CN104651509B - Osteosarcomatous medicine new target drone - Google Patents
Osteosarcomatous medicine new target drone Download PDFInfo
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Abstract
The invention discloses the application that C8orf59 gene diagnoses in preparation and treats in osteosarcomatous medicine.The experiment proves that the expression in osteosarcoma tissue of the C8orf59 gene apparently higher than normal structure, therefore, C8orf59 gene can make osteosarcoma diagnosis more accurately, quickly as diagnosing osteosarcomatous Specific marker.The present invention can substantially be suppressed cell proliferation by the expression of C8orf59 gene in interference osteosarcoma cell line, be migrated, attacks, becomes tumor ability, accelerates apoptosis simultaneously.Therefore the osteosarcomatous gene therapy of C8orf59 gene pairs has potential using value, is expected to be applied to osteosarcomatous treatment, and on this basis, exploitation resists osteosarcomatous new drug, contributes for clinical osteosarcomatous preventing and treating.
Description
Technical field
The invention belongs to biomedical sector, relate to a kind of osteosarcomatous medicine new target drone, be specifically related to C8orf59 gene
Application in preparation diagnosis osteosarcoma product and preparation treatment osteosarcoma medicine.
Background technology
Osteosarcoma is derived from the malignant tumor of mesenchymal tissue, and its principal causative is characterized as that the tumor cell of proliferation in vivo is straight
Connect formation immaturity bone or osteoid tissue.It it is a kind of modal primary malignant tumor of human skeletal system.Typical kindred
Tumor is a kind of rare (accounting for the 0.2% of whole malignant tumor) high carcinogenic malignant tumor, the most annual each million people of its sickness rate
In have three examples.Osteosarcoma mainly appears on longer skeleton and least a portion of soft tissue.Its principal pathogenetic crowd concentrates on 10
～20 years old teenager, there is sickness rate high, the rate of transform is high in early days, cures the features such as survival rate is low.X-ray, tomography skill
Art, nuclear magnetic resonance, NMR, Angiography and dynamic bone scintigraphy technology etc. are widely used in the diagnosis of this disease, tumor occurs
Degree and type of surgery judgement etc..But utilize above-mentioned clinical means to carry out osteosarcomatous diagnosis, frequently result in patient's
The state of an illness is delayed, and misses optimal treatment period.Therefore finding a kind of osteosarcoma early diagnosis marker is problem demanding prompt solution.
High flux transcript profile order-checking (RNA-sep) is the technology carrying out degree of depth order-checking in transcript profile level, order-checking
Technology application in transcriptome analysis is concentrated mainly on the structure of gene expression profile, the discovery of new gene, little molecule non-coding
Build and the discovery of new micro RNA genes, the protein coding gene of rna expression spectrum annotate and such as fusion
In the transcript profile researchs such as transcript.Data are in initial process is substantially similar, next grinds for different
Studying carefully purpose can select different bioinformatics softwares to calculate.
Utilize large scale sequencing technology directly the cDNA sequence become by mRNA reverse transcription to be checked order, produce number with ten million
The reads quantity of meter, so that the transcriptional level of one section of special genome area can directly pass through comparison to this gene
The reads number in group region is weighed.This technology is commonly used to build the gene expression profile of a certain particular organization or cell, by aligning
Often tissue and pathological tissues carry out the analysis of gene expression profile, obtain the gene of differential expression in two kinds of tissues, are on the one hand
The generation of study of disease and the molecular mechanism of development provide new thinking, and on the other hand diagnosis and treatment for disease provide new
Diagnosis marker or drug target.
Utilize the order-checking screening of high flux transcript profile and the closely-related gene of osteosarcoma, not only contribute to study osteosarcomatous
Occur and the pathomechanism of development, and new diagnosis marker or new medicine target can be provided for diagnosis and treatment osteosarcoma
Point.
Summary of the invention
The present invention utilizes high throughput sequencing technologies to find the expression in osteosarcoma tissue of the C8orf59 gene and in normal bone
Expression in tissue there are differences, compared with normal bone tissues, C8orf59 gene up-regulated in osteosarcoma tissue, passes through
The expression of detection C8orf59 gene, it is possible to judge whether experimenter suffers from osteosarcoma.
An object of the present invention is the application providing C8orf59 gene in preparation diagnoses osteosarcomatous product.
The two of the purpose of the present invention are the application providing C8orf59 gene in osteosarcomatous medicine is treated in preparation.
To achieve these goals, present invention employs following technical scheme:
The invention provides the osteosarcomatous product of a kind of diagnosis, the osteosarcomatous product of described diagnosis is become by detection experimenter
In osteocyte, the expression of C8orf59 gene judges whether experimenter suffers from osteosarcoma.
Further, the osteosarcomatous product of described diagnosis includes: with RT-PCR, real-time quantitative PCR, immune detection, the most miscellaneous
Hand over or the osteosarcomatous product of gene chip diagnosis.Described RT-PCR diagnoses osteosarcomatous product and at least includes a pair specific amplified
The primer of C8orf59 gene；Described real-time quantitative PCR diagnoses osteosarcomatous product and at least includes a pair specific amplified
The primer of C8orf59 gene；Described immune detection diagnoses osteosarcomatous product and includes: be combined with C8orf59 protein-specific
Antibody；Described in situ hybridization diagnoses osteosarcomatous product and includes: with the probe of the nucleic acid array hybridizing of C8orf59 gene；
The osteosarcomatous product of described gene chip diagnosis includes: with the probe of the nucleic acid array hybridizing of C8orf59 gene.
The primer using the specific amplified C8orf59 gene comprised in RT-PCR diagnosis osteosarcoma product is to utilize
Primer5.0 primer-design software or other conventional online software designs of primer form.Those skilled in the art are come
Saying, the amplimer going out can be used in RT-PCR experiment according to known sequential design is to need not pay creative work i.e.
Attainable.Therefore the primer of the specific amplified C8orf59 gene of the RT-PCR of the present invention refer to all can be at RT-PCR
The primer sequence of Successful amplification C8orf59 gene in experiment.In specific embodiments of the present invention, for RT-PCR experiment
C8orf59 gene amplification primer sequence is as follows: forward primer: 5 '-TACGGCTCGTGAGCGGAAT-3 ' (SEQ ID NO:1)；
Reverse primer: 5 '-GGGATAAGTGATTTAATATCC-3 ' (SEQ ID NO:2).
It is root that described real-time quantitative PCR diagnoses the primer of the specific amplified C8orf59 gene that osteosarcomatous product comprises
Form according to the conventional online software design of QPCR primer.To those skilled in the art, according to known sequential design
The amplimer going out can be used in QPCR experiment is that to need not pay creative work the most attainable.Therefore the present invention's is real-time
The primer of the specific amplified C8orf59 gene of quantitative PCR refer to all can QPCR test in Successful amplification C8orf59 base
The primer sequence of cause.In specific embodiments of the present invention, for QPCR experiment C8orf59 gene amplification primer sequence such as
Under: forward primer: 5 '-ATTCCTCAGCAGCGTCAT-3 ' (SEQ ID NO:3)；Reverse primer: 5 '-
GCATCACCAGTATATTACAACAGA-3 ' (SEQ ID NO:4).
With the probe of the nucleic acid array hybridizing of C8orf59 gene can be DNA, RNA, DNA-RNA chimera, PNA or its
Its derivant.The length of described probe does not limit, as long as completing specific hybrid and purpose nucleotide sequence specificity knot
Closing, any length can.The length of described probe can be as short as 25,20,15,13 or 10 bases longs.Equally, described probe
Length can grow to 60,80,100,150,300 base pairs or longer, the most whole gene.Due to different probe length pair
Hybridization efficiency, signal specificity have different impacts, the length of described probe to be typically at least 14 base pairs, the longest general the most not
More than 30 base pairs, optimal with 15-25 base pair with the length of purpose nucleotide sequence complementary.Described probe self-complementary
Sequence is most preferably less than 4 base pairs, in order to avoid affecting hybridization efficiency.
The antibody being combined with C8orf59 protein-specific can be polyclonal antibody, it is also possible to is monoclonal antibody.Above-mentioned
Antibody can obtain from commercial channels, it is possible to use prepared by a series of methods known in the art.Such as, by the people of purification
It is interior to produce polyclonal antibody that C8orf59 albumen or its antigen fragment are injected into animal body.Equally, people's C8orf59 egg is expressed
White or its cell of antigen fragment may also be used for animal being caused immunity and producing antibody.Monoclonal antibody can use hybridoma skill
Prepared by art.The antibody of C8orf59 albumen includes the antibody that can prevent C8orf59 protein function, it is also possible to be not affect people
The antibody of C8orf59 protein function.Each antibody-like can be by causing immunity to the segment of people's C8orf59 albumen or functional domain
And produce, and people's C8orf59 protein product and fragment thereof can produce with recombination method or synthesize with Peptide synthesizer.With
The protein bound antibody of C8orf59 of non-modified form, it is possible to use the gene produced in prokaryotic cell such as E.coli produces
Thing carrys out immune animal and obtains.Be combined with the such as glycosylation of post translational modification form or phosphorylation C8orf59 albumen or polypeptide is anti-
Body, it is possible to use the gene outcome produced in eukaryotic cell such as yeast or insect cell is carried out immune animal and obtained.
The invention provides the application in preparation diagnoses osteosarcomatous product of the C8orf59 gene.
The osteosarcomatous product of described diagnosis judges to be subject to by the expression of C8orf59 gene in detection experimenter's osteoblast
Whether examination person suffers from osteosarcoma.The osteosarcomatous product of described diagnosis includes: with RT-PCR, real-time quantitative PCR, immune detection, former
Position hybridization or the osteosarcomatous product of gene chip diagnosis.Described RT-PCR diagnose osteosarcomatous product at least include a pair special
The primer of amplification C8orf59 gene；Described real-time quantitative PCR diagnoses osteosarcomatous product and at least includes a pair specific amplified
The primer of C8orf59 gene；Described immune detection diagnoses osteosarcomatous product and includes: be combined with C8orf59 protein-specific
Antibody；Described in situ hybridization diagnoses osteosarcomatous product and includes: with the probe of the nucleic acid array hybridizing of C8orf59 gene；
The osteosarcomatous product of described gene chip diagnosis includes: with the probe of the nucleic acid array hybridizing of C8orf59 gene.
The primer using the specific amplified C8orf59 gene comprised in RT-PCR diagnosis osteosarcoma product is to utilize
Primer5.0 primer-design software or other conventional online software designs of primer form.Those skilled in the art are come
Saying, the amplimer going out can be used in RT-PCR experiment according to known sequential design is to need not pay creative work i.e.
Attainable.Therefore the primer of the specific amplified C8orf59 gene of the RT-PCR of the present invention refer to all can be at RT-PCR
The primer sequence of Successful amplification C8orf59 gene in experiment.In specific embodiments of the present invention, for RT-PCR experiment
C8orf59 gene amplification primer sequence is as follows: forward primer: 5 '-TACGGCTCGTGAGCGGAAT-3 ' (SEQ ID NO:1)；
Reverse primer: 5 '-GGGATAAGTGATTTAATATCC-3 ' (SEQ ID NO:2).
It is root that described real-time quantitative PCR diagnoses the primer of the specific amplified C8orf59 gene that osteosarcomatous product comprises
Form according to the conventional online software design of QPCR primer.To those skilled in the art, according to known sequential design
The amplimer going out can be used in QPCR experiment is that to need not pay creative work the most attainable.Therefore the present invention's is real-time
The primer of the specific amplified C8orf59 gene of quantitative PCR refer to all can QPCR test in Successful amplification C8orf59 base
The primer sequence of cause.In specific embodiments of the present invention, for QPCR experiment C8orf59 gene amplification primer sequence such as
Under: forward primer: 5 '-ATTCCTCAGCAGCGTCAT-3 ' (SEQ ID NO:3)；Reverse primer: 5 '-
GCATCACCAGTATATTACAACAGA-3 ' (SEQ ID NO:4).
With the probe of the nucleic acid array hybridizing of C8orf59 gene can be DNA, RNA, DNA-RNA chimera, PNA or its
Its derivant.The length of described probe does not limit, as long as completing specific hybrid and purpose nucleotide sequence specificity knot
Closing, any length can.The length of described probe can be as short as 25,20,15,13 or 10 bases longs.Equally, described probe
Length can grow to 60,80,100,150,300 base pairs or longer, the most whole gene.Due to different probe length pair
Hybridization efficiency, signal specificity have different impacts, the length of described probe to be typically at least 14 base pairs, the longest general the most not
More than 30 base pairs, optimal with 15-25 base pair with the length of purpose nucleotide sequence complementary.Described probe self-complementary
Sequence is most preferably less than 4 base pairs, in order to avoid affecting hybridization efficiency.
The antibody being combined with C8orf59 protein-specific can be polyclonal antibody, it is also possible to is monoclonal antibody.Above-mentioned
Antibody can obtain from commercial channels, it is possible to use prepared by a series of methods known in the art.Such as, by the people of purification
It is interior to produce polyclonal antibody that C8orf59 albumen or its antigen fragment are injected into animal body.Equally, people's C8orf59 egg is expressed
White or its cell of antigen fragment may also be used for animal being caused immunity and producing antibody.Monoclonal antibody can use hybridoma skill
Prepared by art.The antibody of C8orf59 albumen includes the antibody that can prevent C8orf59 protein function, it is also possible to be not affect people
The antibody of C8orf59 protein function.Each antibody-like can be by causing immunity to the segment of people's C8orf59 albumen or functional domain
And produce, and people's C8orf59 protein product and fragment thereof can produce with recombination method or synthesize with Peptide synthesizer.With
The protein bound antibody of C8orf59 of non-modified form, it is possible to use the gene produced in prokaryotic cell such as E.coli produces
Thing carrys out immune animal and obtains.Be combined with the such as glycosylation of post translational modification form or phosphorylation C8orf59 albumen or polypeptide is anti-
Body, it is possible to use the gene outcome produced in eukaryotic cell such as yeast or insect cell is carried out immune animal and obtained.
The osteosarcomatous product of described diagnosis of the present invention is selected from following group: test kit, chip, filter paper.Mentioned reagent box can
To be the test kit of detection gene transcription level, such as RT-PCR kit, QPCR test kit, it is also possible to be detection gene expression water
Flat test kit, such as ELISA kit.Said chip can be gene chip or protein chip, and it can testing goal base
The expression of cause.
Described RT-PCR kit, in addition to comprising the primer of specific amplified C8orf59 gene, also can comprise PCR buffering
Liquid, dNTPs, MgCl2, Tap archaeal dna polymerase.Described RT-PCR kit also can comprise RNA and extract reagent, and RNase extracts examination
Agent comprises Trizol, chloroform, isopropanol, 75% ethanol.
Described QPCR test kit, in addition to comprising the primer of specific amplified C8orf59 gene, also can comprise SYBRGreen and gather
Polymerase chain reaction system.It is glimmering that SYBR Green polymerase chain reaction system comprises PCR buffer, dNTPs, SYBR Green
Photoinitiator dye.
Preferably, PCR buffer comprises: 25mM KCl, 2.5mM MgCl2,200mM (NH4)2SO4。
Described QPCR test kit also can comprise M-MLV reverse transcription system, and this reverse transcription system comprises: T repeats oligonucleotide
Oligo (dT), reverse transcription reaction liquid, M-MLV reverse transcriptase, RNase inhibitor, dNTPs.
Preferably, reverse transcription reaction liquid comprises: the MgCl of the KCL of the Tris-HCL of 250mM pH8.3,375mM, 15mM2,
The DTT of 50mM.
RNase inhibitor can be selected for the noncompetitive of RNase inhibitor commonly used in the art, preferably escherichia coli expression
The recombiant protein enzyme of suppression RNase.
Described QPCR test kit also can comprise RNA and extract reagent, and RNase extracts reagent and comprises Trizol, chloroform, isopropyl
Alcohol, 75% ethanol.
Described ELISA kit, except comprising in addition to the antibody that C8orf59 protein-specific is combined, also comprises and is coated buffering
Liquid, cleaning mixture, confining liquid, sample loading buffer, reaction terminating liquid, destination protein standard substance.
Preferably, being coated buffer is phosphate buffer PBS, and its composition is 140mM NaCl, 2.7mM KCl, 10mM
Na2HPO4, 1.8mM KH2PO4, pH value is 7.4.
Preferably, cleaning mixture is the PBS containing 1%Tween-20, referred to as PBST, and its composition is PBS, 1%Tween-20.
Preferably, confining liquid is the PBS containing 1%BSA, and its composition is PBS, 1%BSA.
Preferably, the composition of sample loading buffer is 50mM Tris-HCl, 0.5M KCl, 1%BSA, 0.05%Tween-
20, pH value is 8.4.
Preferably, reaction terminating liquid composition is 2M H2SO4。
Present invention also offers the application in osteosarcomatous medicine is treated in preparation of the C8orf59 gene.
Further, the osteosarcomatous medicine of described treatment can be the medicine of suppression human osteosarcoma cell proliferation, can be to promote
The medicine of apoptosis in osteosarcoma cells, can be the medicine of suppression osteosarcoma cell migration, can be suppression osteosarcoma cell invasion and attack
Medicine, can also is that suppression osteosarcoma cell becomes the medicine of tumor.
Further, the osteosarcomatous medicine for the treatment of of the present invention includes: suppress C8orf59 gene table by RNA interfering
The double stranded RNA reached, or tumor vaccine based on C8orf59 antigen protein or for suppressing C8orf59 protein active
Protein.
In the present invention, described RNA interference (RNA interference, RNAi) refers to be highly conserved during evolution
, induced by double-stranded RNA (double-stranded RNA, dsRNA), the phenomenon of the efficient selective degradation of homologous mRNA.Make
With RNAi technology can with specific depletion or close specific gene expression, this technology have been widely used for explore gene function and
Infectious disease and the field of gene of malignant tumor.RNAi based on cell screens in terms of functional gene research
There is many advantages, be mainly manifested in most cell types and can use RNAi method, and relatively easily lower or heavy
The expression of silent any genes of interest.
Can efficiently be rejected in order to ensure C8orf59 gene or reticent, according to the mRNA sequential design of C8orf59 gene
SiRNA specific fragment.SiRNA design according to delivered general design principle (Elbashir et.al 2001,
Schwarz et.al 2003, Khvorova et.al 2003, Reynolds et.al 2004, Hsieh et.al 2004,
Ui-Tei et.al 2004), by online tool complete design, this online tool is: siRNASelectionProgram of
Whitehead Institute (BingbingYuan et.al 2004, http://jura.wi.mit.edu/bioc/
And BLOCK-iTTM RNAi Designer ofINVITROGEN (winner of the 2004Frost& siRNAext/)
Sullivan Excellence in Research Award, https: //rnaidesigner.invitrogen.com/
sirna/).In order to improve the effectiveness of siRNA segment further, the advantage of comprehensive two Photographing On-line instruments is designed for
The siRNA segment of screening.Finally, siRNA sequence is filtered by sequence analysis (NCBI BLAST), to improve siRNA sheet
Disconnected specificity also reduces the effect of missing the target that RNAi disturbs.SiRNA oligonucleotide is by Shanghai JiMa pharmacy Technology Co., Ltd
Learn synthesis.In the detailed description of the invention of the present invention, for the concrete letter of the siRNA sequence (siRNA-C8orf59) of C8orf59
Cease as follows: positive-sense strand 5 '-UGUUCUUGGCCAUUGUCAGAA-3 ', antisense strand 5 '-CUGACAAUGGCCAAGAACAAA-3 '.With
Time design negative control siRNA (siRNA-NC): positive-sense strand: 5 '-CGUACGCGGAAUACUUCGA-3 ' (SEQ ID NO:5)；
Antisense strand: 5 '-UCGAAGUAUUCCGCGUACG-3 ' (SEQ ID NO:6).
Described tumor vaccine refers to excite immunologic function in the patient with the particular matter in tumor tissues, makes patient
Immunologic function can killing tumor cell.Tumor vaccine refers mainly to therapeutic vaccine, obtains the most again with vaccine patient
Method is treated, to control recurrence and the transfer of tumor.Tumor vaccine has individuality, because each patient's gained is swollen
Tumor type, the most different, the material expressed by tumor cell is the most different, and tumor vaccine is by the tumor of patient itself
Making, the tumor vaccine therefore prepared for the tumor that patient is concrete has individuation feature, and its side effect is little, with strong points.
Time prepared by described tumor vaccine, with tumor tissues the aseptic freezen protective of operation method excised tumor patient, with
Extract tumor antigen.Obtaining tumor antigen is the first step, and dendritic cell to be obtained is i.e. thin from the single core of patient blood
Born of the same parents obtain.Carry out again after extraction mononuclearcell screening and In vitro culture, then stimulate dendron shape with tumor antigen in vitro
Cell, enables this dendritic cell premunition information, finally feeds back to internal by the dendritic cell trained, make internal
Produce the immunne response for tumor antigen.
Present invention also offers the above-mentioned siRNA for C8orf59 application in osteosarcomatous medicine is treated in preparation.Institute
State and treat the medicine that osteosarcomatous medicine can be suppression human osteosarcoma cell proliferation, can be the medicine promoting apoptosis in osteosarcoma cells
Thing, can be the medicine that migrates of suppression osteosarcoma cell, can be the medicine of suppression osteosarcoma cell invasion and attack, also suppress osteosarcoma
Cell becomes the medicine of tumor.
Said medicine is in addition to comprising siRNA, it is also possible to comprise the most common pharmaceutical carrier.
The method of application of described medicine can be administered orally, systemic administration (such as, through skin, nasal inhalation or with suppository)
Or parenteral administration (such as, intramuscular, intravenously or subcutaneously).
Described medicine can be made into multiple dosage form, such as tablet, injection, capsule etc..With normal saline or containing glucose and
The aqueous solution of other adjuvant is prepared medicine injection by conventional method.The medicine of tablet and capsule etc, it is possible to by this
Prepared by skilled person's conventional method.
Advantages of the present invention and beneficial effect:
(1) present invention firstly discloses C8orf59 gene relevant to osteosarcoma, C8orf59 gene is expected to become diagnosis bone
The molecular marker of sarcoma, and for studying the thinking that osteosarcomatous molecule mechanism provides new.
(2) method that the mode utilizing detection gene expression diagnoses osteosarcomatous presence or absence is sensitiveer, beneficially disease
Sick diagnosis in early days.
Accompanying drawing explanation
Fig. 1 show utilize RT-PCR detect C8orf59 gene differential expression in osteosarcoma tissue and normal bone tissues；
Fig. 2 show utilize real-time quantitative PCR detect the expression in osteosarcoma tissue and normal bone tissues of the C8orf59 gene
Difference；
Fig. 3 shows and utilizes real-time quantitative PCR to detect the RNA interfering for C8orf59 on transcriptional level to C8orf59 base
Because of the impact expressed；
Fig. 4 utilizes the impact that osteosarcoma cell line U2OS is bred by mtt assay detection C8orf59 gene expression.
Detailed description of the invention
The present invention is further detailed explanation with embodiment below in conjunction with the accompanying drawings.Following example are merely to illustrate this
Invention rather than restriction 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) condition described in, or according to the condition proposed by manufacturer.
The gene that embodiment 1 screening is relevant to osteosarcoma
1.1 sample collection
Each collection 8 example normal bone tissues, the sample of osteosarcoma tissue.
The preparation of 1.2 RNA sample and quality analysis
1.2.1 the preparation of RNA sample
Normal bone tissues and osteosarcoma tissue judge RNA sample quality through agarose gel electrophoresis after RNA extracts, and close
Lattice person may be used for further transcriptome analysis.
1.2.2 the quality analysis (NanoDrop1000 spectrophotometer) of RNA sample
NanoDrop1000 spectrophotometer detection RNA sample, the sample requirement of RNA-seq order-checking: OD260/OD280 is
1.8-2.2。
1.2.3 the quality analysis (Agilent Technologies 2100 Bioanalyzer) of RNA sample
Agilent Technologies 2100 Bioanalyzer detects RNA sample quality, observe 28S rRNA and
18S rRNA master tape substantially, concentration qualified without degraded, RNA Perfection Index reach requirement meet RNA-seq order-checking cDNA literary composition
The requirement that storehouse builds, may be used for library construction and order-checking.
1.3 high flux transcript profile order-checkings
1.3.1 RNA-seq reads section location
First by low-quality reading section remove obtain cleaning read section, then utilize TopHat v1.3.1 will clean fragment and
UCSC H.sapiens mates with reference to genome (hg19), the index built in advance of H.sapiens UCSC hg19 version
Download from TopHat homepage, and as reference genome, when utilizing TopHat to mate with genome, it is allowed to each reading section (acquiescence
To 20) there are multiple coupling site, most 2 mispairing.TopHat sets up possible according to exon region and GT-AG shear signal
Shearing site storehouse, navigates to the reading section not navigating to genome on genome according to these shearing site storehouses.We use
The system default parameter of TopHat method.
1.3.2 transcript abundance assessment
The reading segment file matched is by Cufflinks v1.0.3 process, and Cufflinks v1.0.3 is by RNA-seq sheet
Hop count mesh is standardized calculating the relative abundance of transcript.FPKM value refer to each million order-checking fragments in match specific
The segment number of the exon region of gene 1kb length.The confidence interval of FPKM estimated value is calculated by Bayesian inference method.
The GTF comment file of the reference that Cufflinks uses downloads (Homo_ from Ensembl data base
sapiens.GRCh37.63.gtf)。
1.3.3 the detection of difference expression gene
Ensembl GTF file and the original document mated by TopHat of download are transferred to Cuffdiff,
Cuffdiff uses original matching files to re-evaluate the gene expression abundance of the transcript listed in GTF file, detects difference table
Reach.Only having q value ＜ 0.01 in Cuffidff exports, test display is considered as successfully more just differential expression.
1.4 result
The RNA-seq sequencing result of early stage filters out 979 difference expression genes, the gene that wherein expression raises altogether
472, the gene 507 that expression is lowered.
Embodiment 2 large sample PCR checking candidate gene and osteosarcomatous relation
Consider prior art yet there are no the gene carrying out studying about this gene and osteosarcoma dependency as candidate's base
Cause, considers the result of early stage high flux transcript profile degree of depth order-checking simultaneously, selects C8orf59 gene (its according to the size of P value
Express and raise in osteosarcoma tissue) verify.Collect 30 osteosarcoma tissue, collect 20 normal bone tissues samples simultaneously
This, use PCR to carry out the molecular biology experiment checking of classics, and concrete operation step is as follows:
1, RNA extracts
After collecting sample, frozen mortar tissue being put into pre-cooling after liquid nitrogen, taking-up is ground, sample to be organized
After this is powdered:
1. Trizol, room temperature preservation 5min are added；
2. adding chloroform 0.2ml, use forced oscillation centrifuge tube, fully mix, ambient temperatare puts 5min-10min；
3. draw upper strata aqueous phase (inhaling 70%) after 12000rpm high speed centrifugation 15min in another new centrifuge tube, note not
Protein substance between two-layer aqueous phase to be drawn onto.Move into new pipe, add isopyknic-20 DEG C of pre-cold isopropanols, the most reverse mixed
Even, it is placed in 10min on ice；
4. 12000rpm carefully discards supernatant at a high speed after 15min, adds 75% in the ratio of 1ml/ml Trizol
DEPC washing with alcohol precipitation (4 DEG C of preservations), washing precipitate, vibration mixing, 12000rpm high speed centrifugation 5min at 4 DEG C；
5. discarding ethanol liquid, ambient temperatare puts 5min fully to dry precipitation, adds the water dissolution that DEPC processed and sinks
Form sediment；
6. RNA purity and concentration are measured with Nanodrop2000 ultraviolet spectrophotometer, frozen in-70 DEG C.
2, reverse transcription
With RT Buffer, 1 μ g total serum IgE is carried out reverse transcription and synthesize cDNA.Use 25 μ l reaction systems, each sample
Take 1 μ g total serum IgE as template ribonucleic acid, PCR pipe is separately added into following components: DEPC water, 5 × RT Buffer,
10mmol/L dNTP, 0.1mmol/l DTT, 30 μm mol/l Oligo dT, 200U/ μ l M-MLV, template ribonucleic acid.Hatch for 42 DEG C
1h, 72 DEG C of 10min, of short duration centrifugal.
3, RT-PCR amplification
C8orf59: forward primer is 5 '-TACGGCTCGTGAGCGGAAT-3 ' (SEQ ID NO:1)；
C8orf59: reverse primer is 5 '-GGGATAAGTGATTTAATATCC-3 ' (SEQ ID NO:2)；
β-actin: forward primer is 5 '-CATCCTGCGTCTGGACCT-3 ' (SEQ ID NO:7)；
β-actin: reverse primer is 5 '-GTACTTGCGCTCAGGAGGAG-3 ' (SEQ ID NO:8).
Each composition in reactant mixture: forward primer, reverse primer, 10 × PCR buffer, MgCl2、dNTP、Taq DNA
Polymerase, the amount of cDNA template are respectively 0.2,0.2,0.4,0.4,1.0,1.0,0.2,0.1 and 5 μ L, finally supplement double steaming
Water makes reaction system be 10 μ L.The reaction condition of PCR is as follows: 94 DEG C, 5min denaturation；94 DEG C, 30s degeneration；55 DEG C, 30s moves back
Fire；72 DEG C, 30s extends；Totally 35 circulations, electrophoresis detection pcr amplification product.Amount of DNA, bar is weighed with the brightness of DNA band
Band is the brightest, and representation DNA amount is the most.Make internal reference comparison with β-actin, the brightness of the band of C8orf59 gene amplification product is entered
Row homogenization processes, and compares the ratio of the brightness of C8orf59 gene amplification product in normal bone tissues and osteosarcoma tissue.Result
As it is shown in figure 1, compared with normal bone tissues, C8orf59 gene is at the up-regulated of osteosarcoma tissue, with RNA-sep result one
Cause.
4, QPCR amplification
Using 25 μ L reaction systems, each sample arranges 3 parallel pipes, and all amplified reactions are above to protect
The reliability of card result.Prepare following reaction system: SYBR Green polymerase chain reaction system 12.5 μ L, forward primer (5
μm ol/ μ l) 1 μ L, reverse primer (5 μm ol/ μ l) 1 μ L, template cDNA 2.0 μ L, distilled water 8.5 μ L；Amplification C8orf59 gene
Forward primer sequence be 5 '-ATTCCTCAGCAGCGTCAT-3 ' (SEQ ID NO:3), reverse primer sequences is 5 '-
GCATCACCAGTATATTACAACAGA-3 ' (SEQ ID NO:4)；The preferred GAPDH of house-keeping gene, the forward of amplification house-keeping gene
Primer sequence is 5 '-CTCTGGTAAAGTGGATATTGT-3 ' (SEQ ID NO:9), reverse primer sequences is 5 '-
GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO:10).Operations is all carried out on ice.Amplification program is: 95 DEG C
10min, (95 DEG C of 15s, 60 DEG C of 60s) * 45 circulations.Using SYBR Green as fluorescent marker, glimmering at Light Cycler
The enterprising performing PCR of light real-time PCR reacts, and determines that purpose band, Δ Δ CT method are carried out by melt curve analysis analysis and electrophoresis
Relative quantification.Result as in figure 2 it is shown, compared with normal bone tissues, C8orf59 gene up-regulated in osteosarcoma tissue,
Consistent with RNA-sep result.
The expression change of C8orf59 albumen in embodiment 3 immune detection sample
1. antigen protein obtains
Utilize gene engineering expression: the cDNA sequence of people's C8orf59 gene can be obtained from Genbank data base, pass through
PCR amplification obtains encoder block, inserts in prokaryote or eukaryotic expression vector, expresses C8orf59 albumen, and press gene work
The purification system protein purification of journey expression product.
2. prepared by antibody
Following several method can be used to prepare antibody:
(1) cell fusion method: with the C8orf59 protein immune animal (including rabbit, goat etc.) of above-mentioned preparation, it is thus achieved that spleen
Dirty cell, then with myeloma cell fusion, and monoclonal anti Antibody Production Techniques prepares monoclonal antibody routinely.
(2) utilize phage display storehouse, clone the spleen IgG variable region of immune animal and be expressed as genetic engineering list
Clonal antibody.
(3) utilize the Western Immuno animal of purification, prepare polyvalent antibody.
3. detection
(1) with the antibody (multi-resistance or monoclonal antibody) prepared, osteosarcomatous pathology detection, positive letter are carried out with histochemical method
Number it is osteosarcoma.
(2) taking patients serum, detect by ELISA method, positive reaction is the suspicious patient of osteosarcoma.
(3) using C8orf59 antibody as one of the probe of protein chip, diagnose for osteosarcoma.
The embodiment 4 C8orf59 gene expression impact on osteosarcoma cell physiological activity
The detection of 1.siRNA jamming effectiveness
1.1 cells are cultivated
With human osteosarcoma U2OS cell line as object of study, human osteosarcoma U2OS cell line is purchased from Shanghai cell bank, cell
Cultivate by the IMDM culture medium containing 10% hyclone, in the environment of without penicillin, streptomycin, be 5% in volume fraction
CO2, cellar culture under the conditions of 37 DEG C.U2OS cell is put basis of microscopic observation and is grown to cell attachment, and volume is medium, and size is equal
Even, cell ovalize or irregular shape, kytoplasm is less, and nucleus is relatively big, and kernel is clear.
1.2 siRNA disturb C8orf59 gene expression
SiRNA sequence (siRNA-C8orf59) for C8orf59:
Positive-sense strand 5 '-UGUUCUUGGCCAUUGUCAGAA-3 ' (SEQ ID NO:5)；
Antisense strand is 5 '-CUGACAAUGGCCAAGAACAAA-3 ' (SEQ ID NO:6).
Negative control siRNA sequence (siRNA-NC):
Positive-sense strand is 5 '-CGUACGCGGAAUACUUCGA-3 ' (SEQ ID NO:11)；
Antisense strand is 5 '-UCGAAGUAUUCCGCGUACG-3 ' (SEQ ID NO:12).
By cell by 1 × 104/ hole is inoculated in 24 porocyte culture plates, at 37 DEG C, 5%CO2In incubator, cell is cultivated
24h, in without dual anti-, IMEM culture medium containing 10%FBS, transfection (is purchased from according to lipofectamine 2000
Invitrogen company) description transfection, experiment be divided into negative control group and experimental group (20nM), wherein negative control group
The sequence of siRNA Yu C8orf59 gene is without homology, and concentration is 20nM/ hole.Transfect the most respectively.
The transcriptional level of 1.3 QPCR detection C8orf59 genes
1.3.1 the extraction of cell total rna
Use TRIzol Reagent (Invitrogen Cat.No.15596-018) total RNA extraction reagent, by specification
Offer method extracts the total serum IgE of U2OS cell.Method particularly includes: take cell, rinse 3 times with the PBS that concentration is 0.01M, add
Appropriate TRIzol reagent, room temperature is placed 5min cell lysis, is managed with 1mL/ pipe subpackage to 1.5mL Eppendorf after piping and druming uniformly
In.Often pipe adds 0.2mL chloroform, acutely shakes 15s, and room temperature places 2-3min, 4 DEG C, 12000r/min be centrifuged 15min, by upper
Layer aqueous phase moves in clean Eppendorf pipe, adds 0.5mL isopropanol, mixes gently, and room temperature places 10min, 4 DEG C, 7500r/
Min is centrifuged 10min.Abandoning supernatant, 75% washing with alcohol RNA precipitate, 7500r/min is centrifuged 5min, drying at room temperature RNA precipitate, 5-
Appropriate DEPC water it is dissolved in after 10min.Mass fraction is the integrity of the agarose gel electrophoresis detection RNA sample of 1.0%, application
The RNA extracted is quantitative determined by Bio-Photometer.
1.3.2 reverse transcription step is with embodiment 2.
1.3.3 QPCR amplification step is with embodiment 2.
1.4 result
Result such as Fig. 3 shows, siRNA-C8orf59 can effectively suppress the expression of C8orf59 gene.
The impact of 2.C8orf59 gene pairs human osteosarcoma cell proliferation
Cell proliferation in vitro uses mtt assay detection.
2.1 steps:
By 5 × 103Individual cell spreads into 96 orifice plates overnight incubation, transfects negative control RNA or siRNA-next day
C8orf59, and 0h, 24h, 48h, 72h, 96h, 120h, 144h carry out MTT detection after transfection.First, discard culture supernatant,
Change to the 100 μ L fresh culture containing MTT 0.5mg/ml (Sigma company)；Then 4h is hatched for 37 DEG C；Finally change to 100 μ L
DMSO (Sigma company) the 10min that vibrates.Final absorbance uses 490nm wavelength detecting, draws cell growth curve.
2.2 result
As shown in Figure 4, after suppression C8orf59 expresses, the growth of osteosarcoma U 2OS is substantially suppressed result, shows
C8orf59 gene take part in the breeding of osteosarcoma cell.
The apoptotic impact of 3.C8orf59 gene pairs
Use the flow cytomery apoptotic impact of C8orf59 gene pairs.
3.1 step
Method as described above carries out cell transfecting, after transfection 72h, uses pre-cooling PBS washed cell, then uses
0.25% trypsin digestion cell, stops digestion, uses PBS resuspended in the cell of centrifugal collection, is 1 × 10 by cell quantification6Individual/
Ml, takes the 200 above-mentioned cell suspension of μ L and is placed in Appendorf pipe, adds 10 μ L Annexin-V-FITC mixings, and room temperature is dark
Dyeing 15min is hatched at place, and before upper machine, 5min adds 10mg/L propidium iodide (PI) and dyes 5 μ L.The cell of untransfected siRNA is respectively
With Annexin-V-FITC and PI dyeing for standard quantitative.Two Colour Fluorescence cell streaming meter is carried out with FACS flow cytometer
Number, observing apoptosis cell percentages.
3.2 statistical method
Experiment is all according to being repeated 3 times, and result data is all to represent in the way of mean+SD,
SPSS13.0 statistical software is used to carry out statistical analysis, the t inspection that difference between the two uses, it is believed that as P ＜ 0.05
Time there is statistical significance.
3.3 result
The apoptosis rate of C8orf59 gene interference group is (45.23+2.53) %, the apoptosis rate of negative control group
For (7.33+0.26) %, above-mentioned difference has statistical significance (P ＜ 0.05), and the above results shows to suppress C8orf59 gene
Expression can be obviously promoted the apoptosis of osteosarcoma U 2OS.
The impact of 4.C8orf59 gene pairs osteosarcoma cell transfer ability
Use the impact of cut Healing Experiments detection C8orf59 gene pairs osteosarcoma cell transfer ability.
4.1 concrete steps
(1) select containing the 15 fibrinogenic PBS of μ g/ml coat 5cm size culture dish overnight.
Within (2) second days, add the IMDM containing 10%FBS, use after sterilizing a few hours under ultraviolet light.
(3) U2OS cell method as described above is carried out cell transfecting, after transfection 72h, according to 1 × 106Individual carefully
The standard of born of the same parents carries out inoculated and cultured to cell, and after 24h, whether observation of cell grows uniformly.
(4) scrape with cell in the middle of the cell of culture dish, scratch a vestige, place it in after incubator cultivates 24h
Observation of cell transfer ability in cut, selects 3 samples every time, observes 3 times when of observation.
4.2 statistical method
Experiment is all according to being repeated 3 times, and result data is all to represent in the way of mean+SD,
SPSS13.0 statistical software is used to carry out statistical analysis, the t inspection that difference between the two uses, it is believed that as P ＜ 0.05
Time there is statistical significance.
4.3 result
After Matrigel starts 24h, the meansigma methods of the cut healing rate of C8orf59 gene interference group and negative control group is divided
Not being 28%, 75%, above-mentioned difference has statistical significance (P ＜ 0.05), shows to suppress the expression of C8orf59 gene, kindred
The transfer ability of oncocyte U2OS declines.
The impact of 5.C8orf59 gene pairs osteosarcoma cell invasive ability
Use the impact of Transwell invasion and attack cell algoscopy detection C8orf59 gene pairs osteosarcoma cell invasive ability.
5.1 concrete operation steps are:
(1) in advance Matrigel (Beeton-Diekinson, FranklnLakes, NJ) is diluted to the IMDM of 4 DEG C
200 μ g/ml, the IMDM taking 200 μ L dilutions are placed on the Transwell that aperture is 8 μm.
(2) make micropores whole on polycarbon resin film film will be covered by Matrigel, then it is placed on the condition of 37 DEG C
Under overnight.
(3) it is dried process.
(4) with ultraviolet, the Transwell irradiation 2h got ready being come sterilizing, pre-irradiation adds what a small amount of sterilization treatment was crossed
The IMDM of serum-free carries out aquation to it.
(5) method as described above carries out cell transfecting, after transfection 72h, take U2OS cell then by cell number 3 ×
104Joining in each cell, culture medium selects IMDM.
(6) it is placed in 46 orifice plates cultivating.
(7) 5%CO2, 37 DEG C of incubators hatch 24h.
(8) Matrigel and the cell in that face, upper room, that face of lower room is wiped clean after being taken out by cell with napkin or cotton swab
After fixing 10min with methanol, carry out the dyeing of conventional H E, observe with microscope.
(9) observe according to randomly choosing 3 high power lens visuals field, and the carefully number of cells in face, room under counting cell
As penetrating the cell number of Matrigel, this process is carried out 3 times, calculates the reference frame that its average is used as calculating.
5.2 statistical method
Experiment is all according to being repeated 3 times, and result data is all to represent in the way of mean+SD,
SPSS13.0 statistical software is used to carry out statistical analysis, the t inspection that difference between the two uses, it is believed that as P ＜ 0.05
Time there is statistical significance.
5.3 result
After 24h, C8orf59 gene interference group, by the cell number of Transwell film the most each visual field cell
The meansigma methods of number is 47, and cell number the most each visual field cell number that negative control group is by Transwell film
Meansigma methods be 324, above-mentioned difference has statistical significance (P ＜ 0.05), show suppress C8orf59 gene expression, bone
The invasive ability of sarcoma cell U2OS declines.
6.C8orf59 gene pairs osteosarcoma cell becomes the impact of tumor ability
6.1 steps:
(1) method as described above carries out cell transfecting, by after U2OS cell tryptase enzymic digestion after transfection 72h, prepares
Become cell suspension；
(2) it is inoculated in 6 orifice plates (200 cells/well) after cell counting, the cell inoculated is continued in incubator
Cultivation is more than 50 to cell number in 14 days or most single clone, midway carried out changing liquid observation of cell shape every 3 days
State；Experiment terminates under front fluorescence microscope taking pictures cell clone；
(3) experiment fixes cell with paraformaldehyde when terminating, and after PBS washed cell, Giemsa dyes, and takes pictures.
6.2 result
Plate clone experimental result shows, compared with negative control group, the osteosarcoma of C8orf59 gene expression interference group is thin
Clone's speckle number that born of the same parents U2OS is formed substantially reduces, clones the volume of speckle and is obviously reduced, and the above results shows that C8orf59 silence is led
Cause osteosarcoma cell and form the ability decline of clone.
Embodiment 5 C8orf59 antigen protein is as the application of osteosarcoma vaccine
1. antigen protein obtains
Utilize gene engineering expression: the cDNA sequence of people's C8orf59 gene can be obtained from Genbank data base, pass through
PCR amplification obtains encoder block, uses Peptide synthesizer synthesis C8orf59 antigen polypeptide according to above-mentioned sequence.
The in-vitro separation of (2.DC dendritic cell) and cultivation
Detection in peripheral blood of patients underwent is separating interface mononuclearcell after lymphocytes separating solution Ficoll gradient centrifugation.Cell
After scrubbed 2 times, suspend with serum-free medium AIM-V culture fluid (Gibco BRL company), adjust cell concentration to 3 × 106
Individual/ml, inoculates into 6 well culture plates.At 37 DEG C, 5%CO2In incubator after overnight incubation, piping and druming suspension cell recovery are frozen gently
Deposit, in order to prepare effector lymphocyte.Attached cell culture fluid adds containing 1000U/ml grain single colony stimulating factor and 500U/ml
The AIM-V of interleukin.When cultivating to the 7th day, the DC that results suspend.
The people B that the EBV (Epstein Barr Virus, epstein-barr virus (EB)) of 3.T2 cell or HLA-A2 transfects
The C8orf59 antigen polypeptide of lymphocyte and final concentration of 20 μ g/ml hatches 4h, altogether as target cell after washing.
Identify that the present invention treats osteosarcomatous C8orf59 antigen vaccine, by DC submission C8orf59 the most in the following manner
Antigenic peptides activating effect T cell, generation specificity is for the immunne response of C8orf59 epitope:
(1) during the process of DC cell: DC is resuspended in 2ml AIM-V the most afterwards, and C8orf59 antigenic peptides is added to being somebody's turn to do
The final concentration of 40ug/ml of polypeptide.37 DEG C, 5%CO2Cultivate 18h, after 3000rad lonizing radiation irradiate, wash stand-by.
(2) preparation of effector lymphocyte: target cell prepared by step 3 with irradiate after DC cell mix with 20: 1 and to hatch altogether
6-7 days, adding DC according still further to same ratio, after 6-7 days, repetitive stimulation is once again, action effect T cell.In 0,7,10,
14,19 days, culture supernatant is left and taken respectively, for cytokines measurement.
(3) cytokines measurement: use cytokine detection kits (purchased from Endogen company of the U.S.) detection to cultivate
The cytokine of secretion in clear.
5. test result indicate that, through the effector T cell that the DC of C8orf59 antigenic peptides of the present invention stimulation is activated, can produce
Specificity is for the immunne response of C8orf59 epitope, thus specific killing is combined with this identical C8orf59 antigenic peptides
Target cell, illustrates that C8orf59 antigen protein of the present invention can be as potential clinical treatment of osteosarcoma vaccine.
The explanation of above-described embodiment is only intended to understand the method for the present invention and core concept thereof.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, it is also possible to the present invention is carried out some improvement
And modification, these improve and modify also by the protection domain falling into the claims in the present invention.
Claims (9)
1.C8orf59 gene application in preparation diagnoses osteosarcomatous product, it is characterised in that described product is subject to by detection
In examination person's osteoblast, the expression of C8orf59 gene judges whether experimenter suffers from osteosarcoma.
Application the most according to claim 1, it is characterised in that described product includes: with RT-PCR, real-time quantitative PCR, exempt from
Epidemic disease detection, in situ hybridization or the osteosarcomatous product of gene chip diagnosis.
Application the most according to claim 2, it is characterised in that described RT-PCR diagnoses osteosarcomatous product and at least includes
The primer of a pair specific amplified C8orf59 gene；Described real-time quantitative PCR diagnoses osteosarcomatous product and at least includes a pair spy
The primer of different amplification C8orf59 gene；Described immune detection diagnoses osteosarcomatous product and includes: special with C8orf59 albumen
Property combine antibody；Described in situ hybridization diagnoses osteosarcomatous product and includes: with the nucleic acid array hybridizing of C8orf59 gene
Probe；The osteosarcomatous product of described gene chip diagnosis includes: with the probe of the nucleic acid array hybridizing of C8orf59 gene.
4. according to the application according to any one of claim 1-3, it is characterised in that described product is selected from following group: test kit,
Chip, filter paper.
The application in osteosarcomatous medicine is treated in preparation of the 5.C8orf59 gene.
Application the most according to claim 5, it is characterised in that described medicine is selected from following group: suppression osteosarcoma cell increases
The medicine grown, the medicine promoting apoptosis in osteosarcoma cells, the medicine of suppression osteosarcoma cell migration, the medicine of suppression osteosarcoma invasion and attack
Thing, suppression osteosarcoma cell become the medicine of tumor.
Application the most according to claim 6, it is characterised in that described medicine includes suppressing C8orf59 base by RNA interfering
Because of the double stranded RNA expressed, or tumor vaccine based on C8orf59 antigen protein or be used for suppresses C8orf59 albumen to live
The protein of property.
8. the siRNA suppressing C8orf59 gene expression, it is characterised in that described siRNA sequence is: positive-sense strand such as SEQ
Shown in ID NO:5, antisense strand is as shown in SEQ ID NO:6.
9. the application in osteosarcomatous medicine is treated in preparation of the siRNA described in claim 8.
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| CN105535995B (en) * | 2016-01-07 | 2018-12-07 | 赣南医学院第一附属医院 | A kind of osteosarcoma diagnosis and treatment preparation and its application |
| CN106084009A (en) * | 2016-06-24 | 2016-11-09 | 广州军区广州总医院 | Human osteosarcoma U2 OS cell-specific Binding peptide and application thereof |
| CN107881240B (en) * | 2017-12-27 | 2018-10-02 | 北京泱深生物信息技术有限公司 | The diagnosis and treatment marker of osteosarcoma |
| CN107988374A (en) * | 2018-01-12 | 2018-05-04 | 蚌埠医学院第附属医院（蚌埠医学院附属肿瘤医院） | A kind of and the relevant molecular marker of osteosarcoma and its application |
| CN108251531B (en) * | 2018-03-06 | 2021-02-02 | 杨祚璋 | Application of ENSG00000267549 in judging osteosarcoma metastasis |
| CN108165624A (en) * | 2018-03-09 | 2018-06-15 | 蚌埠医学院第附属医院（蚌埠医学院附属肿瘤医院） | Application of the biomarker in osteosarcoma diagnosis and treatment |
| CN108949768B (en) * | 2018-04-28 | 2021-09-07 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | RAB22A-NoeFs fusion gene line for diagnosing and/or treating osteosarcoma and application thereof |
| CN109655608B (en) * | 2018-12-13 | 2020-02-21 | 杭州多泰科技有限公司 | Exosome protein for osteosarcoma diagnosis and instant detection method thereof |
| CN109628593A (en) * | 2018-12-26 | 2019-04-16 | 中国人民解放军第二军医大学第二附属医院 | A kind of osteosarcoma stem cell molecular marker CD24 and its application |
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