CN110066866A - The biomarker and purposes of the 4th hypotype of osteoarthritis - Google Patents

Abstract

The invention discloses the biomarkers and purposes of the 4th hypotype of osteoarthritis, mainly purposes of the biological substance of immune response and/or angiogenesis in diagnosis osteoarthritis parting.

Description

The biomarker and purposes of the 4th hypotype of osteoarthritis

Technical field

The present invention relates to a kind of based on transcription group to the method and the significant base of disease subtypes of gonitis disease parting The detection of cause and access particularly belongs to using biomarker and diagnoses the parting of osteoarthritis.

Technical background

Osteoarthritis (Osteoarthritis, OA) is the most common musculoskeletal system that global All Countries encounter One of disease.Just there is an example Bones and joints replacement operation for each point half in Europe；The whole world has 65 years old of 50% or more crowd to suffer from Osteoarthritis;In China, 50 years old or more crowd has 25.3% to suffer from osteoarthritis.The clinical symptoms of osteoarthritis be pain and Joint function disturbance.The progressive decline of function of joint leads to activity forfeiture and the function limitation in joint；To make patient Limitation of activity, quality of life decline；About 25% OA patient is unable to complete the main activities in normal life.U.S.'s control and prevention of disease One of center and public health department, Harvard University is research shows that osteoarthritis is to cause five main diseases of the elderly's deformity One of disease.In developed country, the treatment cost of osteoarthritis accounts for the 1.0%-2.5% of domestic total production value.

Have at present to arthritic mechanism hypothesis: " abrasion " is theoretical, related to aging, cartilage cell to grow because The reactivity of son reduces, the exception of mitochondria dysfunction and AGE (advanced glycation end products) Accumulation；Therefore many people think that, OA is with the spontaneous disease of organism aging process；However not all the elderly is OA can be suffered from.With going deep into for research, researcher has found that cartilage cell epimatrix (such as collagen or proteoglycans) can induce non-enzymatic Collagen cross-linking simultaneously shortens proteoglycan molecules.The excessive crosslinking of tropocollagen molecule influences the bio-mechanical property of cartilage, causes soft Bone is stiff, frangible；The shortening and degradation of proteoglycans cause its carbohydrate side chain missing and hydrophily to be lost.In addition, AGEs is horizontal Raising will lead to anabolism decline.But these phenomenons can not explain completely individual occur OA risk and disease into Journey, more and more researches show that the occurrence and development of OA and genetic molecule have close connection.Such as: (1) in encoding histone In terms of gene: the FRZB gene that cartilage and bone development are influenced by participating in Wnt access has been proved to related with OA.With glue Original, which synthesizes relevant gene C OL2A1 mutation, will lead to OA, and COL1A1 mutation can reduce women and suffer from arthritic risk.(2) table See genetic aspect: the change of methylation will affect substrate degradation gene (MMP13, ADAMTS4 etc.), inflammatory factor gene The expression of (IL1b, IL6, IL8 etc.) and other OA associated transcription factors (HOX, SOX9, DIO2 and TGF-beta etc.). The change of histone modification will affect the table of glue protogene (COL2A1) and OA related pathways gene (SOX9, Sirtuin1 etc.) It reaches.(3) in terms of Noncoding gene: studying fewer, be concentrated mainly on tiny RNA (mircoRNA) at present.Such as: miR- 127-5p can inhibit MMP-13 to express, and then generate IL-1b；If inhibit miR-127-5p, MMP-13 expression increase from And lead to OA；MiR-140 can target ADAMTS5, IGFBP-5 (insulin-like growth factor binding Protein 5) etc. genes to influence the generation of OA.Long non-coding RNA (long non-coding RNA, lncRNA) Report in osteoarthritis is also considerably less, and wherein one lncRNA --- UC.343 may target HOXC8, shadow for Fu discovery Ring IL-1b expression.

Although there is a few studies person to classify osteoarthritis, it is based only upon clinical picture and sets out, so existing examines The arthritic goldstandard of knochenbruch is still iconography；Treatment also can only be the conservative symptomatic treatment such as analgesic or adopt in diseased region Take modus operandi.To sum up the molecular studies status of osteoarthritis is it can be found that there is many researchs to have been discovered that osteoarthritis General character signal path has certain enlightenment effect to the treatment of osteoarthritis；But it can not be used to instruct osteoarthritis subgroup The molecular labeling of diagnoses and treatment.And subgroup analysis research is of great significance and has built to promotion given patient group curative effect Found successful example, as in lung cancer EGF-R ELISA (Epidermal growth factor receptor, EGFR) after positive sub-population discovery, EFGR inhibitor brings effective therapeutic effect to hypotype patient.

This just needs the case process and parting of more accurate diagnosis OA, conducive to accurate therapeutic scheme, such as medicine The use of object.

Summary of the invention

The present invention carries out parting from the pathomechanism in gene layer viewpoint osteoarthritis, to osteoarthritis, finds effectively With targeted diagnosis marker, thus for treatment OA provide new standard.

The object of the present invention is to provide a kind of based on transcription group to the method for gonitis disease parting, first with The transcript profile high-flux sequence data of OA patient's cartilaginous tissue, analyze data with bioinformatics method, find four Class osteoarthritis hypotype；And the significant gene of every kind of subgroup is obtained using gene spectrum analysis.Point of these osteoarthritis hypotypes Type discloses the exception in different metabolic path, makes a definite diagnosis the arthritis offer solution of disconnected different subtype to be positive, so as to Fundamentally to treat the disease of osteoarthritis, and not only as traditional method is come using antalgica.

On the one hand, the present invention provides a kind of biomarker substance, these biomarkers with the parting of osteoarthritis or Person's type is directly linked.And these biomarker substances are associated with some metabolic pathways.

The first aspect of the present invention, the present invention provide the life in proteoglycan metabolism and/or chondroitin sulfate metabolic pathway Purposes of the object substance in the parting of diagnosis osteoarthritis.In some modes, if in some proteoglycan metabolisms and/or sulphur Biological substance in aching and limp ossein metabolic pathway changes, such as increases or decreases, then it represents that it is poly- that osteoarthritis belongs to albumen The arthritis of glycometabolism and/or chondroitin sulfate metabolic type.In preferred mode, these biomarker substances are selected from following base One of cause is several: proteoglycan metabolism is related: PCOLCE2, S100B, ITM2C, ACAN, FBXO2, SOD3, SERPINA3, SSR3, DCN, WWP2, ITIH6, TCEAL2, FIBIN, FGFBP2, TSPAN6, PLA2G2A, SMOC2, TUBB2B, STC2, ACAN, DCN, FMOD, CDO1, PRELP, PAPSS2, B3GNT7, CHST3, CHST6, CSPG4, BGN, CSGALNACT1, CHPF；Chondroitin sulfate metabolism related gene: ITM2C, DCN, TMEM59, GALNT18, B3GNT7, CHST3, GALNT8, GALNT15, CSPG4, BGN, CSGALNACT1, CHPF, MAN1A2, ITM2A, EXTL1, RPN2, ITM2B, POFUT2, GOLGA2.In some modes, when the transcriptional level of these genes increases, then it represents that the bone that osteoarthritis belongs to the first hypotype closes Section is scorching.

The second aspect of the present invention provides the biological object on collagen degradation and/or the metabolic pathway of collagenous fibres formation Purposes of the matter in the parting of diagnosis osteoarthritis.In some preferred modes, these biomarker substances are selected from following base One of cause is several: Degradation metabolism of collagen related gene: COL6A, COL5A, COL3A,NBL, DKK,ADAMTS2,ABHD, FNDC, LMNA, S100A, MXRA, ANTXR1；Collagenous fibres form related gene: COL5A, COL3A,GJA, ADAMTS2, TGFB, SOX4, ITGA11, LTBP2, AEBP1.In some modes, when the transcriptional level raising of these genes, then Indicate that osteoarthritis belongs to the osteoarthritis of the second hypotype.

The third aspect of the present invention, the biological substance provided on nerve synapse adjusting and/or ion channel are closed in diagnosis bone Save the purposes in scorching parting.In some preferred modes, these biomarker substances be selected from one of following gene or Person is several: nerve synapse adjusts related gene: GRID2, ADGRL3, NRXN1, LINGO2, IL1RAPL1, ADGRB3, PTPRD, LRRC4C, CTNNA2, MACROD2, PCDH15, SGCZ, CNTNAP2, GRID2, GALNTL6, MT-ND5, LRRTM4, CALN1, CTNNA3, AC007682.1, EYS, ADGRL3, GRM7,；Ion channel related gene: GRID2, CNTNAP2, KCNIP4, DPP10, GRIK2, DLG2, CACNA1A, CNTNAP5, CNTN5, KCNIP4, EYS, GRM7, ADGRL3；In some sides In formula, when the transcriptional level of these genes increases, then it represents that osteoarthritis belongs to the osteoarthritis of third hypotype.

The fourth aspect of the present invention provides the biological substance of immune response and/or angiogenesis in diagnosis osteoarthritis Purposes in parting.In some preferred modes, these biomarker substances are selected from one of following gene or several: Immune-related gene: PECAM1, SYK, STOM, GMFG, TSPAN14, PTPRC, COTL1, ARPC5, CAP1, FCGR2B, CD36, VAMP8, CD14, RHOA, SDCBP, RAC1, CTSB, RAB10, RAP1B, PDXK, CYBA, GM2A, GRN, VCL, GNS, ADAM10, PSAP, ATP6AP2, ASAH1, ACTR2, FTL, PLD1, TCIRG1, NRAS, CST3, RAP2B, COLEC11, DUSP6, MPEG1, STOM, PLVAP, IFI16, ATP6V1B2, ADGRL4, PCDH18, APLNR, C1orf162, ZFP36L1, SLCO2B1, PTPRC, ACSL1, COTL1；Angiogenesis genes: MCAM, SYK, APLNR, EGFL7, NOTCH3, CD34, MMRN2, ECSCR, NRP1, GPX1, JUN, CYP1B1, PLXND1, RHOA, GPNMB, C1GALT1, ENG, MYH9, EPAS1, GNA13, ATP5B, RBPJ, PDGFRA, PGF, MMP2, STAT1, TMSB4X, LAPTM5, LGALS3BP, YWHAB, ZEB2, EGFL7, GMFG, SYK, MCAM, MYL6, KCTD12.In some modes, when the transcriptional level of these genes increases, then it represents that Osteoarthritis belongs to the osteoarthritis of the 4th hypotype.

On the one hand, present invention offer joint fluid biomarker substance belongs to the use in the first hypotype in diagnosis osteoarthritis On the way, the biomarker substance is in SCF, RANTES, IFN or IL18 or several.The present invention provides joint fluid In biomarker substance diagnosis osteoarthritis belong to the purposes in the second hypotype, the biomarker substance is selected from VEGF and/or IL6.The present invention provides joint fluid biomarker substance and belongs to the purposes in the 4th hypotype in diagnosis osteoarthritis, The biomarker substance is selected from MIP, SDF or IL8.

Here can illustrate, the appearance of the mark substance in joint fluid also proves parting of the invention from still further aspect Correctly, the appearance for also further illustrating these mark substances can also be used as a determining method of parting.It is not all The inflammatory factor of the osteoarthritis of hypotype is the same, and the inflammatory factor for being different hypotype is different, just it may be said that The importance of bright parting is associated from different metabolic pathways.

On the other hand, the present invention provides a kind of method for diagnosing osteoarthritis hypotype, and the method includes: offer sample Originally the analysis of biomarker and to sample is carried out, if the marker of analysis belongs to proteoglycan metabolism and/or chondroitin sulfate Mark substance in plain metabolic pathway, then it is assumed that the osteoarthritis belongs to the arthritis of the first hypotype；If the marker of analysis Belong to the mark substance in the metabolic pathway that collagen degradation and collagenous fibres are formed, then it is assumed that the osteoarthritis belongs to the second hypotype Arthritis；It is adjusted and the biological substance on ion channel if the marker of analysis belongs to nerve synapse, then it is assumed that the bone closes Section inflammation belongs to the arthritis of third hypotype；If the marker of analysis belongs to the biological substance of immune response and angiogenesis, Think that the osteoarthritis belongs to the arthritis of the 4th hypotype.

In certain methods, the main body for providing sample is Human Osteoarthritis.In other mode, the biology There is exception in mark substance, to judge the parting of osteoarthritis according to abnormal.This exception be usually and the above metabolin The content of the biological substance of qualitative correlation is higher than the level (not being the patient of osteoarthritis) of normal person.These exception can be egg White, nucleic acid level raising, such as some gene expressions increase, then the parting of some type of osteoarthritis in surface.

In some preferred modes, biomarker substance includes: proteoglycan metabolism correlation: PCOLCE2, S100B, ITM2C, ACAN, FBXO2, SOD3, SERPINA3, SSR3, DCN, WWP2, ITIH6, TCEAL2, FIBIN, FGFBP2, TSPAN6, PLA2G2A, SMOC2, TUBB2B, STC2, ACAN, DCN, FMOD, CDO1, PRELP, PAPSS2, B3GNT7, CHST3, CHST6, CSPG4, BGN, CSGALNACT1, CHPF；And/or with chondroitin sulfate metabolism related gene: ITM2C, DCN, TMEM59, GALNT18, B3GNT7, CHST3, GALNT8, GALNT15, CSPG4, BGN, CSGALNACT1, CHPF, MAN1A2, ITM2A, EXTL1, RPN2, ITM2B, POFUT2, GOLGA2.In some modes, when the transcriptional level of these genes It increases, then it represents that osteoarthritis belongs to the osteoarthritis of the first hypotype.

In some preferred modes, the metabolic pathway of biomarker substance and collagen degradation and/or collagenous fibres formation It is related.In some preferred modes, these biomarker substances are selected from one of following gene or several: collagen degradation Metabolism related gene: COL6A, COL5A, COL3A,NBL, DKK,ADAMTS2,ABHD, FNDC, LMNA, S100A, MXRA, ANTXR1；Collagenous fibres form related gene: COL5A, COL3A,GJA,ADAMTS2, TGFB, SOX4, ITGA11, LTBP2, AEBP1.In some modes, when the transcriptional level of these genes increases, then it represents that the bone that osteoarthritis belongs to the second hypotype closes Section is scorching.

It is related in the adjusting of biomarker substance nerve synapse and/or ion channel in some preferred modes.Some In preferred mode, these biomarker substances are selected from one of following gene or several: nerve synapse adjusts dependency basis Cause: GRID2, ADGRL3, NRXN1, LINGO2, IL1RAPL1, ADGRB3, PTPRD, LRRC4C, CTNNA2, MACROD2, PCDH15, SGCZ, CNTNAP2, GRID2, GALNTL6, MT-ND5, LRRTM4, CALN1, CTNNA3, AC007682.1, EYS, ADGRL3, GRM7,；Ion channel related gene: GRID2, CNTNAP2, KCNIP4, DPP10, GRIK2, DLG2, CACNA1A, CNTNAP5, CNTN5, KCNIP4, EYS, GRM7, ADGRL3；In some modes, when the transcription water of these genes It is flat to increase, then it represents that osteoarthritis belongs to the osteoarthritis of third hypotype.

In some preferred modes, the correlation of the immune response of biomarker substance and/or angiogenesis.Some preferred Mode in, these biomarker substances are selected from one of following gene or several: immune-related gene: PECAM1, SYK, STOM, GMFG, TSPAN14, PTPRC, COTL1, ARPC5, CAP1, FCGR2B, CD36, VAMP8, CD14, RHOA, SDCBP, RAC1, CTSB, RAB10, RAP1B, PDXK, CYBA, GM2A, GRN, VCL, GNS, ADAM10, PSAP, ATP6AP2, ASAH1, ACTR2, FTL, PLD1, TCIRG1, NRAS, CST3, RAP2B, COLEC11, DUSP6, MPEG1, STOM, PLVAP, IFI16, ATP6V1B2, ADGRL4, PCDH18, APLNR, C1orf162, ZFP36L1, SLCO2B1, PTPRC, ACSL1, COTL1；Angiogenesis genes: MCAM, SYK, APLNR, EGFL7, NOTCH3, CD34, MMRN2, ECSCR, NRP1, GPX1, JUN, CYP1B1, PLXND1, RHOA, GPNMB, C1GALT1, ENG, MYH9, EPAS1, GNA13, ATP5B, RBPJ, PDGFRA, PGF, MMP2, STAT1, TMSB4X, LAPTM5, LGALS3BP, YWHAB, ZEB2, EGFL7, GMFG, SYK, MCAM, MYL6, KCTD12.In some modes, when the transcriptional level of these genes increases, then it represents that osteoarthritis belongs to The osteoarthritis of 4th hypotype.

In some modes, the sample be cartilaginous tissue, one of subchondral bone tissue or synovial tissue or It is several.

On the other hand, classifying method of the invention is as follows: based on transcription group to the method for gonitis disease parting and The detecting step of the significant gene of disease subtypes is as follows:

(1) case sample is collected；

(2) cDNA is carried out to sample and builds library, RNA-seq sequencing；

(3) divide group to sample using unsupervised method；

(4) the significant gene and function of hypotype are excavated；

(5) more fabric analysis verifyings divide group；

(6) tissue staining verifying divides group.

Further, the specific implementation of the step (1) are as follows: collect multicenter patient's sample, this research has collected four The knee joint of the Bones and joints replacement operation patient of hospital.It is extracted the cartilaginous tissue of patient, subchondral bone tissue and synovial tissue.

Further, it the specific implementation of the step (2) are as follows: for the cDNA banking process of cartilaginous tissue: chooses each The cartilage of sample, synovial membrane, subchondral bone carry out tissue grinder with grinding pipe and beveller respectively and extract RNA；It is clear after reverse transcription It washes, is enriched with cDNA；Nanodrop measures RIN > 7, and total amount > 500ng sample retains sequencing.

Further, the specific implementation of the step (3) are as follows: low quality number is carried out to the high-throughput transcript profile data of acquisition According to removal, remaining data carry out genome mapping, and in statistical sample gene expression quantity.It is utilized based on gene expression profile non- The method of supervision carries out a point group to sample.The clustering method of SC3 has been used herein, it has been found that points of four groups results have best Silhouette value.And four crowds of patients also have apparent difference in phenotype, and first group of patient's proteoglycan matrix is said and lift a curfew Weight (polysaccharide degradation-type)；Osteophytosis is serious (osteophytosis type) by second group of patient's cartilage；Third group patient age is minimum, table Reveal pain sensitivity (painful)；4th group of patient articular's narrow gaps simultaneously have high inflammatory reaction.

Further, the specific implementation of the step (4) are as follows: screening p value is greater than 0.9 gene less than 0.001, AUROC Significant gene as hypotype.

Further, the specific implementation of the step (6) are as follows: obtain the express spectra number of patient's subchondral bone and synovial tissue According to, and knitted with the multiple groups in the entire joint of ligand receptor joint analysis and mutually done.It was found that proteoglycan degradation-type patient is kneed It is active in multiple groups are knitted and mutually done that extracellular matrix forms access；The Degradation metabolism of collagen access of collagen explanation type patient is matched in synovial membrane It is significant that body-cartilage receptor mutually does expression；The bone differentiation of high inflammation type patient and bone remoulding sending down the fishbone as receptor it is mutual do in table Up to significant.These discoveries confirm the consistency of hypotype significant access and hypotype phenotype above.

Further, the specific implementation of the step (7) are as follows: Safarinin O dyeing and histogenic immunity Histochemical staining experimental verification hypotype metabolic pathway.The destination gene expression that the present invention has detected 4 OA subgroups is horizontal.SO dyeing knot Fruit shows the expression of proteoglycans (Aggrecan), compares the coloration result of four groups, compared with normal sample, four groups There is proteoglycans synthesis to reduce, obvious polysaccharide metabolic disturbances characteristic is presented in C1.Finally, the present invention has selected MMP13, cynapse Plain (Synapotopsin), tri- mark substances of CD34 carry out immunohistochemical staining experiment, wherein MMP13 is indication The biomarker substance (marker) of cartilage matrix degradation, the MMP13 expression obvious compared with other three groups displays of C2 group result increase The phenomenon that；Synapotopsin is marker relevant to nerve, and Synapotopsin is in the higher expression of C3；CD34 is indication The important biomolecule mark substance of inflammation, we have found that histochemical staining shows that C4 has the characteristics that high inflammation occurs.With grouping result Unanimously, C1 is mainly shown as polysaccharide metabolic disorder, and C2 is mainly shown as chondrin.It can be seen that these MMP13, Synapotopsin, CD34 can also be used as one of the important indicator of inflammation parting, this is because the exception of these markers with Parting is closely connected, and without one of the important mark substance of metabolic pathway exception.

Detailed description of the invention

Fig. 1 is analysis flow chart diagram.

Fig. 2 is the result figure of hypotype significant gene and significant metabolic pathway.

Fig. 3 is SO and histochemical staining verifying.

Fig. 4 is protein science verifying analysis result figure.

Specific embodiment

In order to more specifically describe the present invention, with reference to the accompanying drawing and specific embodiment is to technical solution of the present invention It is described in detail.These explanations are only to show how the present invention realizes, can not limit specific range of the invention.

The present invention is based on transcription groups to the method for gonitis disease parting and the discovery of the significant gene of disease subtypes Specific step is as follows:

Examples of implementation 1: case is collected

Multicenter patient's sample is collected, this research has collected the knee joint of the Bones and joints replacement operation patient of four hospitals.It extracts The cartilaginous tissue of patient, subchondral bone tissue and synovial tissue.

Examples of implementation 2: cDNA is carried out to sample and builds library, RNA-seq sequencing

The acquisition and processing (samples of examples of implementation 1) of tissue samples: the present invention is using surgery trepan from directly checking previously Erosion and intact cartilage below region obtain 0.5cm diameter, the subchondral bone trabecular bone core of 1cm depth.It removes remaining soft Bone tissue.Bone is rapidly frozen in liquid nitrogen immediately, and carries out cryogrinding using the cooling grinder of liquid nitrogen.Cartilage takes the complete of abrasion Layer cartilaginous tissue is rapidly frozen in liquid nitrogen and carries out cryogrinding using the cooling grinder of liquid nitrogen.It takes near abrasion cartilage Synovial membrane is equally freezed and is ground.

RNA separation and reverse transcription: using RNeasy Fibrous Tissue Mini Kit(Qiagen, Duesseldorf, Germany) according to the explanation of manufacturer total serum IgE is separated from homogenised tissue.Pass through Nano-Drop (NanoDrop Technologies, DE, USA) measures RNA concentration and purity in Bioanalyzer 2100(Agilent Technologies, CA, USA) in assessment RNA integrality, so as to then be used only RIN(RNA integrality numerical value) be equal to or Greater than those of 6 samples.

By the dNTP mixture of total serum IgE and 1 μ l the oligo-dT primer (a kind of random primer) being anchored and 1 μ l (10mM, Fermentas it) mixes, is denaturalized 5 minutes at 72 DEG C.7 microlitre of first chain reaction mixture (contains 0.50 μ lSuperScriptII Reverse transcriptase (Invitrogen), 0.25 μ lRNA enzyme inhibitor (Clontech), 2 μ lSuperscriptII First- Strand Buffer(Invitrogen), 0.25 μ lDTT(Invitrogen), 1 μ l is by glycine betaine (Sigma), 0.9 μ lMgCl2 (Sigma) it is added in each sample with 0.1 μ l nuclease-free water (Gibco).By incubating 90 minutes at 42 DEG C, 10 are then carried out A circulation (50 DEG C 2 minutes, 42 DEG C 2 minutes) carries out reverse transcription reaction.Finally, making reverse transcriptase by incubating 15 minutes at 70 DEG C Inactivation.Then 10 the second chains of μ l synthesis 2 μ l of buffer, 1 μ l, cDNA 100ng of enzymatic mixture and water is being added to end reaction body Product carries out the second chain synthesis after being 20 μ l.Reactant is incubated 150 minutes at 16 DEG C, is then purified with AMPure XP pearl.

The preparation in transcript profile library: 5 nanogram cDNA are carried out with Nextera DNA sample reagent preparation box (Illumina) Label reaction, 2.5 μ l2 × Tagment DNA buffers and 1.25 μ lTagmentDNA enzymes are added, final volume is 5 μ l.It will Label reaction is incubated for 10 minutes at 55 DEG C.Then whole volume is used for restricted circulation collection PCR's and 3.75 μ l Nextera PCR primer mixture (NPM), 1 primer of Index (N7xx) of 1.25 μ l and the PCR primer mixture of 1.25 μ l (PPC).It is following to carry out the second wheel amplification: 72 DEG C 3 minutes, 98 DEG C 30 seconds, then 5 circulations (98 DEG C 10 seconds, 63 DEG C 30 seconds, 72 DEG C 3 minutes).Purified with the AMPure XP magnetic bead of 1:1 ratio, and sample is loaded to High-Sensitivity DNA The quality in library is checked on chip, while being quantified with Qubit high sensitivity DNA kit (Invitrogen).It will be literary Library is diluted to the final concentration of 2nM and merging, and 10pmol is sequenced on Illumina HiSeq 2500.

Examples of implementation 3: divide group to sample using unsupervised method

Low quality data removal is carried out to the high-throughput transcript profile data of acquisition: 1) being removed with FASTX_TOOLKIT low-quality Data, sequence of the content less than 80% of base of the removal quality greater than 20.2) select total sequence reads 30 ten thousand to two hundred Sample between ten thousand.Remaining data carry out genome mapping, and in statistical sample gene expression quantity.Selection is being greater than 10 The gene occurred in sample.Express spectra based on these genes carries out a point group to sample using non-supervisory method.It uses herein The clustering method of SC3, it has been found that dividing four groups of results has best silhouette coefficient (Silhouette) value.

3.1 excavate the significant gene of hypotype

Screen significant gene of gene of the p value less than 0.0001, AUROC greater than 0.9 as hypotype.Present invention discover that significant Significant gene mainly has and (is specifically shown in Fig. 2A and 2B.1-4)

First hypotype:PCOLCE2, S100B, ITM2C, C2orf40, ACAN, FBXO2, SOD3, SERPINA3, SSR3, DCN, WWP2, ITIH6, TCEAL2, FIBIN, FGFBP2, TSPAN6, PLA2G2A, SMOC2, TUBB2B, STC2。

Second hypotype:TGFB, COL6, NBL, DKK, GJA, COL5, ABHD, FNDC, LMNA, S100A, COL3, S100A, MXRA。

Third hypotype:LRRC4C, CTNNA2, MACROD2, PCDH15, SGCZ, CNTNAP2, GRID2, CNTNAP5, CNTN5, GALNTL6, MT-ND5, KCNIP4, LRRTM4, CALN1, CTNNA3, AC007682.1, EYS, GRM7, ADGRL3。

4th hypotype:TMSB4X, PECAM1, LAPTM5, LGALS3BP, COLEC11, DUSP6, MPEG1, STOM, YWHAB, PLVAP, IFI16, ZEB2, ATP6V1B2, EGFL7, GMFG, SYK, MCAM, MYL6, KCTD12, ADGRL4, PCDH18, APLNR, C1orf162, ZFP36L1, SLCO2B1, PTPRC, ACSL1, COTL1.

The corresponding function of the gene of these different subtypes is analyzed respectively, it is found that different hypotypes corresponds to different generations Reason approach.Analysis is carried out to their function and finds that the mark sexual function access of different subtype is: the first hypotype: proteoglycan generation It thanks or chondroitin sulfate is metabolized；Second hypotype: collagen degradation or collagenous fibres composition；Third hypotype: nerve synapse adjust or from Subchannel；4th hypotype: immune response or angiogenesis.

It is analyzed from the transcriptional level of the above gene, finds four hypotypes significantly distinguished, then it is enterprising from gene level The different types of division of osteoarthritis is gone.In addition, different gene pairs answers different metabolic pathway or biology intracorporal Biological substance circulating path is different.So these genes can be used as the biomarker substance of parting, when discovery patient is that bone closes When section is scorching, analyze whether these genes express in articular cartilage tissue there are the height of the above gene or rate of rotation is horizontal It increases, then can carry out the differentiation of hypotype according to above-mentioned standard.

For example, when detecting discovery PCOLCE2, S100B, ITM2C, C2orf40, ACAN, FBXO2, SOD3 in cartilaginous tissue, SERPINA3, SSR3, DCN, WWP2, ITIH6, TCEAL2, FIBIN, FGFBP2, TSPAN6, PLA2G2A, SMOC2, TUBB2B, The rate of rotation level of a gene or multiple genes in STC2 increases, then it represents that the arthritis belong to proteoglycan metabolism or/ With arthritis caused by chondroitin sulfate metabolic problems.It is appreciated that being found when being detected in cartilaginous tissueTGFB, COL6, NBL, DKK, GJA, COL5, ABHD, FNDC, LMNA, S100A, COL3, S100A, MXRAIn a gene or multiple genes turn Record is horizontal to be increased, then it represents that pass caused by the arthritis belongs to collagen degradation and/or collagenous fibres composition path goes wrong Section is scorching.More it is appreciated that when detecting discovery LRRC4C, CTNNA2, MACROD2, PCDH15, SGCZ in cartilaginous tissue, CNTNAP2, GRID2, CNTNAP5, CNTN5, GALNTL6, MT-ND5, KCNIP4, LRRTM4, CALN1, CTNNA3, The transcriptional level of a gene or multiple genes in AC007682.1, EYS, GRM7, ADGRL3 increases, then it represents that the joint Inflammation belong to nerve synapse adjust and ion channel there is a problem caused by arthritis.It is further appreciated that more it is appreciated that working as Discovery TMSB4X, PECAM1, LAPTM5, LGALS3BP, COLEC11, DUSP6, MPEG1, STOM are detected in cartilaginous tissue, YWHAB, PLVAP, IFI16, ZEB2, ATP6V1B2, EGFL7, GMFG, SYK, MCAM, MYL6, KCTD12, ADGRL4, A gene or multiple bases in PCDH18, APLNR, C1orf162, ZFP36L1, SLCO2B1, PTPRC, ACSL1, COTL1 The transcriptional level of cause increases, then it represents that the arthritis belongs to immune response and angiogenesis there is a problem caused by joint It is scorching.

Specific typing data see the table below:

The relevant gene of 1: the first hypotype of table

Marker gene	Under indicatrix Area AUROC	P after correction Value	Description	Biological process (GO)	Classification
						PCOLCE2	1.21E-14	0.95	Precollagen C- endopeptidase reinforcing agent	GO:0016504: peptide zymoexciter activity；GO:0010559: the tune of glycoprotein biosynthetic process Section；GO:0030198: extracellular matrix composition	Proteoglycan metabolism
S100B	9.52E-14	0.94	S100 calbindin B.	GO:0005509: calcium binding；GO:0005539: glycosaminoglycan combines	Proteoglycan metabolism
						ITM2C	1.55E-13	0.94	Integrated membrane protein 2C	GO:1903510: mucopolysaccharide metabolic process；GO:0030204: chondroitin sulfate metabolic process	Chondroitin sulfate metabolism
ACAN	4.76E-13	0.93	Chondroitin sulfate proteoglycan core egg It is white	GO:0030246: carbohydrate combines；GO:0006022: glycosaminoglycan metabolic process；GO: 0030203: glycosaminoglycan metabolic process	Proteoglycan metabolism；Chondroitin sulfate metabolism
						FBXO2	1.68E-12	0.92	F-Box albumen 2	GO:0030246: carbohydrate combines；GO:1903510: mucopolysaccharide metabolic process	Proteoglycan metabolism
SOD3	1.96E-12	0.92	Superoxide Dismutase 3	GO:0051216: cartilage development；GO:0010559: the adjusting of glycoprotein biosynthetic process	Proteoglycan metabolism
						SERPINA3	2.12E-12	0.92	Serpin family protein 3	GO:0005578: albuminous cell epimatrix；GO:0006027: glycosaminoglycan catabolic process	Proteoglycan metabolism
DCN	4.79E-12	0.92	Decorin	GO:0006022: glycosaminoglycan metabolic process；GO:0009100: glycoprotein metabolism process；GO: 0030204: chondroitin sulfate metabolic process	Proteoglycan metabolism；Chondroitin sulfate metabolism
						WWP2	4.79E-12	0.92	WW containing E3 ubiquitin protein ligase Structural domain	GO:0001190: activating transcription factor activity；GO:0001085:RNA polymerase II transcription factor In conjunction with	Proteoglycan metabolism
ITIH6	4.97E-12	0.92	Inter-Alpha inhibitor H5 sample egg It is white	GO:0006022: glycosaminoglycan metabolic process；GO:0009100: glycoprotein metabolism process；	Proteoglycan metabolism
						SMOC2	8.36E-11	0.90	The modularization calbindin of secretion	GO:0030198: extracellular matrix composition；GO:0050654: chondroitin sulfate proteoglycan metabolism Process	Proteoglycan metabolism
STC2	1.16E-10	0.90	Stanniocalcin GAP-associated protein GAP 2	GO:0070482: the reaction to oxygen level；GO:0005539: glycosaminoglycan combines	Proteoglycan metabolism
						FMOD	2.41E-10	0.89	Keratin sulfate proteoglycans fiber Adjust element	GO:0008194:UDP glycosyl transferase activity；GO:0009100: glycoprotein metabolism process；GO: 0030204: chondroitin sulfate metabolic process	Proteoglycan metabolism；Chondroitin sulfate metabolism

The relevant gene of 2: the second hypotype of table

TGFBI	1.35E-05	0.89	Transforming growth factor β induction	GO:0005578: albuminous cell epimatrix；GO:0005518: collagen combines	Collagenous fibres are formed
						COL6A	1.80E-05	0.89	Collagen VI type Alpha	GO:0030574: collagen catabolic process；GO:0032963: collagenic supersession process；GO: 0001503: ossification	Degradation metabolism of collagen；Collagenous fibres are formed
NBL	2.86E-05	0.88	DAN family bmp antagonist	GO:0007178: transmembrane receptor protein serine/threonine kinase signal path	Degradation metabolism of collagen
						DKK	3.94E-05	0.88	Dickkopf WNT signal pathway inhibitor	GO:0071559: the reaction to transforming growth factor β	Degradation metabolism of collagen
GJA1	6.00E-05	0.88	Gap junction protein Alpha	GO:0007517: muscular organ development	Collagenous fibres are formed
						COL5A	0.000186051	0.86	Collagen V-type Alpha	GO:0030574: collagen catabolic process；GO:0030199: collagenous fibril composition	Degradation metabolism of collagen；Collagenous fibres are formed
LMNA	0.000227153	0.86	Lamin A/C	GO:0010463: mesenchymal cell proliferation	Degradation metabolism of collagen
						COL3A	0.000412635	0.85	Collagen type III Alpha	GO:0031589: cell substrate adherency；GO:0030199: collagenous fibril composition；GO:0044420: thin Extracellular matrix components	Degradation metabolism of collagen；Collagenous fibres are formed
S100A10	0.000470572	0.85	S100 calbindin A.	GO:0007160: Cell-matrix adhesion	Degradation metabolism of collagen；Collagenous fibres are formed
						MXRA5	0.000955334	0.84	Matrix remodeling GAP-associated protein GAP	GO:0071559: the reaction to transforming growth factor β	Degradation metabolism of collagen；Collagenous fibres are formed
SOX4	0.001684379	0.84	SRY(Sex Determination region Y)-Box 4	GO:0001501: shell system development	Collagenous fibres are formed
						ANTXR1	0.001737043	0.84	ANTXR cell adhesion molecule	GO:0030198 extracellular matrix composition；GO:0005518 collagen combines	Degradation metabolism of collagen；Collagenous fibres are formed
ITGA11	0.001906274	0.83	Integrin subunit Alpha	GO:0007160: Cell-matrix adhesion；GO:0001503: ossification	Collagenous fibres are formed
						LTBP2	0.003962057	0.82	Potential transforming growth factor β binding protein	GO:0005578: albuminous cell epimatrix；GO:0071559: the reaction to transforming growth factor β	Collagenous fibres are formed
AEBP1	0.005507051	0.82	AE binding protein	GO:0001501: shell system development	Collagenous fibres are formed

Table 3: the relevant gene of third hypotype

LRRC4C	1.25E-05	1.00	Repeat region 4C rich in leucine	GO:0098794: postsynaptic；GO:0097060: synaptic membrane	Nerve synapse is adjusted
						CTNNA2	3.89E-05	1.00	Cadherin GAP-associated protein GAP	GO:0050807: cynapse composition is adjusted；GO:0030424: aixs cylinder；GO:0048813: dendron form occurs	Nerve synapse is adjusted
PCDH15	5.92E-07	1.00	Protocadherin GAP-associated protein GAP	GO:0098742 passes through cell-cell adherence of plasma membrane adhesion molecule	Nerve synapse is adjusted
						GRID2	4.85E-05	1.00	Ionotropic glutamate receptor Delta type subunit 2	GO:0051965: the positive regulation of positive synapse assembly；GO:0007416: synapse assembly	Nerve synapse is adjusted；Ion channel
CNTNAP	9.99E-05	1.00	Contactin GAP-associated protein GAP	GO:0034705: potassium channel compound；GO:0034702: ion channel complex；GO:0030424: aixs cylinder	Ion channel
						LRRTM4	2.46E-06	0.99	Cross-film neuron is repeated rich in leucine	GO:0045211: postsynaptic membrane	Nerve synapse is adjusted；Ion channel
KCNIP4	0.000123 233	0.99	Potassium voltage-gated channel interaction protein 4	GO:0034705: potassium channel compound；GO:0034703: cationic channel complex；GO:0099106: ion is logical Road regulator activity	Ion channel
						GRM7	1.06E-05	0.99	Glutamic acid metabolism receptor	GO:0042734: presynaptic membrane；GO:0035249: cynapse transmitting, Glutamatergic	Nerve synapse is adjusted；Ion channel

The relevant gene of 4: the four hypotype of table

PECAM1	3.87E-07	0.99	Blood platelet and endothelial cell adhesion molecule	GO:0042119: neutrophil activation；GO:0002446: neutrophil cell mediates immune；GO: 0002576: blood platelet threshing	Immune response；Angiogenesis
						TMSB4X	3.89E-07	0.99	Extrasin beta 4 X is chain	GO:0003779: actin combines；GO:0030335: the positive regulation of cell migration	Angiogenesis
LAPTM5	1.48E-06	0.97	Lysosomal protein cross-film 5	GO:0005774: tonoplast；GO:0110053: the adjusting of actin filament tissue；GO:0031589: cell Substrate adherency	Angiogenesis
						LGALS3BP	1.52E-06	0.97	Agglutinin galactoside combines solubility 3- to combine Albumen	GO:0034774: secretory granules lumen；GO:0060205: cytoplasmic vesicle chamber；GO:0072562: blood particulate	Immune response；Angiogenesis
COLEC11	1.61E-06	0.97	Assemble subfamily member	GO:0006898: receptor mediated endocytosis	Immune response
						DUSP6	2.35E-06	0.97	Dual specificity phosphatase enzyme	GO:0001933: the negative regulator of protein phosphorylation；GO:0070371:ERK1 and ERK2 cascade	Immune response
STOM	2.60E-06	0.97	Erythrocyte membrane protein band	GO:0042119: neutrophil activation；GO:0002446: neutrophil cell mediates immune	Immune response
						YWHAB	2.71E-06	0.96	Tyrosine 3-monooxygenase/tryptophan 5- is mono- to be added Oxygenase activated protein β	GO:0045296: cadherin combines；The adjusting of GO:0061013:mRNA catabolic process；GO: 0050839: cell adhesion molecule combines	Immune response；Angiogenesis
PLVAP	1.55E-06	0.96	Plasmalemma vesicle associated protein	GO:0098589: diaphragm area；GO:0030335: the positive regulation of cell migration；GO:0034612: bad to tumour The reaction of necrosis factor	Immune response
						IFI16	4.35E-06	0.96	Interferon gamma inducible protein 16	GO:0045088: innate immune response is adjusted；GO:0031349: the positive adjusting of defense reaction	Immune response
ZEB2	5.37E-06	0.96	Zinc finger Homeobox	GO:0022604: cellular morphology is adjusted；GO:0001667:ameboidal type cell migration；GO: 0010631: migration of epithelial cells	Angiogenesis
						EGFL7	5.30E-06	0.95	Blood vessel endothelium element	GO:0001525: angiogenesis；GO:0001935: endothelial cell proliferation	Angiogenesis
ATP6V1B2	6.36E-06	0.95	ATP enzyme H+ transports V1 subunit B2	GO:1901652: the reaction to peptide；GO:0005774: tonoplast	Immune response
						SYK	2.96E-06	0.95	Spleen related tyrosine kinases	GO:0042119: neutrophil activation；GO:0001525: angiogenesis；GO:0007596: blood is solidifying Gu	Immune response；Angiogenesis
GMFG	6.76E-06	0.95	Glia maturation factor Gamma	GO:0002446: neutrophil cell mediates immune；GO:0003779: actin combines	Immune response；Angiogenesis
						MCAM	6.62E-06	0.95	Melanoma cells adhesion molecule	GO:0005925: focal adhesion；GO:0001525: angiogenesis；GO:0030335: the positive regulation of cell migration	Angiogenesis
MYL6	7.85E-06	0.95	Myosin light chain	GO:0015629: actin cytoskeleton	Angiogenesis

It also absolutely proves above, it is to belong to that we can diagnose specific arthritis with the raising of the transcriptional level of these mark substances In the arthritis of which kind, belong to the arthritis of what hypotype, after finding the mechanism and basic physiology principle of morbidity, to be Successive treatment provides strong help and support.

It is appreciated that the gene level found above is only the portion gene level in these 4 different physiological paths Variation, then explanation is only the variation of these gene levels and causes arthritic parting different.It is understood that Abnormal variation has occurred in this 4 kinds different physiological paths, then illustrates that these variations belong to and cause one of arthritic factor, such as Proteoglycan metabolism or/and chondroitin sulfate metabolic disorder or collagen degradation and/or collagenous fibres group exception, nerve synapse Adjust and/or ion channel occur it is abnormal or, immune response and/or angiogenesis are abnormal, then one kind occur in these exceptions Or it is several, then it is the pathogenesis of osteoarthritis.Here exception be usually it is relevant to these metabolic pathways directly or The horizontal abnormality of the biological substance of detection and show, such as the change of albumen, gene level that these metabolic pathways are first closed Change and shows.It compares it is appreciated that these exception refers to normal non-Human Osteoarthritis.These bases The raising of the transcriptional level of cause will have a direct impact on the variation of the associated biomolecule substance of different metabolic approach, usually quantity and work Property variation, it is abnormal that these variations show that corresponding metabolic pathway occurs, so as to cause the different partings of osteoarthritis, i.e., Different pathogenesis provides accurate diagnostic result for later period efficient diagnosis.

It not only include gene level or protein level (including enzyme so the biological substance of these different physiology courses Level) the perhaps raising of other substances or the reduction of some substances, all be diagnose arthritis parting a kind of biomarker Substance.This is readily appreciated that, gene level occurs abnormal, and the exception of gene transcription level generally can all be caused (such as to compare normal person Increase), so as to cause the exception of protein level.The exception of protein level be usually enzyme activity perhaps the exception of quantity or The exception of precursor substance, these all occur from the metabolic pathway of Cucumber the abnormal biological substance of performance, thus Show as the metabolic disorder of Cucumber.Certainly, the horizontal exception of proteoglycan or chondroitin sulfate itself (such as concentration Level increase) be also illustrate osteoarthritis at Anttdisease Mechanism.That is, with proteoglycan metabolism or/and chondroitin sulfate generation Thank exception or collagen degradation and/or collagenous fibres groups is abnormal, nerve synapse is adjusted and/or ion channel occur it is abnormal or The biological substance that person, immune response and/or angiogenesis first close extremely, such as directly related substance (enzyme, genetic transcription water It is flat) or the substance (precursor substance of metabolic pathway etc.) of indirect correlation all can serve as the mark substance of arthritis parting, Arthritic parting is diagnosed using these mark substances.This solves traditional understanding, and traditional technology does not all find pass The scorching cause of disease of section is also far from being parting, let alone effective treatment, so generalling use anodyne to be relieved pain, reaches Less than the purpose treated at all.What this point will be appreciated that.So these biological substances not only include the invention demonstrates that tool The raising of body gene transcription level further includes the horizontal exception of corresponding albumen (such as enzyme), can also include precursor substance Horizontal exception.Here "horizontal" can be the meaning that quantity, activity or both have concurrently.

In addition, by drug therapy or mitigate arthritis ache also from change these exceptions, to effectively control Treat arthritis.Generally and normally in contrast here exception, for osteoarthritis, is just referring to not no osteoarthritis Crowd is normal person person, so, exception here refers to arthritic crowd, to be sentenced by different abnormal indexes The disconnected specifically arthritis in what type or hypotype, so that be conducive to the later period carries out drug therapy to the ill.Then selection is proper for treatment When mode allow abnormal approach of acting on behalf of to restore normal, so as to fundamentally reaching treatment osteoarthritis or alleviate pain Bitterly, it is solved rather than just using anodyne.

Examples of implementation 4: protein science verifying divides group

We are confirmed whether that there are four the inflammation of OA hypotype is anti-using the protein factor expression quantity in ELISA detection patient's synovial membrane It answers identical.Specific step is as follows:

According to the manufacturer's instructions, pass through multiple assay technology ProcartaPlex Human Cytokine Panel The synovia of (eBioscience, Inc., San Diego, CA, USA) measurement OA patient.Pass through Luminex LX100 TM (Luminex, Austin, TX, USA) multiple assay detection system reads measurement.Measure following cell factor: BDNF, Eotaxin / CCL11, EGF, FGF-2, GM-CSF, NGF β, IFN α, IL-1 β, IL-1 α, IL-2, IL-4, IL-5, IL-7, IL-8/ CXCL8, IL-9, IL-10, IL-12 p70, IL-13, IL-15, IL-17A, IL-21, IL-22, IL-23, IL-27, IL-31, IP-10/CXCL10, MIP-1 α/CCL3, RANTES/CCL5, TNF α, TNF β/LTA, PDGF-BB, PLGF, VEGF-D. It is specifically shown in shown in Fig. 4 .1-4.6.

Interpretation of result: we have found that RANTES(Fig. 4 .3 in first crowd of patient), IFN γ and SCF(Fig. 4 .3) it is contour Expression, these protein functions are all related to induction neutrophil leucocyte.The contour expression of VEGFA in second crowd of patient, IL6, explanation The immune response of second crowd of patient is related to B cell.Third group is because related not showing particularly to be immunized to nerve The characteristics of reaction.4th group of patient MIP1 α, SDF1 α and IL8 high expression, illustrate that the immune response of the 4th crowd of patients and T are thin Cell phase is closed.Wherein, C1 indicates that the inflammatory reaction figure of the patient of the first hypotype, C2 indicate the inflammatory reaction of the patient of the second hypotype Figure, C3 indicate that the inflammatory reaction figure of the patient of third hypotype, C4 indicate the inflammatory reaction figure of the patient of the 4th hypotype.It (is specifically shown in Fig. 4 .1-4.6)

Examples of implementation 5: tissue staining verifying divides group

The experiment of histogenic immunity histochemical staining:

1) slice is dewaxed with dimethylbenzene, through graded ethanol to aquation: dimethylbenzene (I) 60min → dimethylbenzene (II) 60min → diformazan Benzene: ethyl alcohol (1:1) 30min → 100%(I) ethyl alcohol 90sec → 100%(II) ethyl alcohol 90sec → 90% ethyl alcohol 90sec → 2min is washed in 80% ethyl alcohol of ethyl alcohol 90sec → 70% ethyl alcohol of 90sec → 50% 90sec → distillation；

2) slice is immersed in antigen retrieval buffers progress hot repair and answers, and 65 DEG C, overnight (> 12h)；

3) room temperature is naturally cooled to, PBS is washed 3 times, each 5min；

4) 0.3% H₂O₂Covering samples, room temperature inactivate 10min, and PBS is washed 3 times, each 5min；

5) 0.2% TritonX-100 Covering samples, room temperature 15min rupture of membranes, PBS are washed 3 times, each 5min；

6) plus 2% BSA confining liquid, room temperature close 90min；

7) 1%BSA dilutes primary antibody.It is added primary antibody, Covering samples, being placed at room temperature for half an hour in wet box is placed on 4 DEG C and (is not added overnight Antibody is as negative control), rewarming half an hour after refrigerator took out in second day；

8) PBS is washed three times, each 5min；

9) secondary antibody is diluted.Secondary antibody, Covering samples, room temperature 2h is added；

10) PBS is washed three times, each 5min；

11) DAB mixed liquor is configured.DAB mixed liquor is added, covers sample, microscopically observation colour developing；

12) PBS is washed three times, each 5min；

13) hematoxylin contaminates core.Hematoxylin dyes 2min30s, after tap water is sufficiently washed, 1% hydrochloride alcohol color separation 1sec；

14) tap water is sufficiently washed, and 0.1% ammonium hydroxide returns blue 30sec, is placed in tap water

15) conventional dehydration, transparent, mounting: 70% ethyl alcohol of ethyl alcohol 3-4sec → 80% ethyl alcohol of 3-4sec → 90% 3-4sec → 100% ethyl alcohol (I) ethyl alcohol of 2min → 100% (II) 2min → dimethylbenzene: ethyl alcohol (1:1) 5min → dimethylbenzene (I) 10min → bis- Toluene (II) 10min → resinene sealing.

Safarinin O &Fastgreen dyeing

1) slice of patient's cartilaginous tissue is dewaxed with dimethylbenzene, through graded ethanol to aquation: dimethylbenzene (I) 60min → dimethylbenzene (II) 60min → dimethylbenzene: ethyl alcohol (1:1) 30min → 100%(I) ethyl alcohol 90sec → 100%(II) ethyl alcohol 90sec → 90% The ethyl alcohol of ethyl alcohol 90sec → 80% ethyl alcohol of 90sec → 70% ethyl alcohol of 90sec → 50% 90sec → distillation wash 2min；

2) hematoxylin contaminates core.Hematoxylin dyes 2min30sec, after tap water is sufficiently washed, 1% hydrochloride alcohol color separation 1sec；

3) tap water is sufficiently washed, and 0.1% ammonium hydroxide returns blue 30sec, is placed in tap water

4) 0.02%Fastgreen dyes 8min；

5) tap water washes away excess dyestuff, sucks excessive moisture；

6) color separation in 1% acetic acid, 3-4sec；

7) tap water thoroughly washes > 2min；

8) 0.1% Safrain-O dyes 6min；

9) tap water washes away excess dyestuff；

10) conventional dehydration, transparent, mounting: 70% ethyl alcohol of ethyl alcohol 3-4sec → 80% ethyl alcohol of 3-4sec → 90% 3-4sec → 100% ethyl alcohol (I) ethyl alcohol of 2min → 100% (II) 2min → dimethylbenzene: ethyl alcohol (1:1) 5min → dimethylbenzene (I) 10min → bis- Toluene (II) 10min → resinene sealing.

As a result (it is specifically shown in Fig. 3 A-3B):

Pass through safranin O dyeing (safranin O staining, SO dyeing) and immunohistochemical staining (Immunohistochemical staining, IHC staining) have detected 4 osteoarthritis (Osteoarthritis, OA) destination gene expression of subgroup is horizontal.Wherein, SO dyeing can by make cartilage matrix the matrix egg of upper red detection cartilage White polysaccharide synthesis is horizontal.Red is deeper to illustrate that proteoglycan level is higher, and indication cartilage matrix is more normal.Immunohistochemistry dye Color, which is specific antibody by marking with color developing agent, to be passed through antigen-antibody reaction in histocyte in situ and histochemical is in Colour response carries out qualitative, positioning, quantitative determination to corresponding antigens, and yellowish-brown is presented in coloration result, and yellowish-brown is generally presented on carefully The periphery of karyon, yellow is deeper, and the explanation marker representation is higher.In this study, we pass through to normal group and osteoarthritis Four subgroups carry out SO dyeing, and all relatively normal group of four subgroup SO dyeing of osteoarthritis is shallow, illustrate the slice sample of osteoarthritis Cartilage matrix wreck.Wherein a group (C1) coloration result is most shallow, illustrates a group polysaccharide metabolic disorder.We select simultaneously Mmp-13 (Matrix metallopeptidase 13, MMP13), Synapotopsin, CD34 tri- Marker carries out immunohistochemical staining experiment, and wherein MMP13 is the marker of indication cartilage collagen degradation, MMP13 Dyeing is deeper, illustrates that its expression is higher, i.e. cartilage collagen degradation is more serious, and two groups of (C2) immunohistochemical staining results are compared with it The phenomenon that obvious MMP13 expression of his three groups of displays increases；Synapotopsin is marker relevant to nerve, Synapotopsin dyeing is deep to illustrate that the group is related to nerve, Synapotopsin higher expression in three groups (C3)；CD34 is The important marker of indication inflammation, we have found that histochemical staining shows that four groups (C4) have the characteristics that high inflammation occurs.With us Grouping result it is consistent, C1 is mainly shown as polysaccharide metabolic disorder, and C2 is mainly shown as that cartilage collagen is degraded, and C3 is neural phase It closes, C4 is inflammation correlation.It can be seen that the present invention collects multicenter clinical sample, one kind has been invented based on transcription group to knee The detection of method and the disease subtypes significant gene and access of arthritis disease parting.

The above-mentioned description to embodiment is for the ease of those skilled in the art it will be appreciated that and using this Invention.Those skilled in the art obviously easily can make various modifications to above-described embodiment, and described herein one As principle be applied in other embodiments without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, this Field technical staff announcement according to the present invention, the improvement made for the present invention and modification all should be in protection models of the invention Within enclosing.

In the case where lacking any element specifically disclosed herein, limitation, may be implemented illustrated and described herein Invention.Used terms and expressions method is used as the term of explanation rather than limits, and is not intended in these terms and table Up to any equivalent for excluding shown and described feature or part thereof in the use of method, and it should be realized that various remodeling exist It is all feasible in the scope of the present invention.It is therefore to be understood that although specifically being disclosed by various embodiments and optional feature The present invention, but the modifications and variations of concept as described herein can be used by those of ordinary skill in the art, and recognize It is fallen into for these modifications and variations within the scope of the present invention of the appended claims restriction.

It is described herein or record article, patent, patent application and every other document and can electronically obtain The content of information to a certain extent in full include herein by reference, just as each individual publication by specific and single Solely point out by reference.Applicant retains from any of any this article, patent, patent application or other documents And all material and information are incorporated into the right in the application.

Claims (11)

1. purposes of the biological substance of immune response and/or angiogenesis in diagnosis osteoarthritis parting.

2. purposes according to claim 1, the biomarker substance is selected from one of following gene or several: PECAM1, SYK, STOM, GMFG, TSPAN14, PTPRC, COTL1, ARPC5, CAP1, FCGR2B, CD36, VAMP8, CD14, RHOA, SDCBP, RAC1, CTSB, RAB10, RAP1B, PDXK, CYBA, GM2A, GRN, VCL, GNS, ADAM10, PSAP, ATP6AP2, ASAH1, ACTR2, FTL, PLD1, TCIRG1, NRAS, CST3, RAP2B, COLEC11, DUSP6, MPEG1, STOM, PLVAP, IFI16, ATP6V1B2, ADGRL4, PCDH18, APLNR, C1orf162, ZFP36L1, SLCO2B1, PTPRC, ACSL1, COTL1；MCAM, SYK, APLNR, EGFL7, NOTCH3, CD34, MMRN2, ECSCR, NRP1, GPX1, JUN, CYP1B1, PLXND1, RHOA, GPNMB, C1GALT1, ENG, MYH9, EPAS1, GNA13, ATP5B, RBPJ, PDGFRA, PGF, MMP2, STAT1, TMSB4X, LAPTM5, LGALS3BP, YWHAB, ZEB2, EGFL7, GMFG, SYK, MCAM, MYL6, KCTD12 or the corresponding protein substance of these genes.

3. purposes according to claim 2, wherein when the transcriptional level raising of these genes or protein substance level liter It is high, then it represents that osteoarthritis belongs to the osteoarthritis of the 4th hypotype.

4. purposes according to claim 1, the biomarker substance is MIP, SDF or IL8.

5. purposes according to claim 1, the biomarker substance is CD34.

6. a kind of method for diagnosing main body osteoarthritis parting, this method comprises: the biological object of immune response and/or angiogenesis Whether matter there is exception, if there is exception, then it represents that belongs to the osteoarthritis of the 4th hypotype.

7. according to the method described in claim 6, wherein the main body suffers from osteoarthritis.

8. according to the method described in claim 6, the biomarker substance is selected from one of following gene or several: PECAM1, SYK, STOM, GMFG, TSPAN14, PTPRC, COTL1, ARPC5, CAP1, FCGR2B, CD36, VAMP8, CD14, RHOA, SDCBP, RAC1, CTSB, RAB10, RAP1B, PDXK, CYBA, GM2A, GRN, VCL, GNS, ADAM10, PSAP, ATP6AP2, ASAH1, ACTR2, FTL, PLD1, TCIRG1, NRAS, CST3, RAP2B, COLEC11, DUSP6, MPEG1, STOM, PLVAP, IFI16, ATP6V1B2, ADGRL4, PCDH18, APLNR, C1orf162, ZFP36L1, SLCO2B1, PTPRC, ACSL1, COTL1；MCAM, SYK, APLNR, EGFL7, NOTCH3, CD34, MMRN2, ECSCR, NRP1, GPX1, JUN, CYP1B1, PLXND1, RHOA, GPNMB, C1GALT1, ENG, MYH9, EPAS1, GNA13, ATP5B, RBPJ, PDGFRA, PGF, MMP2, STAT1, TMSB4X, LAPTM5, LGALS3BP, YWHAB, ZEB2, EGFL7, GMFG, SYK, MCAM, MYL6, KCTD12。

9. according to the method described in claim 6, the biomarker substance is CD34.

10. according to the method described in claim 6, wherein, the sample is cartilaginous tissue.

11. according to the method described in claim 6, wherein the sample is joint fluid, the wherein biomarker in joint fluid Substance is MIP, SDF or IL8.