CN106916892A - CDHR3 as diagnosing tumor mark application - Google Patents

CDHR3 as diagnosing tumor mark application Download PDF

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Publication number
CN106916892A
CN106916892A CN201710134575.6A CN201710134575A CN106916892A CN 106916892 A CN106916892 A CN 106916892A CN 201710134575 A CN201710134575 A CN 201710134575A CN 106916892 A CN106916892 A CN 106916892A
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cdhr3
osteosarcoma
genes
expression
albumen
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杨承刚
任静
向常娟
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Beijing Medintell Bioinformatic Technology Co Ltd
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Beijing Medintell Bioinformatic Technology Co Ltd
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    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N2800/00—Detection or diagnosis of diseases
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Abstract

The present invention relates to CDHR3 as the application of diagnosing tumor mark, applications of the CDHR3 as osteosarcoma diagnosis marker, CDHR3 activators in clinical treatment of osteosarcoma is more particularly related to.Osteosarcoma is low due to the incidence of disease, is not furtherd investigate, and the molecular marker of current osteosarcoma is simultaneously few.The present invention carries out high-flux sequence to osteosarcoma case sample, with reference to bioinformatic analysis and molecular biology experiment, pick out the gene C DHR3 closely related with osteosarcoma, it is expected to turn into osteosarcoma diagnosis marker, for its clinical diagnosis provides reference, for osteosarcoma early diagnosis provides Clinical Basis.

Description

CDHR3 as diagnosing tumor mark application
Technical field
The present invention relates to biomedicine field, and in particular to CDHR3 as diagnosing tumor mark application, more specifically It is related to applications of the CDHR3 as osteosarcoma diagnosis marker, CDHR3 activators in clinical treatment of osteosarcoma.
Background technology
The CDHR3 assignments of genes gene mapping in the 7q22.3 regions of chromosome, be it is a kind of comprising six outer calcium mucin domains across Memebrane protein, it can mediate sticking for homology cell, participate in the processes such as cell differentiation, cell-cell interaction.Research shows it The gene played an important role in pole asthma in early days are formed, shows that it is related to osteosarcoma but without report.
Osteosarcoma is that one kind originates from the mesenchymal Primary Malignant Bone Tumor of bone, and age of onset is generally 8-25 Sui, society Can harmfulness greatly, there is morning and incidence is high in its hematogenous metastasis, progress is rapid, and oneself has small turn when making a definite diagnosis for 80% patient Move stove.With the development of the treatment methods such as chemotherapy, surgical technic, bone remoulding, overall survival obtains larger raising within 5 years. But, the treatment of osteosarcoma in recent years runs into bottleneck, especially osteosarcoma early stage transfer on, i.e., the early diagnosis of osteosarcoma into It is clinical problem demanding prompt solution.
The research of tumor markers makes it possible the early diagnosis of osteosarcoma.The presence or quantitative change of tumor markers can To point out the property of tumour, tissue generation, cell differentiation, cell function so as to understanding tumour, to help the diagnosis of tumour, divide Class, Index for diagnosis and treatment are instructed.But due to the osteosarcoma incidence of disease is low, and about 0.3/ ten thousand, cause sample to be collected difficult, do not have Have and furtherd investigate, the molecular marker of current osteosarcoma is simultaneously few.The present invention carries out high pass measurement to osteosarcoma case sample Sequence, with reference to bioinformatic analysis and molecular biology experiment, picks out the gene C DHR3, CDHR3 closely related with osteosarcoma It is expected to turn into osteosarcoma diagnosis marker, is that Patients with Osteosarcoma brings hope for its clinical diagnosis provides reference.
The content of the invention
It is an object of the invention to provide a kind of osteosarcoma diagnostic preparation, said preparation detection CDHR3 genes and/or CDHR3 The expression of albumen.
Preparation it is an object of the invention to provide detection CDHR3 genes or albumen is in osteosarcoma diagnostic reagent is prepared Using.
Further, in osteosarcoma illness group by the expression and normal control tissue or cancer of CDHR3 genes or albumen Tissue is compared, and expression is low.
Further, expression of the detection CDHR3 genes or albumen in biological sample is by detecting CDHR3 parts Or the part or all of amino acid sequence of whole mRNA and/or detection coding CDHR3 albumen is carried out.
Preferably, biological sample is the biological sample from patient, including cell, tissue etc., can be blood or Person's urine etc., preferably peripheral blood.
Further, osteosarcoma diagnostic preparation is using PCR kit for fluorescence quantitative, method for gene chip, sequence measurement detection The expression of CDHR3 genes in osteosarcoma biological sample.Preferably, it is special containing a pair in described PCR kit for fluorescence quantitative The primer of specific amplification CDHR3 genes；Described genetic chip includes the probe with the nucleic acid array hybridizing of CDHR3 genes. It is furthermore preferred that PCR kit for fluorescence quantitative is contained within the sense primer and anti-sense primer of specific detection CDHR3 genes, upstream Primer sequence is SEQ ID NO.1, and downstream primer sequence is SEQ ID NO.2.
Fluorescence quantitative PCR method is the specific probe by fluorescent dye or fluorescence labeling, and PCR primer is marked Tracking, real time and on line monitoring course of reaction can be analyzed with reference to corresponding software to product, calculate testing sample template Initial concentration.
Genetic chip is also called DNA microarray (DNA microarray), can be divided into three kinds of main Types：1) it is fixed on poly- Nucleic acid probe or cDNA fragments on compound substrate (nylon membrane, nitrocellulose membrane etc.) surface, generally with the target of isotope marks Gene is hybrid with it, and is detected by radiography technology.2) DNA probe array on a glass is fixed with point sample method, Hybridized by the target gene with fluorescence labeling and detected.3) oligonucleotide probe being directly synthesized on the hard surfaces such as glass Array, the target gene hybridization with fluorescence labeling is detected.
Further, osteosarcoma diagnostic preparation detects the expression of CDHR3 albumen in biological sample using immunization method.It is preferred that , immunization method is that ELISA is detected and/or collaurum detection.
Further, the ELISA method of detection CDHR3 albumen is to use ELISA detection kit.Antibody in the kit Commercially available CDHR3 monoclonal antibodies or polyclonal antibody can be used.
Further, ELISA detection kit includes：The solid phase carrier of coating CDHR3 antibody, enzyme labelled antibody, the substrate of enzyme, Protein standard substance, negative controls, dilution, cleaning solution, enzyme reaction terminate liquid etc..
Further, collaurum detection uses colloidal gold immunochromatographimethod technology or collaurum percolation.Further, described glue Detection zone (T) specking on body gold detection kit nitrocellulose filter has the anti-CDHR3 antibody, quality control region (C) specking to have immune Globulin IgG.
Enzyme-linked immunosorbent assay (ELISA) will known antigen or antibody absorption in surface of solid phase carriers, make enzyme mark The technology that the antigen-antibody reaction of note is carried out in solid phase surface.ELISA detection kit can according to testing goal and operating procedure Be divided into indirect method, double-antibody method, competition law, double site one-step method, prize law survey IgM antibody, using Avidin and biotin ELISA.Chromogenic substrate may be selected horseradish peroxidase (HRP) or alkaline phosphatase (AP) in ELISA detection kit.
Immune colloid gold detection technique：(1) immune colloid gold light microscopic decoration method cell suspension smear or histotomy, can use The antibody of colloid gold label is dyeed, and can also be strengthened with silver-colored developer solution and marked on the basis of colloid gold label, makes to be reduced Silver atoms be deposited on marked gold grain surface, the sensitiveness of colloid gold label can be remarkably reinforced.(2) immune colloid gold electricity Mirror decoration method can be combined with the antibody of colloid gold label or antiantibody with negative staining Virus Sample or tissue ultra-thin section, then be carried out Negative staining.Can be used for the observation of morphology of virus and Viral diagnosis.(3) dot immunogold filtration assay application miillpore filter makees carrier, first By antigen or antibody point on film, sample to be checked is added after closing, with the corresponding antigen of antibody test of colloid gold label after washing Or antibody.(4) with ribbon be fixed on film for specific antigen or antibody by colloidal gold immunity chromatography, colloid gold label examination Agent (antibody or monoclonal antibody) is adsorbed on pad, after sample to be checked is added in the sample pad of test strips one end, is passed through Capillarity is moved forward, and is reacted to each other after the colloid gold label reagent on dissolving pad, when be moved to fixed antigen or During the region of antibody, there is specific binding and be trapped in thing to be checked, be gathered in detection therewith again with the conjugate of gold marked reagent Take, colour developing result can be observed by the naked eye.
It is an object of the invention to provide a kind of preparation for treating osteosarcoma, promotion CDHR3 genes are contained in the preparation Transcription or expression reagent or compound.Further, described CDHR3 genes and/or the expression product of gene are in kindred Low expression in tumor tissue.
Those skilled in the art know promotion gene and its expression of expression product generally can be using the one kind in following methods And/or it is several：The promoter of activated gene, the albumen of activated gene expression or the factor, import and promote genetic transcription or expression Carrier.
Preferably, the carrier containing transcription or the expression for promoting CDHR3 genes and/or activation in the preparation for the treatment of osteosarcoma The promoter of CDHR3 genes and/or the albumen or the factor of activation CDHR3 gene expressions.
Preparation it is an object of the invention to provide above-mentioned treatment osteosarcoma is in clinical treatment of osteosarcoma medicine or reagent is prepared Application.
Acceptable carrier is the carrier generally utilized in preparation in pharmacy of the invention, and the carrier includes lactose (lactose), dextrose (dextrose), sucrose (sucrose), sorbierite (sorbitol), mannitol (mannitol), shallow lake Powder, acacia gum, calcium phosphate, alginates (alginate), gel (gelatin), calcium silicates, microcrystalline cellulose, polyethylene Pyrrolidones (polyvinylpyrrolidone), cellulose (cellulose), water, syrup, methylcellulose (methyl Cellulose), methyl hydroxybenzoate (methyl hydroxybenzoate), propyl hydroxy propyl benzoate (propyl Hydroxybenzoate), talcum, magnesium stearate (stearic acid magnesium) and mineral oil (mineral oil) Deng, but it is not limited to this.
Pharmacy acceptable carrier of the invention can also be comprising lubricant, wetting agent, sweet in addition to mentioned component Taste agent, flavouring agent, emulsifying agent, suspending agent, preservative etc..The suitable carrier and preparation permitted in pharmacy are recorded in thunder in detail Bright Deng Shi pharmacy pandect.
Pharmaceutical composition of the invention can by it is oral or it is parenteral be administered, during as non-oral administration, can lead to Intravenous injection is crossed, intranasal injection, local injection, intraventricular injection, spinal cavity is injected, hypodermic injection, intraperitoneal injection, percutaneously The modes such as administration are administered.
The suitable dosage of pharmaceutical composition of the invention according to preparation ways, administering mode, patient year The factor of age, body weight, sex, morbid state, food, administration time, method of administration, drainage rate and draw property etc and can be with Carry out various prescriptions, generally, skilled practitioner can be easily determined by and prescription to desired treatment or prevent it is effective to Pharmaceutical quantities.
Pharmaceutical composition of the invention can easily be implemented according to general technical staff of the technical field of the invention Method, carried out using receptible carrier in pharmacy and/or excipient formulation such that it is able to unit dose form Prepare or interior prepare in the multicapacity container.Now, formulation be the solution of oiliness or aqueous medium, suspension or Emulsion form, or can also be extract, powder agent, granule, tablet or capsule form, dispersion can also be included Agent or stabilizer.
It is an object of the invention to provide a kind of gene detecting kit for detecting osteosarcoma, the kit detects gene CDHR3, using special sense primer and anti-sense primer, upstream primer sequence is SEQ ID NO.1, and downstream primer sequence is SEQ ID NO.2。
Further, the PCR kit is suitable for presently, there are all types fluorescence quantitative gene extender of in the market, spirit Sensitivity is high, it is quantitative quick and precisely, good stability, have a good application prospect.
Further, above-mentioned PCR kit for fluorescence quantitative component includes：Specific primer, internal control primer, quantitative fluorescent PCR Reaction solution.The internal reference is GAPDH.
Described kit also includes RNA extraction agents.
It is an object of the present invention to provide a kind of osteosarcoma protein detection kit, described detection kit detection CDHR3 Albumen.Further, described kit also includes other detection reagents.
It is an object of the present invention to provide a kind of genetic chip for detecting osteosarcoma, described genetic chip includes and CDHR3 The probe of the nucleic acid array hybridizing of gene.
Brief description of the drawings
Fig. 1 is RT-PCR amplification curve diagrams
Fig. 2 is RT-PCR solubility curve figures
Fig. 3 is the CDHR3 relative expressions spirogram in tissue samples
Fig. 4 is the CDHR3 relative expressions spirogram in blood sample
Specific embodiment
With reference to specific embodiment, the present invention is expanded on further, is only used for explaining the present invention, and it is not intended that to this The limitation of invention.It will be understood by those skilled in the art that：Can in the case where principle of the invention and objective is not departed from So that these embodiments are carried out with various changes, modification, replacement and modification, the scope of the present invention is limited by claim and its equivalent It is fixed.The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition or according to the bar proposed by manufacturer Part examinations.
The high-flux sequence of embodiment 1 and analysis
10 osteosarcoma case samples and 5 normal control tissues are collected respectively, wherein 5 in 10 osteosarcoma case samples The primary sample of example, 5 transfer samples.RNA extractions are carried out, agarose gel electrophoresis after RNA extractions can be preliminary from electrophoresis result Judge that whether up-to-standard the RNA sample extracted is, if can be used for further transcriptome analysis.And then pass through NanoDrop1000 spectrophotometers detect the extraction situation of RNA sample, the sample requirement of RNA-seq sequencings：OD260/OD280 It is 1.8-2.2.
Microarray dataset is the high-flux sequence platforms of HiSeq 2500 of Illumina companies, carries out high flux transcript profile depth Sequencing, we carry out total evaluation to the quality of sequencing data with Fast-QC softwares after sequencing, including the mass value of base divides Cloth, the position distribution of mass value, G/C content, PCR duplication contents, frequency of kmer etc..In differential gene table When analysis, according to the FPKM values for obtaining, differential screening is carried out using internationally recognized algorithm EBSeq.Wherein, during screening, LOG2FC>1 or<-1,FDR<0.05.In order to be better understood from the function of difference expression gene, we enter to difference expression gene Gene Onlogy and signal path analysis are gone, and functional annotation and protein interaction net have been carried out to difference expression gene Network is analyzed, in view of the result of data above analysis, with reference to document, we have screened and have lowered the obvious gene of expression in osteosarcoma group CDHR3。
The osteosarcoma group of embodiment 2 and normal group CDHR3 expression conditions
One material and method
1st, material
Collect 17 osteosarcoma tissue samples, 28 osteosarcoma blood samples, the control of 6 normal bone tissues, 20 it is normal Blood sample is compareed, and it is grouped and is numbered.
Tissue extraction：During patient's initial surgery, tissue samples are extracted in art under sterile working, every piece to be cut into soya bean big It is small, it is put into cryopreservation tube rapidly, and be placed in liquid nitrogen as early as possible after taking-up.
Blood is extracted：Patient gathers 3-5 milliliters of the periphery blood specimen of case in time after confirming, EDTA anticoagulant tubes are put into rapidly In, -80 DEG C of refrigerators are placed in as early as possible.
2nd, method
The extraction of 2.1 osteosarcoma and normal control total serum IgE
UsingReagent (invitrogen, article No. 15596-018) carries out sample rna extraction, experimental implementation Carried out by product description.
After extraction, RNA purity and concentration are measured with Nanodrop2000 ultraviolet specrophotometers, frozen in -70 DEG C.RNA Quality judging standard：The OD260/OD280 values of RNA samples are between 1.7-2.2；Total serum IgE electrophoresis pattern has clearly 28S, 18S Band；70 DEG C of water-baths be incubated 1 hour after electrophoresis pattern and water-bath insulation before collection of illustrative plates no significant difference.
2.2 reverse transcriptions synthesize cDNA
Using PrimeScriptTM RT reagent Kit with gDNA Eraser(Perfect Real Time) (article No.：RR047B cDNA reverse transcriptions) are carried out, genomic DNA reaction is removed first, 5 × gDNA Eraser are added in test tube Buffer 2.0uL, gDNA Eraser 1.0uL, TotalRNA 1ug, plus Rnase Free H20 makes cumulative volume to 10ul, water Heat 2min in bath, then by PrimeScript RT Enzyme Mix I 1.0uL, RT Primer Mix 1.0uL, 5 × PrimeScript Buffer 2 (for Real Time) 4.0uL, RNase Free dH20 4.0uL, add above-mentioned after mixing Common 20uL is mixed together in test tube, 37 DEG C of heating 15min, 85 DEG C of heating 5sec, the cDNA of synthesis need long-term guarantor in water-bath When depositing, please preserved in -20 DEG C or lower temperature.
2.3 Real-Time PCR
2.3.1 instrument and analysis method
With the type quantitative real time PCR Instruments of ABI 7300, useMethod carries out the relative quantitative assay of data.
2.3.2 design of primers
Using online primer-design software, synthesized by invitrogen companies after design of primers.Specific primer sequence is as follows：
Table 1
Operating process is as follows：
(1) reaction system：
Table 2
Reagent Usage amount
2×SuperReal PreMix Plus 10μl
Sense primer (10uM) 0.6μl
Anti-sense primer (10uM) 0.6μl
2μl
DNA profiling 2ul
Sterile purified water 4.8ul
Amplification program is：95 ° of 15min, (95 DEG C of 10sec, 55 DEG C of 30sec, 72 DEG C of 32sec) × 40 circulations, 95 DEG C 15sec, 60 DEG C of 60sec, 95 DEG C of 15sec).
(2) primer screening
After each sample cDNA is mixed, 3 times of gradient dilutions are carried out as template, sample respectively takes 2 μ l and makees template after dilution, Expanded (be shown in Table 3) with genes of interest primer and reference gene primer respectively, while carrying out melt curve analysis point at 60-95 DEG C Analysis, according to amplification efficiency is high and the unimodal principle of solubility curve carries out primer screening.
Table 3
(3) sample RealTimePCR detections
Make template by 2 μ l are taken after 3 times of dilutions of each sample cDNA, entered with genes of interest primer and reference gene primer respectively Row amplification.Simultaneously solubility curve analysis is carried out at 60-95 DEG C.
Two experimental results
Real-time quantitative PCR amplification curve flex point understands that amplification curve entirety collimation is good, shows the amplification effect of each reaction tube Rate is close, and the limit is flat and nothing raises up now, and exponent phase slope is larger, illustrates that amplification efficiency is (see Fig. 1) higher；Sample is expanded Product solubility curve is all unimodal, illustrates that amplified production only has one, is specific amplification (see Fig. 2)；According to the phase of qRT-PCR To quantitative equation：2-ΔΔCt, compare CDHR3 genes in osteosarcoma tissue and normal structure, osteosarcoma blood sample and normal control Expression in blood sample.Result shows：QRT-PCR stable amplification results, in tissue samples, CDHR3 is in osteosarcoma The expression of group is 0.15 times (being specifically shown in Fig. 3) of Normal group, in blood sample, tables of the CDHR3 in osteosarcoma group It is 0.53 times (being specifically shown in Fig. 4) of Normal group up to level, result above demonstrates the whole of high flux transcript profile expression data Close the result of analysis CDHR3 low expressions in osteosarcoma group.
The present invention filters out osteosarcoma related gene CDHR3 using high-flux sequence, and binding molecule biological experiment is confirmed CDHR3 is osteosarcoma diagnosis and treatment mark.
SEQUENCE LISTING
<110>Beijing Yang Shen biology information technologies Co., Ltd
<120>CDHR3 as diagnosing tumor mark application
<160> 4
<170> PatentIn version 3.3
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<213>Artificial sequence
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ggagcgagat ccctccaaaa t 21
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ggctgttgtc atacttctca tgg 23

Claims (10)

1. a kind of osteosarcoma diagnostic preparation, it is characterised in that osteosarcoma diagnostic preparation detects the table of CDHR3 genes and/or albumen Up to level.
2. osteosarcoma diagnostic preparation according to claim 1, it is characterised in that in osteosarcoma illness group CDHR3 genes or The expression of albumen is compared with normal control tissue or cancer beside organism, and expression is low.
3. osteosarcoma diagnostic preparation according to claim 1, it is characterised in that detection CDHR3 genes or albumen are in biology The expression in product that imitates be part by detecting the part or all of mRNA of CDHR3 and/or detection coding CDHR3 albumen or Whole amino acid sequences are carried out.
4. osteosarcoma diagnostic preparation according to claim 3, it is characterised in that biological sample is the life from patient Thing sample, including cell, preferably tissue, peripheral blood.
5. osteosarcoma diagnostic preparation according to claim 1, it is characterised in that osteosarcoma diagnostic preparation uses fluorescent quantitation PCR kit, genetic chip, high-flux sequence detect the expression of CDHR3 genes；Or using ELISA kit, colloid gold test paper Bar detects the expression of CDHR3 albumen.
6. osteosarcoma diagnostic preparation according to claim 5, it is characterised in that contain in PCR kit for fluorescence quantitative To the primer of specific amplification CDHR3 genes；Genetic chip includes the probe with the nucleic acid array hybridizing of CDHR3 genes； Contain CDHR3 monoclonal antibodies or polyclonal antibody in ELISA kit, colloidal gold strip.
7. application of the osteosarcoma diagnostic preparation described in claim 1-6 any one in osteosarcoma diagnostic reagent is prepared.
8. a kind of preparation for treating osteosarcoma, it is characterised in that contain the examination of transcription or the expression for promoting CDHR3 genes in preparation Agent or compound.
9. preparation according to claim 8, it is characterised in that containing promoting CDHR3 in the preparation of the treatment osteosarcoma The carrier of transcription or the expression of gene and/or the promoter of activation CDHR3 genes and/or the albumen of activation CDHR3 gene expressions Or the factor.
10. application of the preparation described in claim 8-9 any one in clinical treatment of osteosarcoma medicine is prepared.
CN201710134575.6A 2017-03-08 2017-03-08 CDHR3 as diagnosing tumor mark application Pending CN106916892A (en)

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