards, is limited with covering bottom of bottle, is placed it in 37 DEG C of constant incubators later 2-3 minutes；

(10) it is observed with inverted microscope, when cell starts to bounce back, and space between cells starts to increase, then shows cartilage Cell starts to dissociate；

(11) digestion in HANKS liquid termination culture bottle is added, featheriness bottom of bottle cartilage cell generally requires repeatedly more It is secondary just adherent cartilage cell to be made to completely fall off；

(12) cell suspension is centrifuged with the speed of 1200rpm, after ten minutes discards supernatant；

(13) cell suspension is made and counts, then carries out secondary culture according to the ratio of 1:3.

Two, experimental result

Normal chondrocyte is generally in 24 hours or so beginning adherent growths, and attached cell can achieve after 48 hours 90% or so, bottom of bottle can be covered within 4 to 7 days under normal circumstances, and adherent cartilage cell has endochylema abundant, flat in polygonal Shape can have multiple protrusions, the karyon that cell space center is round or oval, it is seen that the cartilage of 2 to 3 kernels, some regions is thin Born of the same parents are in colony growth.When culture to the third generation, cartilage cell starts to dedifferente, and this trend dedifferented can be with algebra Increase and enhance, the variation of fibrocyte sample is gradually appeared when to forth generation, is embodied in: form is in spindle shape；Edge Protrusion is more and elongated；Endochylema increases but nucleus offset from center and kernel are loose.

5 RNAi of embodiment interferes TSPAN15 expression and the influence to cartilage cell

One, material

(1) cell origin

The cartilage cell that embodiment 4 is cultivated.

(2) siRNA design and synthesis

According to Photographing On-line software siDirect version 2.0 (http://design.rnai.jp/), according to gene Sequence designs corresponding siRNA, particular sequence is shown in Table 3 referring to NCBI:NM_012339.3 (TSPAN15).Conjunction is sent to after design The synthesis of Cheng company.

3 siRNA sequence list of table

Two, experimental method

(1) expression of RNA AF panel cartilage cell TSPAN15 gene

Cell grouping and transfection

1. cell is grouped

C group: blank control group；C1 group: transfection liposome group；C2 group: nonspecific siRNA group is transfected；S1, S2, S3 Group: the siRNA group of specificity is transfected.

2. transfection

According to Lipofectamine^TMThe step of 2000Transfection Reagent is provided carries out.

(1) cartilage cell synchronizes: the day before transfection takes first generation cartilage cell to test, and is added into 5% In FBS DMEM in high glucose culture medium, continue in 37 DEG C of 5%CO₂It is inside cultivated, so that all cartilage cells be made to be in synchronous shape State reduces test interference；

(2)5×10⁴For cell inoculation in 6 orifice plates, these are used for the cell quantity of initial vaccination, should be able to be in 24 hours Cell confluency is set to reach 70%；

(3) prepare siRNA-Lipofectamine^TM2000 compounds:

A. 5ul LipofectamineTM 2000 is diluted with 250u1Opti-MEM, mixed gently, be incubated for 5 points at room temperature Clock.

B. experiment each group take respectively 7.5u1siRNA be added 250u1Opti-MEM I in be diluted, and gently shake by It is mixed；

C. it is incubated for after five minutes, by diluted siRNA and Lipofectamine^TM20 are incubated at room temperature after 2000 mixing Minute.

(4) cell, culture medium and siRNA-Lipofectamine is added in each hole in culture plate^TM2000 is multiple Close object.Then culture plate is gently shaken, them are sufficiently mixed；

(5) 37 DEG C are placed on, CO₂It is incubated for 48 hours in incubator, in fluorescence microscopy microscopic observation cell transfecting quantity, inspection Survey transfection efficiency；

(6) transfection efficiency is the ratio of the cell quantity in fluorescope and the light microscopic visual field, and the transfection efficiency of cell reaches 90% or more, it can just carry out subsequent experiment.Calculation formula is as follows:

Cell quantity × 100% under the transfection efficiency=cell that fluoresces quantity/same field of view

3. the variation of application Real-time PCR method detection transfection front and back TSPAN15 gene expression

(1) building of standard curve: being chosen at 1 bottle of cartilage cell normally cultivated in 50mI culture bottle, extracts RNA, surveys Determine RNA concentration and purity, carry out reverse transcription reaction, ten times of the DNA profiling dilutions that reaction is generated obtain being equivalent to 10⁴- 10⁰The DNA profiling of copies/ul is separately added into TSPAN15 gene primer and internal control primer, prepares 25u1 reaction system, uses Real-time PCR amplification instrument carries out pcr amplification reaction.Obtain the standard curve of TSPAN15 and internal reference.

(2) variation of Real-time PCR method detection transfection front and back TSPAN15 gene expression: group of cells is extracted RNA measures RNA concentration and purity, carries out reverse transcription reaction, every group of DNA profiling is simultaneously into the Real- of TSPAN15 and internal reference Time PCR reaction, experiment is in triplicate.

(3) agarose gel electrophoresis is carried out to PCR product.

Three, experimental result

The siRNA and control siRNA of 3 TSPAN15 genes transfect first generation cartilage cell respectively, as the result is shown a large amount of Green fluorescence is found in cartilage cell, it was demonstrated that the transfection that siRNA has been obtained in cartilage cell, then in fluorescope and light microscopic Lower observation cartilage cell's quantity, carries out the detection of transfection efficiency, transfection efficiency reaches 90% or more as the result is shown.Real-time PCR the results show that transfect nonspecific siRNA group to TSPAN15 gene expression in cartilage cell without obvious inhibiting effect, and Blank control group no difference of science of statistics, 3 TSPAN15 gene siRNA groups of transfection are all to TSPAN15 gene table in cartilage cell Up to certain inhibiting effect is played, wherein TSPAN15-siRNA2 and TSPAN15-siRNA3 inhibitory effect is most obvious, and inhibiting rate reaches 64% and 72%, it is specifically shown in Fig. 1.

The proliferative conditions of 6 mtt assay of embodiment detection cartilage cell

One, mtt assay experimental procedure

1. being added about 1 × 10 according to every hole in 96 orifice plates⁴Cell, then in 37 DEG C of 5%CO₂Under conditions of culture it is 24 small When；

2. according to experimental group, (blank control group, cartilage cell add nonspecific siRNA group, cartilage cell to add TSPAN15-siRNA3 group, every group of 6 repetitions) continue culture until being suitble to detection；

3. then at 37 DEG C, 5%CO₂And 100% continue under conditions of humidity to be incubated for appropriate time；

4. 5 × MTT is diluted to 1 × MTT with Dilution Buffer simultaneously；

5. 1 × MTT of 50ul is added in every hole, and it is incubated for 4 hours under the conditions of 37 DEG C；

6. after supernatant is sucked out, also needing that 150u1DMSO is added in every hole, and place it on plate shaker and shaken It is even；

7. microplate reader wavelength is set as 570nm, the optical density in each hole is detected.

8. the calculating of cell proliferation rate: cell proliferation rate %=(by prospect hole OD value/control cell OD value) × 100%, In by prospect hole OD value=instrument connection OD value-background OD value (no cell but the complete medium for having MTT), control cell OD value For the OD value for normally cultivating cell hole.

Three, experimental result

It was sampled on node at 0,12,24,48 and 72 hour respectively, opposite cartilage cell's control group, cartilage cell Jia Feite For anisotropic siRNA group cell Proliferation without significant difference, cartilage cell adds TSPAN15-siRNA3 group to be proliferated in 0-24 hour cell It is unobvious, but as time increases, chondrocyte proliferation rate dramatically increases on 48,72 hours nodes, and difference has system Meter learns meaning (P < 0.05), as a result as shown in Fig. 2 and table 4.

The proliferation rate (%) of 4 each group cartilage cell of table

Time	Blank control group	Nonspecific siRNA group	TSPAN15-siRNA3 group
				0h	99.273±0.674	99.273±0.771	99.273±0.528
12h	99.562±0.985	98.852±1.562	102.011±1.058
				For 24 hours	99.681±1.024	99.746±1.974	104.323±0.972
48h	99.398±1.387	100.023±0.956	129.885±1.103
				72h	99.775±1.916	99.546±0.849	140.011±2.007

Claims

1. the preparation of the expression product of detection TSPAN15 gene and/or gene answering in preparation Osteoarthritis diagnostic preparation With.

2. application according to claim 1, which is characterized in that the Osteoarthritis diagnostic preparation uses fluorescent quantitation PCR kit, genetic chip, immunization method detect the expression of TSPAN15 gene in Osteoarthritis tissue.

3. application according to claim 2, which is characterized in that containing a pair of special in the PCR kit for fluorescence quantitative The primer of specific amplification TSPAN15 gene；It include the spy with the nucleic acid array hybridizing of TSPAN15 gene in the genetic chip Needle.

4. application according to claim 3, which is characterized in that contain specificity in the PCR kit for fluorescence quantitative The upstream primer and downstream primer of TSPAN15 gene are detected, upstream primer sequence is SEQ ID NO.9, and downstream primer sequence is SEQ ID NO.10。

5. application according to claim 1, which is characterized in that the Osteoarthritis diagnostic preparation uses immunization method Detect the expression product of TSPAN15 gene in Osteoarthritis synovial tissue.

6. application according to claim 5, which is characterized in that the immunization method is ELISA detection and/or colloidal gold Detection.

7. reagent or compound containing transcription or the expression for inhibiting TSPAN15 gene are in the preparation of preparation treatment Osteoarthritis In application, which is characterized in that inhibit TSPAN15 gene transcription or expression reagent or compound be inhibit TSPAN15 base The siRNA of transcription or the expression of cause.

8. application according to claim 7, which is characterized in that the preparation of the treatment Osteoarthritis contains in following One group or several groups of siRNA:SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 With SEQ ID NO.6.

9. the compound containing the expression product for inhibiting TSPAN15 gene and/or TSPAN15 gene promotes cartilage cell in preparation The application being proliferated in preparation, which is characterized in that inhibit the compound of the expression product of TSPAN15 gene and/or TSPAN15 gene For inhibition TSPAN15 gene and/or the siRNA of the expression product of TSPAN15 gene.

10. application according to claim 9, which is characterized in that containing following in the promotion chondrocyte proliferation preparation In one group or several groups of siRNA:SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6.