CN106755549A

CN106755549A - Application of the molecular marker in osteoarthritis

Info

Publication number: CN106755549A
Application number: CN201710155724.7A
Authority: CN
Inventors: 张晓非
Original assignee: TIANJIN HOSPITAL TIANJIN CITY
Current assignee: TIANJIN HOSPITAL TIANJIN CITY
Priority date: 2017-03-16
Filing date: 2017-03-16
Publication date: 2017-05-31
Anticipated expiration: 2037-03-16
Also published as: CN106755549B

Abstract

The invention discloses a kind of application of molecular marker in osteoarthritis, the molecular marker is FGGY.The present invention is experimentally confirmed, FGGY genes up-regulated in the synovial tissue of Human Osteoarthritis, and the expression based on this by detecting FGGY can be used for the diagnosis of osteoarthritis.

Description

Application of the molecular marker in osteoarthritis

Technical field

The invention belongs to biomedicine field, it is related to application of the molecular marker in osteoarthritis, the specific molecule Mark is FGGY.

Background technology

Osteoarthritis is a kind of chronic joint diseases.It is with the denaturation of articular cartilage, the destruction of cartilaginous tissue and joint The hyperplasia of surrounding sclerotin is characterized.The incidence of osteoarthritis increases with advancing age, and it is a kind of common old age Property joint disease, be also the elderly's pain and the Etiological disabled.Research in worldwide shows the 60 of 60-65% Year, above old man suffered from Symptomatic Osteoarthritis, and 80% such patient activity is limited.

With the fast-developing of China's economic, the raising of trophic level and the improvement of medical level, the average longevity of resident Life is extended and gradually steps into aging society；Therefore become more important for the prevention and control of chronic disease.Bones and joints It is scorching that the social concern of such as disabled, moving obstacle and pain is brought to society as a kind of chronic disease, further increase medical treatment and The burden of nursing.

Although at present research think that the morbidity of osteoarthritis is relevant with age, sex, and with inherent cause and environmental factor Etc. closely related, but the pathogenesis of osteoarthritis is still indefinite.Osteoarthritis morbidity generally involves knee joint, early symptom compared with Gently, easy delay diagnosis, have clinically been in middle and advanced stage during most of Human Osteoarthritis to hospital admission, patients with terminal is past Toward that can only be treated by joint replacement surgery, huge spirit and financial burden is brought to patient and family members.Therefore, Bones and joints The early diagnosis of inflammation, therapeutic effect are monitored and prognosis evaluation plays an important roll for the preventing and treating of osteoarthritis.At present, Bones and joints Scorching diagnosis still relies on clinical symptoms and x-ray imaging diagnosis, and K-L classifications are still to judge the osteoarthritis order of severity, in advance Afterwards with the Main Basiss for instructing osteoarthritis treatment.In theory, molecular marker is the discovery that medical diagnosis on disease, examination of curative effect and pre- After assess most sensitive, maximally effective index.Therefore, the new osteoarthritis molecular marker of searching and drug target are osteoarthritis Key issue urgently to be resolved hurrily in early prevention and treatment.

Molecular targeted therapy refers to the abnormal signal of regulation and control disease related gene, intervention disease on cellular and molecular level Path or blocking energy pathway, the molecular target that searching can be intervened have great importance to the targeted therapy of osteoarthritis.

The content of the invention

In order to make up the deficiencies in the prior art, diagnose and treat for osteoarthritis it is an object of the invention to provide one kind Molecular marker.

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

The invention provides the application of FGGY genes, the product for preparing diagnosis osteoarthritis.

Further, the product diagnoses osteoarthritis by determining the expression of FGGY in sample.

Further, FGGY up-regulateds in osteoarthritis.

Further, the product is included by by sequencing technologies, nucleic acid hybridization technique, nucleic acid amplification technologies or immune survey The change of fixed method detection FGGY genes and/or its expression product.

Further, the nucleic acid amplification technologies are selected from PCR, reverse transcriptase polymerase chain reaction, transcription Jie Amplification, ligase chain reaction, strand displacement amplification and the amplification based on nucleotide sequence led.

The invention provides a kind of product for diagnosing osteoarthritis, the product can be by detecting FGGY genes in sample The change of gene and/or its expression product diagnoses osteoarthritis.

Further, the product includes chip, kit or preparation.

Further, the product includes chip, kit or preparation.Wherein, the chip includes genetic chip, protein Chip；The genetic chip includes solid phase carrier and is fixed on the oligonucleotide probe of solid phase carrier, and the oligonucleotides is visited Pin includes the oligonucleotide probe for FGGY genes for detecting FGGY gene transcription levels；The protein-chip includes Solid phase carrier and be fixed on solid phase carrier FGGY albumen specific antibody or part；The kit includes genetic test Kit and protein immunization detection kit；The gene detecting kit includes the examination for detecting FGGY gene transcription levels Agent；The protein immunization detection kit includes the specific antibody or part of FGGY albumen.

Genetic chip of the present invention or gene detecting kit can be used to detect including the multiple including FGGY genes The expression of gene (for example, multiple genes related to osteoarthritis).The protein chip or protein immunization detection reagent Box can be used to detect the table including the multiple protein (such as multiple protein related to osteoarthritis) including FGGY albumen Up to level.Multiple marks of osteoarthritis are detected simultaneously, the accuracy rate of osteoarthritis diagnosis is greatly improved.

The invention provides application of the FGGY genes in the pharmaceutical composition for preparing treatment osteoarthritis.

Further, described pharmaceutical composition includes FGGY genes and/or the lower adjustment of its expression product.The lower bag of adjusting Include and suppress the material of FGGY gene expressions, suppress the material of FGGY gene expression product stability, and/or suppress FGGY gene tables Up to the material of its lytic activity.

Preferably, the lower adjustment is the siRNA or the antibody for FGGY albumen for FGGY genes.

Further, the described pharmaceutical composition also other drugs including anti-Bones and joints, the combination between medicine is conducive to disease The effective treatment of disease.

The advantages of the present invention：

Present invention firstly discovers that molecular marker FGGY and osteoarthritis that development occurs is related, by detecting subject The change of FGGY, can be used for the diagnosis of osteoarthritis.

The present invention provides theoretical foundation for the Mechanism Study of osteoarthritis, is that the clinical diagnosis and treatment of osteoarthritis are carried A kind of supplementary means is supplied.

Brief description of the drawings

Fig. 1 is the differential expression situation map in osteoarthritic tissue using high-flux sequence detection gene；

Fig. 2 is the expression figure in Human Osteoarthritis using QPCR detection FGGY genes.

Specific embodiment

The present invention, by a large amount of screenings, is found that in osteoarthritis FGGY is in first by in-depth study extensively Existing specificity low expression.

FGGY

The albumen of FGGY gene codes can have with carbohydrate such as phosphorylation ribulose, ribitol, L-arabinose alcohol Research finds that in some crowds the gene polynorphisms are relevant with sporadic ALS.

Can be used for FGGY polynucleotides of the invention or albumen (polypeptide) sequence includes the FGGY of various sources and species, and And the FGGY including wild type, saltant type, or its active fragment.In some embodiments of the present invention, the FGGY sources In the mankind, the coded sequence or amino acid sequence of a kind of representational people FGGY are respectively such as SEQ ID NO.1 and SEQ ID NO.2 It is shown.

Polynucleotides of the invention can be DNA form or rna form.DNA form includes cDNA, genomic DNA or people The DNA of work synthesis.DNA can be single-stranded or double-strand.DNA can be coding strand or noncoding strand.

Polypeptide of the invention can be recombinant polypeptide, natural polypeptides, synthesis polypeptide, preferably recombinant polypeptide.It is of the invention many Peptide may also include or not include the methionine residues of starting.

The polynucleotides for encoding the mature polypeptide of FGGY include：The coded sequence of encoding mature polypeptide；Mature polypeptide Coded sequence and various additional coding sequences；The coded sequence (and optional additional coding sequence) and non-coding of mature polypeptide Sequence.Term " polynucleotides of coded polypeptide " can be included encoding the polynucleotides of this polypeptide, or also include attached Plus the polynucleotides of coding and/or non-coding sequence.

The invention further relates to the variant of above-mentioned polynucleotides, its coding has many of identical amino acid sequence with the present invention The fragment of peptide or polypeptide, analogs and derivatives.The variant of this polynucleotides can be the natural allelic variant for occurring or The variant that non-natural occurs.These nucleotide variants include substitution variants, Deletion variants and insert variation.Such as this Known to field, allelic variant is an alternative forms for polynucleotides, it be probably one or more nucleotides substitution, Missing is inserted, but will not be from the function of substantially changing its coded polypeptide.

The invention further relates to the nucleic acid fragment with above-mentioned sequence hybridization, including the just nucleic acid fragment with antisense.Such as this Literary used, the length of " nucleic acid fragment " at least contains 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 cores Thuja acid, more than preferably at least 100 nucleotides.Nucleic acid fragment can be used for the amplification technique (such as PCR) of nucleic acid determining and/or Separate the polynucleotides of coding FGGY albumen.

People FGGY nucleotides full length sequence of the invention or its fragment can generally use PCR TRAPs, recombination method or artificial The method of synthesis is obtained.For PCR TRAPs, can be according to published relevant nucleotide sequence, especially ORFs sequence Arrange to design primer, and made with commercially available cDNA storehouses or the cDNA storehouses as prepared by conventional method well known by persons skilled in the art It is template, expands and obtain relevant sequence.When sequence is more long, it is often necessary to carry out twice or multiple PCR is expanded, then again will be each The secondary fragment for amplifying is stitched together by proper order.Once obtain relevant sequence, it is possible to recombination method come large quantities of Amount ground obtains relevant sequence.This is typically to be cloned into carrier, then is transferred to cell, then by conventional method from after propagation Isolated relevant sequence in host cell.

Host cell and carrier

It is of the present invention carry gene carrier be various carriers known in the art, such as commercially available carrier, including plasmid, Clay, bacteriophage, virus etc..Host cell can be prokaryotic, such as bacterial cell；Or low eukaryotic, such as yeast Cell；Or higher eucaryotic cells, such as mammalian cell.Representative example has：Escherichia coli, the bacterium of streptomyces is thin Born of the same parents；Fungal cell's such as yeast；Plant cell；The insect cell of fruit bat S2 or Sf9；The zooblast of CHO, COS or 293 cells Deng.

Converting host cell with recombinant DNA can be carried out with routine techniques well known to those skilled in the art.When host is original When core biology is such as Escherichia coli, the competent cell that can absorb DNA can be harvested after exponential phase of growth, use CaCl₂Method treatment, institute With the step of it is generally well-known in the art.Another method is to use MgCl₂.If desired, conversion can also use the side of electroporation Method is carried out.When host is eucaryote, following DNA transfection methods are can select：Calcium phosphate precipitation, conventional mechanical methods are such as Microinjection, electroporation, liposome packaging etc..

Recombinant polypeptide in the above methods can be expressed or be secreted into extracellular in the cell or on cell membrane.Such as Fruit need, can utilize its physics, chemistry and other characteristics be separated by various separation methods and purification of Recombinant albumen.This A little methods are well-known to those skilled in the art.The example of these methods is included but is not limited to：Conventional renaturation process, use Protein precipitant processes (salting-out method), centrifugation, the broken bacterium of infiltration, super treatment, ultracentrifugation, sieve chromatography (gel filtration), suction Attached chromatography, ion-exchange chromatography, the combination of high performance liquid chroma- tography (HPLC) and other various LC technologies and these methods.

Molecular marker

Term " molecular marker " is its expression in tissue or cell and normal or healthy cell in the present invention Or the expression of tissue compares any gene or albumen for changing.

Herein comprising the methods availalbe any in the prior art for detection molecules marker expression.Molecular marker of the present invention The expression of thing can be detected (e.g., RNA transcript) or protein level in nucleic acid level.It is intended to determine by " detection expression " The quantity of the expression product of RNA transcript or its molecular marker gene or presence.Therefore, " detection expression " is comprising a molecule mark Will thing is determined to be expressed, can not being detected expression, and expression is in low-level, expression in normal level or the reality of overexpression Example.In order to determine low expression, the examined body sample can compare with the corresponding body sample from Healthy People.That That is, " normal " level of the expression is the expression of molecular marker, such as from human subject or not by bone Molecular marker expression in the cervical tissue sample of the sufferer of afflicted with osteoarthritis.This sample can be with normalized form Present.In certain embodiments, the determination of molecular marker overexpression need to not compare body sample and corresponding from Healthy People Body sample.

Detection method

Nucleic acid amplification technologies of the present invention are selected from PCR (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), the amplification (TMA) of transcriptive intermediate, ligase chain reaction (LCR), strand displacement amplification (SDA) and based on nucleic acid sequence The amplification (NASBA) of row.Wherein, PCR is needed RNA reverse transcriptions into DNA (RT-PCR) before amplification, and TMA and NASBA directly expand Increase RNA.

Generally, PCR is circulated using the annealing of denaturation, primer pair and opposite strand and the multiple of primer extend, with index side Formula increases the copy number of target nucleic acid sequence；Then be used for for reverse transcriptase (RT) to prepare complementary DNA (cDNA) from mRNA by RT-PCR, Then cDNA is expanded by PCR to produce multiple copies of DNA；TMA is in the temperature of substantial constant, ionic strength and pH Under the conditions of autocatalytically synthesize multiple copies of target nucleic acid sequence, multiple RNA copies of wherein target sequence are autocatalytically given birth to Into other copy, TMA optionally includes using blocking, part, dwell section and other modification parts, to improve TMA processes Sensitivity and the degree of accuracy；Two groups of complementary DNA oligonucleotides that LCR is hybridized using the adjacent area with target nucleic acid.DNA few nucleosides Acid is covalently attached in the multiple circulations of repetition of thermal denaturation, hybridization and connection by DNA ligase, to produce detectable double-strand Connection oligonucleotide product；SDA is circulated using the multiple of following steps：Primer sequence pair is moved back with the opposite strand of target sequence Fire, primer extend is carried out in the case where there is dNTP α S to produce (hemiphosphorothioated) of the thiophosphorylation of double-strand half Primer extension product, the nicking of endonuclease that semi-modified restriction enzyme enzyme recognition site is carried out mediation, and from cutting The polymerase-mediated thing extension drawn that mouth 3' ends are carried out is put with replacing existing chain and producing for next round primer annealing, nicking and chain The chain for changing, so as to the geometry for causing product is expanded.

Probe

" probe " refers to the molecule of the expression that can be used in measurement specific gene.Exemplary probe includes PCR primer And gene specific DNA oligonucleotide probe, the micro probe array being for example fixed on microarray substrate, quantitative nucleic acid enzyme are protected Shield inspection probe and the molecular barcode probe for connecting and the probe being fixed on pearl.

Term " probe " refers to that what can be combined with the particular sequence of another molecule or subsequence or other parts divides in the present invention Son.Unless otherwise noted, " probe " is often referred to be matched and another polynucleotides (often referred to as " target multinuclear by complementary base Thuja acid ") combine polynucleotide probes.Lack sufficient sequence according to the preciseness of hybridization conditions, probe energy and with the probe mutual The target polynucleotide of benefit property is combined.Probe can make direct or indirect mark, and its scope includes primer.Crossing system, including, but It is not limited to：Solution, solid phase, mixed phase or in situ hybridization determination method.

As probe, it is possible to use fluorescence labeling, radio-labeled, biotin labeling etc. are carried out to cancer detection with polynucleotides The label probe of mark.The labeling method of polynucleotides is in itself known.Can check by the following method in sample whether There is subject nucleic acid：Fixed subject nucleic acid or its amplified matter, are hybridized with label probe, are washed, and then determine with The mark of solid phase binding.Alternatively, cancer detection polynucleotides can be also fixed, subject nucleic acid is hybrid with it, then application mark The detections such as note probe are incorporated into the subject nucleic acid in solid phase.In this case, many nucleosides are used in the cancer detection being incorporated into solid phase Acid is also referred to as probe.The method that subject nucleic acid is determined using polynucleotide probes is also known in this area.Can enter as follows Row the method：Polynucleotide probes are made to be connect (preferably within ± 4 DEG C) near Tm or its with subject nucleic acid in buffer solution Touch for hybridizing, wash, the template nucleic acid for then determining the label probe of hybridization or being combined with solid phase probe.

The polynucleotides used as probe be preferably sized to 18 or more nucleotides, more preferably 20 or More nucleotides, and coding region total length or less.When being used as primer, the polynucleotides are preferably sized to 18 Or more nucleotides, and 50 or more Oligonucleotide.These probes have mutual with the specific base sequence of target gene The base sequence of benefit.Here, so-called " complementation ", as long as hybridization, can not be complete complementary.These polynucleotides are usual Have more than 80%, preferably more than 90%, more preferably more than 95%, particularly preferred 100% relative to the specific base sequence Homology.These probes can be DNA, or RNA, furthermore it is possible to be to lead in one part or whole nucleotides Cross the polynucleotides that the artificial replacement nucleic acid such as PN, LNA, ENA, GNA, TNA is obtained.

Chip

Term " chip " is also referred to as " array ", refers to the solid support of the nucleic acid comprising connection or peptide probes.Array is usual Comprising the various different nucleic acid or peptide probes that substrate surface is connected to according to different known locations.These arrays, also referred to as " microarray ", can generally produce these arrays using mechanical synthesis methods or light guiding synthetic method, and the light guiding is closed The combination of photolithography method and solid phase synthesis process is incorporated into method.Array can include flat surface, or can be pearl Nucleic acid or peptide in son, gel, polymer surfaces, the fiber of such as optical fiber, glass or any other suitable substrate.Can be with Certain mode carrys out array of packages, so as to allow to carry out the diagnosis of global function device or the manipulation of other manner.

Antibody

In the present invention, term " antibody " refers to the natural or synthetic antibody of selective binding target antigen.The term bag Include polyclonal and monoclonal antibody.In addition to complete immunoglobulin molecules, the fragment of those immunoglobulin molecules or poly- It is " anti-that the mankind of the immunoglobulin molecules of compound and selective binding target antigen or humanization form are also included within term In the range of body ", as long as they show desired BA." monoclonal antibody " refers to from a group substantially homogeneity The antibody that obtains of antibody, that is, each antibody for constituting colony is identical and/or combine same epitope, except production monoclonal antibody During it is issuable may become external, such variant is general with indivisible presence.Such monoclonal antibody is typically wrapped The antibody comprising the polypeptide sequence for combining target is included, wherein target Binding peptide sequence is by including in many polypeptide sequences of comforming Select what single target Binding peptide sequence was obtained in interior process.

Monoclonal antibody also includes " chimeric " antibody, wherein a part for heavy chain and/or light chain and derived from particular species Or the corresponding sequence that belongs in the antibody of specific antibodies classification or subclass is identical or homologous, and the remainder of chain with derived from another One species or the corresponding sequence belonged in the antibody of another antibody isotype or subclass are identical or homologous, and this antibody-like piece Section, as long as they show desired BA.

Polyclonal antibody includes antibody obtained by the immune FGGY protein of animal (for example, mouse) to producing human antibody. After chimeric antibody or humanized antibody is prepared for, the amino acid in variable region (for example, FR) and/or constant region can be used Other amino acid substitutions etc..

Pharmaceutical composition

Pharmaceutical composition of the invention includes the lower adjustment of FGGY genes and/or its expression product, when being carried out in treatment During using (administration), expression or the activity of FGGY genes and/or albumen can be suppressed, so as to suppress osteoarthritis.Of the invention In specific embodiment, the lower adjustment includes suppressing the material of FGGY gene expressions, suppresses FGGY gene expression products stabilization Property material, and/or suppress the material of FGGY gene expression products activity.

These can be generally configured in nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein pH 5-8 is ordinarily be about, preferably pH is about 6-8, although pH value can have with the property for being configured material and illness to be treated Changed.The pharmaceutical composition for having configured can be administered by conventional route, including but be not limited to be administered orally, non-stomach Intestinal canal administration, it is administered or passes through the storage medicine being implanted into dress by sucking spray delivery, local administration, rectally, nasal administration, cheek Put administration.In the present invention term parenteral route include subcutaneous, intracutaneous, intravenous, intramuscular, in joint, intra-arterial, intrasynovial, chest In bone, bring up in interior, damage location and intracranial injection or infusion techniques.As long as destination organization can be reached.

Pharmaceutical composition in the present invention, the FGGY albumen of the present invention containing safe and effective amount or its lower adjustment and pharmacy Upper acceptable carrier or excipient.This kind of carrier is included but is not limited to：Salt solution, buffer solution, glucose, water, glycerine, ethanol And combinations thereof.Pharmaceutical preparation should match with administering mode.Pharmaceutical composition of the invention can be made into injection form, for example The aqueous solution with physiological saline or containing glucose and other assistant agents is prepared by conventional method.Such as tablet and capsule it Class pharmaceutical composition, may be carried out by conventional means preparation.Pharmaceutical composition such as injection, solution, tablet and capsule are preferably in nothing Prepared under the conditions of bacterium.The dosage of active component is the effective dose for the treatment of.

Pharmaceutical composition of the invention can be used for lowering endogenic FGGY hyperproteosises, by reducing FGGY albumen Expression weakens the function of FGGY albumen, so as to treat because FGGY albumen increases caused osteoarthritis.

Medicine of the invention can also can be with master with the drug combination of other treatment osteoarthritis, other therapeutic compound The active component wanted is administered simultaneously, or even is administered simultaneously in same composition.Can also with single composition or with it is main The different dosage form of active component individually give other therapeutic compounds.The Fractional of main component can be with other Therapeutic compound is administered simultaneously, and other dosage can be administered alone.Over the course for the treatment of, can be according to the serious journey of symptom The physiologic response of degree, the frequency of recurrence and therapeutic scheme, adjusts the dosage of pharmaceutical composition of the present invention.

Term " treatment " refers to for the purpose of healing, improvement, stabilization or prevention disease, pathological state or illness in the present invention The medical supervision carried out to patient.The term includes active treatment, i.e., specially for the purpose of improving disease, pathological state or illness Treatment, and also including etiological treatment, the i.e. treatment for the purpose of removing relevant disease, pathological state or the cause of disease of illness. Additionally, the term also includes palliative treatment, that is, it is designed for controlling for relief of symptoms rather than cure diseases, pathological state or illness Treat；Prophylactic treatment, i.e., at utmost reducing or partially or completely suppress the development of relevant disease, pathological state or illness For the purpose for the treatment of；And supportive treatment, i.e., it is mesh to improve relevant disease, pathological state or illness for supplementing another kind Specific therapy treatment.

In the present invention term " sample " include any cell, tissue or body fluid sampling, including but not limited to, organize or The source of cell sample can come from the solid tissue of fresh, freezing and/or the organ or tissue's sample for preserving, or living tissue Check or aspirate, blood or any blood constituent；Body fluid such as celiolymph, amniotic fluid, peritoneal fluid or interstitial fluid.Tissue samples Can be the cell or cell line of primary or in vitro culture.Alternatively, tissue or cell sample are obtained from diseased tissue/organ. Tissue samples may include the compound of the tissue nature mixing, such as preservative, anticoagulant, buffer, fixative, battalion Support agent, antibiotic or similar compound.

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 technique 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) described in condition, or according to the condition proposed by manufacturer.

Embodiment 1 screens the gene marker related to osteoarthritis

1st, sample collection

It is each to collect 6 surrounding normal synovial tissues and Osteoarthritic Synovium tissue, confirmed through pathological diagnosis, all trouble Person is preoperative not to receive any type for the treatment of.The sample that operation is cut freezes in liquid nitrogen, the equal informed consent of patient, above-mentioned all The acquirement of sample is by the agreement of the committee of organizational ethics.

2nd, the preparation of RNA sample

Taking-up freezes the tissue samples in liquid nitrogen, tissue samples is put into the mortar of precooling and is ground, according to Specification in kit is extracted and separates RNA.It is specific as follows：

1) 1ml TRIzol are added into glass homogenate bottle in super-clean bench and (180 DEG C of homogenate bottle baking oven is dried 4 in advance Individual hour), homogenate bottle to be pressed onto instrument, the tissue for weighing 50-100mg is put into glass homogenate bottle, and rotating speed is adjusted into 1500 Turn left the right side, beginning is homogenized in ice-water bath, often grind 30s, stop 30s, repeatedly 3-4 times.Sample volume is not to be exceeded TRIzol volumes 10%.

2) sample of TRIzol will be added in (15-30 DEG C) placement 10min of room temperature, nucleic acid-protein compound is divided completely From.

3) 1ml TRIzol add 200 μ l chloroforms, acutely vibrate 2min, are rocked once every 1min, after 5-6 times then quiet Only 7min.

4) 4 DEG C, 12000rpm is centrifuged 15min.Sample is divided into three layers：Bottom is yellow organic phase, and upper strata is colourless aqueous phase With an intermediate layer.RNA is main in water phase, and water phase volume is about the 60% of TRIzol used.

5) repeat step 3) and 4).

6) upper strata aqueous phase is transferred in new EP pipes and (tries not to be drawn to intermediate layer in order to avoid polluting).Add 500 μ l Isopropanol, room temperature places 10min.

7) 4 DEG C, 12000rpm is centrifuged 15min, white precipitate occurs in ttom of pipe after centrifugation.On carefully being removed with pipettor Clearly.

8) the cold ethanol of 1ml 75%, concussion washing precipitation are added.4 DEG C, 7500rpm is centrifuged 5min, carefully discards supernatant.

9) repeat step 8).

10) by EP pipes back-off in sucking unnecessary moisture on filter paper, and with the liquid (rifle in the 10 μ careful suction pipes of l pipette tips Head should not contact RNA), EP pipes room temperature is placed into 5min.

11) water (DEPC water) of the 40 μ l without RNase is added, after the RNA that will be extracted dilutes 5 times, OD is detected with Nanodrop Value and concentration, carry out mark on pipe；It is placed in -80 DEG C of Refrigerator stores.

3rd, high flux transcript profile sequencing

3.1 RNA-seq reads are positioned

First by low-quality read removal obtain clean read, then using TopHat v1.3.1 will clean fragment and UCSC H.sapiens reference genes group (hg19) is matched, the index of the advance structure of H.sapiens UCSC hg19 editions Downloaded from TopHat homepages, and as reference gene group, when being matched with genome using TopHat, it is allowed to each read (acquiescence Sites, most 2 mispairing are matched to 20) there is multiple.TopHat sets up possible according to exon region and GT-AG shear signals Shearing site storehouse, navigates on genome the read for not navigating to genome according to these shearing site storehouses.We use The system default parameter of TopHat methods.

3.2 transcript abundances are assessed

By Cufflinks v1.0.3 treatment, Cufflinks v1.0.3 are by RNA-seq pieces for the read file for matching Hop count mesh is standardized the relative abundance for calculating transcript.FPKM values refer to it is every 1,000,000 sequencing fragment in match it is specific The segment number of exon region gene 1kb long.The confidential interval of FPKM estimates is calculated by Bayesian inference method. The GTF comment files of the reference that Cufflinks is used download (Homo_ from Ensembl databases sapiens.GRCh37.63.gtf)。

The detection of 3.3 difference expression genes

Cuffdiff is transferred to by the Ensembl GTF files of download and by the original document that TopHat is matched, Cuffdiff re-evaluates the gene expression abundance of the transcript listed in GTF files using original matching files, detects difference table Reach.The only q values ＜ 0.01 in Cuffidff outputs, test display is considered as successfully more just differential expression.

4th, result

RNA-seq results show that expression quantity of the FGGY genes in Human Osteoarthritis is significantly higher than the expression of normal person Amount.

The differential expression of the QPCR sequence verification FGGY genes of embodiment 2

1st, large sample QPCR checkings are carried out to FGGY gene differential expressions.Selected according to the sample collection mode in embodiment 1 Select normal synovial tissue and each 80 of Osteoarthritic Synovium tissue.

2nd, RNA extraction steps are as described in Example 1.

3rd, reverse transcription：(use FastQuant cDNA the first chain synthetic agent box (article No.s：KR106 mRNA reversions) are carried out Record)

Removal genomic DNA reaction first, adds 5 × gDNA Buffer 2.0,1 μ g of μ l, TotalRNA in test tube, Plus Rnase Free ddH₂O makes cumulative volume to 10 μ l, 42 DEG C of heating 3min in water-bath, then by 10 × Fast RT Buffer μ l, the RNase Free ddH of 2.0 μ l, RT Enzyme Mix, 1.0 μ l, FQ-RT Primer Mix 2.0₂The μ l of O 5.0, mixing After to add and be mixed together totally 20 μ l in above-mentioned test tube, 42 DEG C of heating 15min, 95 DEG C of heating 3min, the cDNA of synthesis in water-bath When needing long-term preservation, please preserved in -20 DEG C or lower temperature.

3) PCR

With SuperReal PreMix Plus (SYBR Green) (article No.：FP205), expanded, experimental implementation is by product Product specification is carried out.

1) design of primers

Coded sequence according to FGGY genes and GAPDH genes in Genebank designs QPCR amplimers, by Bo Maide Biotech firm synthesizes.Specific primer sequence is as follows：

FGGY genes：

Forward primer is 5 '-GGACATATTCAGCAGAGA-3 ' (SEQ ID NO.3)；

Reverse primer is 5 '-AGCACTTGGTTTCCTATT-3 ' (SEQ ID NO.4).

The primer sequence of house-keeping gene GAPDH is：

Forward primer：5’-GGAGCGAGATCCCTCCAAAAT-3’(SEQ ID NO.5)

Reverse primer：5’-GGCTGTTGTCATACTTCTCATGG-3’(SEQ ID NO.6)

2) 20 μ l PCR reaction systems are prepared according to table 1：

The PCR reaction systems of table 1

3) PCR reaction conditions：：95 ° of 15min, (95 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 32s) × 40 circulations, 95 DEG C of 15s, 60 DEG C of 60s, 95 DEG C of 15s.Using SYBR Green as fluorescent marker, carried out on Light Cycler quantitative real time PCR Instruments PCR reacts, and is analyzed by melt curve analysis and electrophoresis determines purpose band, and Δ Δ CT methods carry out relative quantification, and each sample is carried out 3 times Repeat to test.

5th, statistical method

With GAPDH as internal reference, the experiment of Osteoarthritic Synovium tissue and normal synovial tissue fluorescence quantitative RT-RCR is calculated As a result, statistical analysis is carried out using SPSS18.0 statistical softwares, difference between the two is checked using t, with P<0.05 has Significant difference.

6th, result

As shown in Fig. 2 compared with control group, FGGY genes express significantly rise in osteoarthritic tissue, difference has Statistical significance (P<0.05) it is, consistent with RNA-sep results.

The explanation of above-described embodiment is only intended to understand the method for the present invention and its core concept.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.

SEQUENCE LISTING

<110>Tianjin Hospital, Tianjin City

<120>Application of the molecular marker in osteoarthritis

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 1728

<212> DNA

<213>People source

<400> 1

atgtctggtg gagaacagaa accagagagg tactatgtgg gtgtggacgt tggaacaggc 60

agtgtccgtg cagctctggt ggaccagagt ggggtcctgt tggcttttgc agaccagcca 120

attaagaatt gggagcccca gttcaaccac catgagcagt cctccgagga catctgggct 180

gcgtgctgtg ttgtcacaaa gaaagttgta caagggattg atttaaacca aattcgagga 240

cttgggtttg atgccacgtg ttctctggtt gttttggata agcagtttca cccattacca 300

gtcaaccagg aaggggattc ccatcgaaac gtcatcatgt ggctggacca tcgagcagtc 360

agtcaagtta acaggatcaa tgagaccaag cacagtgtcc tccagtacgt cgggggggtg 420

atgtctgtgg aaatgcaggc cccgaaactt ctgtggctga aagagaactt gagagagatt 480

tgctgggata aggcgggaca tttctttgat ctcccggact tcttatcgtg gaaggcaaca 540

ggtgtcacag cacggtctct ctgctccctg gtgtgtaagt ggacatattc agcagagaaa 600

ggctgggacg acagtttctg gaaaatgatt ggtttggaag actttgttgc agataattac 660

agcaaaatag gaaaccaagt gctacctcct ggagcttctc ttggaaatgg gctcacacca 720

gaggcagcaa gagaccttgg ccttctccct gggattgcgg tcgcagcttc actcattgat 780

gcccatgcag gaggactagg agtgattggg gcagatgtga gagggcacgg cctcatctgt 840

gaggggcagc cagtgacgtc acggctggct gtcatctgtg gaacgtcttc ttgtcacatg 900

gggatcagca aagacccgat ttttgtacca ggcgtctggg ggccttattt ctcagccatg 960

gtacctgggt tctggctgaa tgaaggtggt cagagcgtta ctggaaaatt gatagaccac 1020

atggtacaag gccatgctgc ttttccagaa ctacaagtaa aggccacagc cagatgccag 1080

agtatatatg catatttgaa cagtcacctg gatctgatta agaaggctca gcctgtgggt 1140

ttccttactg ttgatttaca tgtttggcca gatttccatg gcaaccggtc tcccttagca 1200

gatctgacac taaagggcat gagaaccact ggatatctgt atattccggc tttggcagcg 1260

ttgcactctc ccagttctct actctcccct caggtcaccg gattgaaact gtctcaggac 1320

cttgatgatc ttgccattct ctacctggcc acagttcaag ccattgcttt ggggactcgc 1380

ttcattatag aagccatgga ggcagcaggg cactcaatca gtactctttt cctatgtgga 1440

ggcctcagca agaatcccct ttttgtgcaa atgcatgcgg acattactgg catgcctgtg 1500

gtcctgtcgc aagaggtgga gtccgttctt gtgggtgctg ctgttctggg tgcctgtgcc 1560

tcaggggatt tcgcttctgt acaggaagca atggcaaaaa tgagcaaagt tgggaaagtt 1620

gtgttcccga gactacagga taaaaaatac tatgataaga aataccaagt attcctgaag 1680

ctggttgaac accagaagga gtatttggcg atcatgaatg atgactga 1728

<210> 2

<211> 575

<212> PRT

<213>People source

<400> 2

Met Ser Gly Gly Glu Gln Lys Pro Glu Arg Tyr Tyr Val Gly Val Asp

1 5 10 15

Val Gly Thr Gly Ser Val Arg Ala Ala Leu Val Asp Gln Ser Gly Val

20 25 30

Leu Leu Ala Phe Ala Asp Gln Pro Ile Lys Asn Trp Glu Pro Gln Phe

35 40 45

Asn His His Glu Gln Ser Ser Glu Asp Ile Trp Ala Ala Cys Cys Val

50 55 60

Val Thr Lys Lys Val Val Gln Gly Ile Asp Leu Asn Gln Ile Arg Gly

65 70 75 80

Leu Gly Phe Asp Ala Thr Cys Ser Leu Val Val Leu Asp Lys Gln Phe

85 90 95

His Pro Leu Pro Val Asn Gln Glu Gly Asp Ser His Arg Asn Val Ile

100 105 110

Met Trp Leu Asp His Arg Ala Val Ser Gln Val Asn Arg Ile Asn Glu

115 120 125

Thr Lys His Ser Val Leu Gln Tyr Val Gly Gly Val Met Ser Val Glu

130 135 140

Met Gln Ala Pro Lys Leu Leu Trp Leu Lys Glu Asn Leu Arg Glu Ile

145 150 155 160

Cys Trp Asp Lys Ala Gly His Phe Phe Asp Leu Pro Asp Phe Leu Ser

165 170 175

Trp Lys Ala Thr Gly Val Thr Ala Arg Ser Leu Cys Ser Leu Val Cys

180 185 190

Lys Trp Thr Tyr Ser Ala Glu Lys Gly Trp Asp Asp Ser Phe Trp Lys

195 200 205

Met Ile Gly Leu Glu Asp Phe Val Ala Asp Asn Tyr Ser Lys Ile Gly

210 215 220

Asn Gln Val Leu Pro Pro Gly Ala Ser Leu Gly Asn Gly Leu Thr Pro

225 230 235 240

Glu Ala Ala Arg Asp Leu Gly Leu Leu Pro Gly Ile Ala Val Ala Ala

245 250 255

Ser Leu Ile Asp Ala His Ala Gly Gly Leu Gly Val Ile Gly Ala Asp

260 265 270

Val Arg Gly His Gly Leu Ile Cys Glu Gly Gln Pro Val Thr Ser Arg

275 280 285

Leu Ala Val Ile Cys Gly Thr Ser Ser Cys His Met Gly Ile Ser Lys

290 295 300

Asp Pro Ile Phe Val Pro Gly Val Trp Gly Pro Tyr Phe Ser Ala Met

305 310 315 320

Val Pro Gly Phe Trp Leu Asn Glu Gly Gly Gln Ser Val Thr Gly Lys

325 330 335

Leu Ile Asp His Met Val Gln Gly His Ala Ala Phe Pro Glu Leu Gln

340 345 350

Val Lys Ala Thr Ala Arg Cys Gln Ser Ile Tyr Ala Tyr Leu Asn Ser

355 360 365

His Leu Asp Leu Ile Lys Lys Ala Gln Pro Val Gly Phe Leu Thr Val

370 375 380

Asp Leu His Val Trp Pro Asp Phe His Gly Asn Arg Ser Pro Leu Ala

385 390 395 400

Asp Leu Thr Leu Lys Gly Met Arg Thr Thr Gly Tyr Leu Tyr Ile Pro

405 410 415

Ala Leu Ala Ala Leu His Ser Pro Ser Ser Leu Leu Ser Pro Gln Val

420 425 430

Thr Gly Leu Lys Leu Ser Gln Asp Leu Asp Asp Leu Ala Ile Leu Tyr

435 440 445

Leu Ala Thr Val Gln Ala Ile Ala Leu Gly Thr Arg Phe Ile Ile Glu

450 455 460

Ala Met Glu Ala Ala Gly His Ser Ile Ser Thr Leu Phe Leu Cys Gly

465 470 475 480

Gly Leu Ser Lys Asn Pro Leu Phe Val Gln Met His Ala Asp Ile Thr

485 490 495

Gly Met Pro Val Val Leu Ser Gln Glu Val Glu Ser Val Leu Val Gly

500 505 510

Ala Ala Val Leu Gly Ala Cys Ala Ser Gly Asp Phe Ala Ser Val Gln

515 520 525

Glu Ala Met Ala Lys Met Ser Lys Val Gly Lys Val Val Phe Pro Arg

530 535 540

Leu Gln Asp Lys Lys Tyr Tyr Asp Lys Lys Tyr Gln Val Phe Leu Lys

545 550 555 560

Leu Val Glu His Gln Lys Glu Tyr Leu Ala Ile Met Asn Asp Asp

565 570 575

<210> 3

<211> 18

<212> DNA

<213>Artificial sequence

<400> 3

ggacatattc agcagaga 18

<210> 4

<211> 18

<212> DNA

<213>Artificial sequence

<400> 4

agcacttggt ttcctatt 18

<210> 5

<211> 21

<212> DNA

<213>Artificial sequence

<400> 5

ggagcgagat ccctccaaaa t 21

<210> 6

<211> 23

<212> DNA

<213>Artificial sequence

<400> 6

ggctgttgtc atacttctca tgg 23

Claims

The application of 1.FGGY genes, it is characterised in that the product for preparing diagnosis osteoarthritis.
2. application according to claim 1, it is characterised in that the product is by determining the expression of FGGY in sample To diagnose osteoarthritis.
3. application according to claim 2, it is characterised in that FGGY up-regulateds in osteoarthritis.
4. the application according to claim any one of 1-3, it is characterised in that the product is included by by the way that skill is sequenced The method detection FGGY genes of art, nucleic acid hybridization technique, nucleic acid amplification technologies or immunoassays and/or the change of its expression product Change.
5. application according to claim 4, it is characterised in that the nucleic acid amplification technologies be selected from PCR, Reverse transcriptase polymerase chain reaction, the amplification of transcriptive intermediate, ligase chain reaction, strand displacement amplification and based on nucleotide sequence Amplification.
6. a kind of product for diagnosing osteoarthritis, it is characterised in that the product can by detect in sample FGGY genes and/ Or the change of its expression product diagnoses osteoarthritis.
7. product according to claim 6, it is characterised in that the product includes chip, kit or preparation.
8. product according to claim 7, it is characterised in that the chip, kit or preparation include being directed to FGGY bases The antibody or part of the specific primer, probe or the albumen for FGGY codings of cause.
Application of the 9.FGGY genes in the pharmaceutical composition for preparing treatment osteoarthritis.
10. application according to claim 9, it is characterised in that described pharmaceutical composition includes FGGY genes and/or its table Up to the lower adjustment of product.