CN106544430A - A kind of molecular marked compound of detection carcinoma of prostate and its application - Google Patents
A kind of molecular marked compound of detection carcinoma of prostate and its application Download PDFInfo
- Publication number
- CN106544430A CN106544430A CN201610962167.5A CN201610962167A CN106544430A CN 106544430 A CN106544430 A CN 106544430A CN 201610962167 A CN201610962167 A CN 201610962167A CN 106544430 A CN106544430 A CN 106544430A
- Authority
- CN
- China
- Prior art keywords
- prostate
- carcinoma
- c2cd4d
- mrna
- expression
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57434—Specifically defined cancers of prostate
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Oncology (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Hospice & Palliative Care (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Public Health (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of molecular marked compound of detection carcinoma of prostate, expressing protein and its fragment, analogs and derivatives of the molecular marked compound for the C2CD4D or mRNA.The present invention further discloses the method whether the molecular marker analyte detection carcinoma of prostate correlation mRNA expressions are lowered.The invention also discloses a kind of test kit of prostate cancer diagnosis, the test kit includes the primer and description of specific amplification carcinoma of prostate correlation mRNA, and the carcinoma of prostate correlation mRNA is C2CD4D.The C2CD4D of the present invention makes prostate cancer diagnosis, more accurately, quickly as the molecular marked compound of detection carcinoma of prostate.And therapy target and important evidence are provided for clinical practices such as gene therapy, Drug therapys.
Description
Technical field
The present invention relates to genetic engineering field, and in particular to by the use of C2CD4D related to carcinoma of prostate as detection prostate
The molecular marked compound of cancer and its application.
Background technology
Carcinoma of prostate is one of common male reproductive system malignant tumor.In world wide, prostate-cancer incidence exists
Second is occupied in all malignant tumor of male, the sickness rate of carcinoma of prostate alreadys exceed pulmonary carcinoma in the U.S., become first harm
The tumor of men's health.The sickness rate of Asia carcinoma of prostate is well below American-European countries, but ascendant trend is presented in recent years, and increases
Length is rapider than European and American developed countries.Patients with prostate cancer is mainly elderly men, typically at 50 years old with sequela, 95%
The elderly men of more than 60 years old is born in, incidence rate constantly increases with age.Carcinoma of prostate early stage is more without any disease
Shape, even if uncomfortable, is also not enough to cause the attention of patient, when tumor increases urethra, and often increases with prostate
Life is mutually obscured.Find that metastasis focus just finds carcinoma of prostate first in the patient of China about 80%.Now, pathological changes are up to evening
Phase, prognosis malas.
The clinical diagnostic modalities of carcinoma of prostate mainly have serum PSA (PSA) detection, rectum to refer at present
Inspection, the detection of endorectal ultrasonography ripple, living tissue pathologic finding etc..PSA (Prostate Specific Antigen) is prostata tissue
Specific antigen, by prostate anatomical structure understand PSA by prostate duct enter blood and urine in, some cases or
Under physiological conditions, PSA can be entered in blood, such as prostatitis, urine retention, prostatitis adenosis, prostatic hyperplasia and prostate
Cancer etc., therefore by detecting that Serum PSA level has certain false positive rate predicting carcinoma of prostate.From the beginning of 1991, inspection
Surveying PSA contents in serum is used to predict carcinoma of prostate that higher than 4ng/mL, content to be considered that carcinoma of prostate is positive, sensitivity 79%,
Specificity 20%-59%, average sensitivity about 33%, in concentration 4-10ng/mL gray area part, specificity be it is minimum, at this moment
Generally require to be determined by invasive aspiration biopsy, considerable distress and spirit and financial burden are brought to patient.So
PSA can not preferably as diagnosis of prostate cancer mark.Digital rectal examination is most simple, most economical practical method, main
The forefinger passed through by doctor touches prostate, to find many asymptomatic patients with prostate cancer, it is possible to obtain and examine in early days
Chance that is disconnected and effecting a radical cure.But all there is limitation in the method for the above.The limitation of such as digital rectal examination is mainly at 4 aspects：(1)
It is when patient's prostate lump is little, easy to cause missed diagnosis；(2) enlargement of some patientss carcinoma of prostate is not obvious, but is already belonging in late period, be difficult root
Control；(3) can not be detected using this when patient's rectum has illness；(4) may have when doctors experience is not enough fail to pinpoint a disease in diagnosis or mistaken diagnosis can
Energy.
Recent studies indicate that, mRNA is closely related with carcinoma of prostate, they may participate in tumors generations, development and
Transfer, so pathogenesis, early diagnosiss, individualized treatment, the detection of transfer and prognosis to tumor etc. may have corresponding
Effect.
The content of the invention
In order to realize the early diagnosiss of carcinoma of prostate, individualized treatment, it is an object of the invention to provide it is a kind of it is new before
Row adenocarcinoma mRNA, and expressing protein and its fragment, the analogs and derivatives of the mRNA.
It is another object of the present invention to carcinoma of prostate correlation mRNA purposes before detection in row adenocarcinoma.
For achieving the above object, present invention firstly provides a kind of molecular marked compound of detection carcinoma of prostate, the molecule mark
Expressing protein and its fragment, analogs and derivatives of the note thing for the C2CD4D or mRNA, the described C2CD4D's or mRNA
Expressing protein and its fragment, the analogs and derivatives down-regulated expression in carcinoma of prostate biological sample.
Further, the invention provides a kind of method for detecting whether carcinoma of prostate correlation mRNA expressions are lowered.
(1) expression of carcinoma of prostate correlation mRNA in testing sample is detected, wherein carcinoma of prostate correlation mRNA
It is C2CD4D；
(2) expression of carcinoma of prostate correlation mRNA in cell to be measured is compared with a control, so that it is determined that prostate
Whether cancer expression is lowered.
Preferably, the testing sample is cell tissue sample.
Preferably, the detection is by genechip detection method, quantitative fluorescent PCR, protein chip detection method, immunity
Method or Ag-Ab detection method are carried out.Preferably, the immunization method is that ELISA is detected and/or gold colloidal detection.
Preferably, the ELISA method is to use ELISA detection kit, and the test kit includes：Coating C2CD4D monoclonals
The solid phase carrier of antibody, enzyme labelled antibody, the substrate of enzyme, protein standard substance, negative controls, diluent, cleaning mixture, enzyme reaction end
Only liquid etc..The colloidal gold method is to use detection kit, described antibody can adopt commercially available C2CD4D monoclonal antibodies or
Polyclonal antibody.It is highly preferred that described gold-immunochromatographyreagent reagent for assay box is oozed using colloidal gold immunochromatographimethod technology or gold colloidal
Filter method.It is highly preferred that detection zone (T) specking on described gold-immunochromatographyreagent reagent for assay box nitrocellulose filter has anti-C2CD4D to resist
Body, quality control region (C) specking have immunoglobulin IgG.
Further, the invention provides a kind of individual test kit with carcinoma of prostate probability of detection, the reagent
Box includes the primer and description of specific amplification carcinoma of prostate correlation mRNA, and the carcinoma of prostate correlation mRNA is C2CD4D.
Preferably, the primer has SEQ ID NO:1 and SEQ ID NO:Primer shown in 2.
Preferably, the detection kit contains C2CD4D forward primer SEQ ID NO:1 and reverse primer SEQ ID
NO:2, dry powder, Taq enzyme, dNTP, magnesium ion, and PCR reaction buffers.
Further, the invention provides it is a kind of detection carcinoma of prostate test kit, the test kit include detect prostatitis
The chip and description of adenocarcinoma related gene, the chip are selected from the group：
(1) DNA chip, the chip have the point sample DNA, the point sample DNA in the substrate and point sample point sample area on the substrate
Including the probe for specifically binding to carcinoma of prostate correlation mRNA transcripts, the carcinoma of prostate correlation mRNA is C2CD4D；
(2) protein chip, the chip have the deposited protein in the substrate and point sample point sample area on the substrate, the point sample
Albumen includes specifically binding to relating to prostate cancers because of the antibody of expression product, and the carcinoma of prostate correlation mRNA is
C2CD4D。
Further, the invention provides a kind of preparation for treating carcinoma of prostate, contains in the preparation and promote C2CD4D
The reagent of the reagent and promotion C2CD4D expression products of expression；The reagent of the promotion C2CD4D expression includes promoting mRNA transcriptions
Reagent, promote the reagent of mRNA translations, the reagent for promoting C2CD4D protein contents to improve；The promotion C2CD4D expression products
Reagent include promoting the reagent of C2CD4D expression stabilities, promote the reagent of C2CD4D expression products activity, promote C2CD4D
The reagent of expression product function.
Those skilled in the art know promotion mRNA and its expression of expression product can generally adopt the one kind in following methods
And/or it is several：The promoter of activating molecules mark, the albumen of activating molecules marker expression or the factor, import and promote molecule
The carrier that mark is transcribed or expressed.
Preferably, it is described treatment carcinoma of prostate preparation in containing promote C2CD4D transcription or expression carrier and/or
The promoter of activation C2CD4D and/or the albumen or the factor of activation C2CD4D expression.
The preparation of the treatment carcinoma of prostate that the present invention is provided on the one hand for supplement endogenouss C2CD4D albumen disappearance or
Deficiency, by the expression for improving C2CD4D albumen, so as to treat carcinoma of prostate caused by C2CD4D protein delations.On the other hand can
For promoting the activity or function of C2CD4D albumen, so as to treat carcinoma of prostate.
Beneficial effects of the present invention are as follows：
The invention discloses a kind of C2CD4D related to carcinoma of prostate, and it is further characterized by the C2CD4D's or mRNA
Expressing protein and its fragment, analogs and derivatives are lowered in expression in prostate cancer.Prostate is detected using the mRNA
Cancer can not only fast and effectively accomplish early diagnosiss, and provide treatment for clinical practices such as gene therapy, Drug therapys
Target spot and important evidence.
Description of the drawings
C2CD4D protein expression situations in carcinoma of prostate forming process in Fig. 1 WB analysis mice bodies.
Specific embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.If not specializing, embodiment
In the conventional meanses that are well known to those skilled in the art of technological means used, reagent used can be commercially available.
The experimental technique of unreceipted actual conditions in embodiment, usually this area conventional method, such as according to normal condition
Such as Sambrook et al., molecular cloning, laboratory manual (third edition) (Science Press, 2002) in the condition, or according to
Condition proposed by reagent manufacturing firm.
The present inventor carries out high flux transcript profile to the mRNA of prostate cancer tissue sample and cancer beside organism's sample
Sequencing, carries out genescreen by bioinformatics method, picks out candidate mRNA C2CD4D, in existing research not
C2CD4D related to carcinoma of prostate report, further, inventor has carried out molecular biology method checking, it was confirmed that C2CD4D
The down-regulated expression in prostate gland cancer cell, its related preparations can be used to treat carcinoma of prostate.
The C2CD4D of the present invention is known mRNA before making the present invention, and its essential information is as follows：Genbank accession number：
NCBI reference sequences:NM_001136003.1, from human genome, its nucleotides sequence is classified as SEQ ID NO.5.
The present invention also adopts RT-PCR method and Western Blotting methods to detect above-mentioned mRNA in carcinoma of prostate group
The expression with Carcinoma side normal tissue is knitted, and demonstrates mRNA down-regulated expressions in carcinoma of prostate.
Fluorescence quantitative PCR method is the probe by fluorescent dye or fluorescently-labeled specificity, and PCR primer is marked
Tracking, real time and on line monitoring course of reaction can be analyzed to product with reference to corresponding software, calculate testing sample template
Initial concentration.The appearance of quantitative fluorescent PCR, greatly simplifies the process of detection by quantitative, and is truly realized absolute quantitation.
The appearance of various detecting systems, makes the selectivity of experiment higher.Automation mechanized operation improves work efficiency, and reaction is quick, repetition
The good, sensitivity of property is high, high specificity, result are clear.
Western Blotting methods are Western immunoblottings, are, by Protein transfer to film, then to utilize anti-
Body is detected.The method uses polyacrylamide gel electrophoresis, and detected material is protein, and " probe " is antibody, " aobvious
Color " is resisted with the two of labelling.Through the detached protein examples of PAGE, solid phase carrier (such as cellulose nitrate film) is transferred to
On, solid phase carrier is with non-covalent bond form adsorbed proteins, and can keep the polypeptide forms and its biologic activity of electrophoretic separation
It is constant.Using the protein or polypeptide on solid phase carrier as antigen, play immunoreation with corresponding antibody, then with enzyme or isotope
The second antibody of labelling reacts, and develops the color through substrate or autoradiography is to detect the specificity genes of interest table of electrophoretic separation
The protein ingredient for reaching.The technology is also widely used in the expression for detecting protein level.
Terms used herein " down-regulated expression " refers to the sequence corresponding to expressed gene, wherein the measurement card of sequence amount
It is bright, from from normal individual or from by stages of prostate cancer determine with the different individualities for having identified morbid state of carcinoma of prostate
Same gene in detached biological sample is compared, the gene from suffer from carcinoma of prostate or by stages of prostate cancer determine
Carcinoma of prostate identified that the expression in the individuality of morbid state in detached biological sample is reduced.According to the present invention,
" down-regulated expression " refer to by the inventive method intensity for hybridization measure at least 1%, 2%, 3%, 4%, 5%, 6%, 7%,
8%th, 9%, 10% or more expression is reduced, and such as 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more
It is low.
1 high-flux sequence of embodiment screens difference expression gene
1st, sample
Tissue specimen is obtained in BJ Union Hospital's urological surgery during taking in October, 2012 in December, 2015
27, the equal Jing pathological examinations of all specimen confirm, wherein 8, cancer beside organism's sample, carcinoma of prostate specimen 19, after numbering
Put -80 DEG C of cryogenic refrigerators to preserve.
2nd, Total RNAs extraction is carried out to tissue samples
UsingReagent (invitrogen, article No. 15596-018) carries out sample rna extraction, experiment behaviour
Work is carried out by product description, and concrete operations are as follows：
After collecting sample, frozen tissue being put in the mortar of pre-cooling after liquid nitrogen, taking-up is ground, sample to be organized
After this is powdered：
1. Trizol, room temperature preservation 5 minutes are added；
2. the imitative 0.2mL of chlorination, uses forced oscillation centrifuge tube, fully mixes, and places -10 minutes 5 minutes under room temperature；
3. 12000rpm high speed centrifugations are drawn upper strata aqueous phase (inhaling 70%) and in another new centrifuge tube pipe, are noted after 15 minutes
The protein substance that should not be drawn onto between two-layer water phase.New pipe is moved into, isopyknic -20 DEG C of pre- cold isopropanols are added, it is fully reverse
Mix, be placed in 10 minutes on ice；
4. 12000rpm carefully discarded supernatant at a high speed after 15 minutes, added 75% in the ratio of 1mL/mL Trizol
DEPC ethanol washes paint precipitation (4 DEG C of preservations), washes paint precipitate, vibration mixing, 12000rpm high speed centrifugations 5 minutes at 4 DEG C；
5. ethanol liquid is discarded, places 5 minutes fully to dry precipitation under room temperature, add the water dissolution that DEPC was processed to sink
Form sediment；
6. RNA purity and concentration are measured with Nanodrop2000 ultraviolet spectrophotometers, it is frozen in -80 DEG C.RNA mass is sentenced
Calibration is accurate：The OD260/OD280 values of RNA samples are between 1.7-2.2；Total serum IgE electrophoresis pattern has clearly 28S, 18S band；
The 70 DEG C of electrophoresis patterns of water bath heat preservation after 1 hour and the collection of illustrative plates no significant difference before water bath heat preservation.
3rd, the quality analysiss of RNA sample
RNA extract after agarose gel electrophoresiies, from electrophoresis result can with preliminary judgement extract RNA sample it is up-to-standard with
It is no, if to can be used for further transcriptome analysis.And then RNA sample is detected by NanoDrop1000 spectrophotometers
Extraction situation, the sample requirement of RNA-Seq sequencings：OD260/OD280 is 1.8-2.2.
4th, high-flux sequence
Microarray dataset is the 2500 high-flux sequence platforms of HiSeq of Illumina companies, carries out high flux transcript profile depth
Sequencing, after sequencing, we use Fast-QC (http://www.bioinformatics.babraham.ac.uk/projects/
Fastqc/) software carries out total evaluation, including the quality Distribution value of base, the position point of mass value to the quality of sequencing data
Cloth, G/C content, PCR duplication contents, frequency of kmer etc..In differential genes expression analysis, according to obtaining
FPKM values, 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 difference expression gene has been carried out Gene Ontology and
Signal path is analyzed, and functional annotation and protein interaction analysis of network are carried out to difference expression gene, in view of above number
According to the result of analysis, with reference to document, we have screened differential expression C2CD4D, and the mRNA is lowered in expression in prostate cancer.
2 RT-PCR of embodiment verifies prostate cancer tissue and cancer beside organism's C2CD4D expressions
1st, material
During 34, prostate cancer tissue sample and 8, cancer beside organism's sample take from BJ Union Hospital 2012 to 2015 years
Carcinoma of prostate sample in urological surgery, is grouped to which and is numbered.All sample standard deviations confirm through pathological examination.
2nd, method
2.1 pairs of tissue samples carry out Total RNAs extraction, with the extracting method of embodiment 1.
2.2 reverse transcriptions synthesize cDNA
UsingIII Reverse Transcriptase (invitrogen, article No. 18080-044)
CDNA reverse transcriptions are carried out, experimental implementation is carried out by product description, and concrete operations are as follows：
Using Reverse Transcriptase kit, converse record synthesis cDNA is carried out with RT Buffer to l μ g total serum IgEs.Using 25 μ L
Reaction system, each sample take 1 μ g total serum IgEs as template ribonucleic acid.It is standby that -20 DEG C of refrigerators are put in the cDNA preservations of acquisition.
2.3 Real-Time PCR
2.3.1 instrument and analysis method
With 7500 type quantitative real time PCR Instruments of ABI, 2 are adopted-ΔΔCtMethod carries out data relative quantitative assay.
2.3.2 design of primers
Using online primer-design software, gene order is with reference to NCBI:NM_001136003.1 (C2CD4D), interior participation in the election
GAPDH, is synthesized by invitrogen companies after design of primers.Concrete primer sequence is as follows：
1 primer sequence of table
Operating process is as follows：
2 Real Time reaction systems of table
| Component | Addition |
| 2×mix | 10μL |
| Forward primer (10uM) | 0.5μL |
| Downstream primer (10uM) | 0.5μL |
| Template | 2μL |
| Add sterile purified water | To 25 μ L |
(1) reaction system：Use PowerGreen PCR Master Mix (invitrogen, article No.
4367659) expanded, experimental implementation is carried out by product description.
Amplification program is：95 DEG C of 5min, (95 DEG C of 15sec, 61 DEG C of 45sec) × 35 circulations.
(2) primer screening
After each sample cDNA is mixed, 5 times of gradient dilutions are carried out as template, after dilution, sample respectively takes 2 μ L and makees template,
Expanded with genes of interest primer and reference gene primer respectively, while melt curve analysis analysis is carried out at 60-95 DEG C, according to expansion
Increasing Efficiency is high and the unimodal principle of solubility curve carries out primer screening.
(3) sample Real Time-PCR detections
Make template by 2 μ L being taken after the 10 times of dilutions of each sample cDNA, entered with genes of interest primer and reference gene primer respectively
Row amplification.Solubility curve analysis is carried out at 60-95 DEG C simultaneously.
3rd, experimental result
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 higher；Sample amplified production is molten
Solution curve is all unimodal, illustrates that amplified production only has one, is specific amplification；According to the relative quantification formula of qRT-PCR：2-ΔΔCt× 100%, compare expressions of the C2CD4D in prostate cancer tissue and cancer beside organism.As a result show：QRT-PCR expands
Increase result to stablize, wherein C2CD4D suffers from 49% in the expression only cancer beside organism in tissue, above knot in carcinoma of prostate
Fruit demonstrates the result of confluence analysiss C2CD4D down-regulated expressions in patients with prostate cancer of high flux transcript profile expression data.
3 gene chip of embodiment verifies prostate cancer tissue and cancer beside organism's sample C2CD4D expressions
1st, the acquirement of material, with embodiment 2.
2nd, the extraction of total serum IgE, with the method for embodiment 1.
3rd, gene chip checking
After the linearized amplification of total serum IgE, cy3-UTP labellings, the cRNAs after fluorescent labeling adopt RNEASY Mini Kit
Purification, carries out fragmentation process to the good cRNAs of labelling with the RNA Fragmentation Reagents of Amhion.Using U.S.
People's full genome chip of expression spectrum (4x 44K genes) of Agilent companies of state, 65 DEG C of hybridization 17h in chip hybridization stove, then
Eluting, dyeing, finally with Agilent DNA MicroarrayScanner scanner scannings.
Chip after hybridization imports data to analysis software, for two groups of ratios Jing after chip scanner reads data point
Natural logrithm absolute value more than 2.0 or the mRNA less than 0.5 as differential expression mRNA.
Data analysiss are carried out using 13.0 statistical softwares of SPSS, group difference compares and adopts one-way analysis of variance method, P<
0.05 difference significance.As a result show, compared with cancer beside organism's sample, the mRNA of C2CD4D in prostate cancer tissue sample
Level is only 48%, and difference has statistical significance (P<0.05).
The structure of 4 carcinoma of prostate mouse model of embodiment
The foundation of carcinoma of prostate mouse model has various methods, such as spontaneous and induction model, xenogenesis implant cast, transgenic
Animal model, Gene Knock-Out Animal Model model etc..Correlational study shows, the cell suspension of PC-3 LNCaP is noted
Enter subcutaneous with mouse armpit.Experimental model of the human tumour graft of nude mice closest to human tumor self character, before being research
The preferable modeling method of row adenocarcinoma.
1st, material
Human prostate tumor LNCaP cell strains are purchased from ATTC companies of the U.S., SPF level nude mices, male, 3-5 week old, body weight
18g-23g, is provided by Concord Hospital's laboratory center.
2nd, method
Laboratory animal is divided into into 6 groups according to table is randomly assigned, 5 is per group, indicate experimental group respectively for Al, A2, A3,
Indicate that matched group is C1, C2, C3 respectively.
2.1 cell culture
In the RPMI1640 culture medium (Gibco) containing 10% hyclone, incubator temperature is protected for LNCaP cell culture
Hold 37 DEG C, 5%CO2Culture environment, changed liquid 1 time every 2 days, when cell is in exponential phase and cell concentration is about 5x107
Individual/mL is standby.
2.2 cell transplantation
The collection after the digestion of 0.25% pancreatin of the LNCaP cells of exponential phase, brine.Take a small amount of cell to enter
Row Trypan Blue detects its existence motility rate, and qualified then concentrating cells to its concentration are close to 1x109Individual/mL.It is anesthetized with ether
Nude mice, cuts off fur in its left and right sides oxter with eye scissorss, exposes skin.The concentrating cells of 300uL are drawn with 1mL syringes
At injection nude mice axillary fossa.Day by day transplantation site is observed to understand incubation period and the speed of growth of tumour growth.Al, A2, A3 group is noted
Entering cell selects liquor 0.3mL, C1, C2, C3 group to inject the normal saline solution of equivalent.
Sampling and process：At the 4th weekend (A1) after modeling, at the 6th weekend (A2), the 8th weekend (A3), de- neck put to death little
Mus, are dissected mice and separate prostate tumor tissue and normal prostate tissue, wiped away the blood taken out at specimen using filter paper, put
In PBS solution, all specimen are inserted into -80 DEG C of Refrigerator stores.
The protein expression level of C2CD4D in 5 WB methods of embodiment detection prostate cancer tissue
First, protein example is prepared and quantitative
1st, RIPA lysates (Beyotime) carry out protein example preparation, and operating procedure is as follows:
Tissue samples are taken out from refrigerator, structural PBS solution are wiped with filter paper, are weighed weight, and tissue is placed in
In mechanical tissue homogenizers, 1:10 tissues add the lysate of respective volume to be homogenized than the bulking value ratio of lysate,
10000-14000g is centrifuged 3-5 minutes, takes supernatant, by 1:1 addition sample buffer strength is placed in 100 degree of water-bath after mixing
Case heating in water bath 3-5 minutes, 10000g are centrifuged 10 minutes, take supernatant, are proceeded in the test tube of another cleaning.
2nd, it is quantitative total protein to be carried out using BCA determination of protein concentration test kit
Health is adopted for century micro BCA protein quantifications test kit (article No.：CW2011), concrete steps are shown in its description.
2nd, SDS- polyacrylamide gel electrophoresis (SDS-PAGE)
1st, protein example degeneration:
A) total protein extraction of phase homogenous quantities according to BCA determination of protein concentration results, is added in each gel well
Thing.According to the ratio that 0.25 microlitre of albumen sample-loading buffer is added per 1 microlitre of protein sample, mixed protein sample and albumen loading
Buffer (5x).
B) 100 DEG C or boiling water bath heating 3-5 minutes, with abundant Denatured protein.
C), after being cooled to room temperature, directly it is loaded in SDS-PAGE glue well.
2nd, prepared by offset plate:
The thick gels of 0.75mm are prepared using the miniature vertical plate electrophoresises device of Bio-Rad companies, book is installed as directed
After glass plate, the separation gel of 5mL 10% is first prepared in small beaker, and formula is as follows：
3 separation gel formula of table
Then plus l mL distilled water is covered encapsulating immediately after mixing, places about 30min after glue polymerization, with steaming under room temperature
Distilled water is washed 2-3 time, then is blotted with filter paper.Then the concentration glue of 2mL 5% is prepared, formula is as follows：
Table 4 concentrates glue formula
| Component | Consumption |
| 30% acrylamide solution | 0.33mL |
| Tris-HCl (1.0M, pH6.8) | 0.25mL |
| 10%SDS | 0.02mL |
| 10%AP | 0.02mL |
| TEMED | 0.002mL |
| Sterilizing ddH2O | It is supplemented to 2mL |
Encapsulating immediately after mixing, inserts sample comb, it is to avoid produce bubble, after gelling is solid, takes out sample comb, afterwards with distillation
Water and 1x protein electrophoresises buffer successively rinse sample well.
3rd, loading and electrophoresis
Gel slab is mounted on electrophoretic apparatuss, lx protein electrophoresises buffer in inside groove, is filled it up with, lx protein electrophoresises delay in water jacket
Rush liquid and should exceed platinum filament, in order loading.Protein quality standard protein gradient is added in the swimming lane of end.Blue dye during electrophoresis
The bottom end expected up to glue stops electrophoresis by nearby.
4th, Western blotting
1st, PAGE gel electrophoretic separation albumen is first carried out according to the method described above.
2nd, in advance with transfer buffer immersion NC films, filter paper, foam rubber cushion.SDS-PAGE takes out gel after terminating, remove dense
Contracting glue, rinses the several seconds in Tris/ glycine buffers, soaks 15-30min in being subsequently placed in transfer buffer.Open electricity to turn
Print folder, special per side pad lastblock soak saturating foam rubber cushion with transfer buffer, then respectively put one piece of saturating filter paper of transfer immersion,
Filter paper it is identical with foam rubber cushion size or with NC films, gel size is identical, and gel is lain on cathode side filter paper, finally will
NC films are lain on gel, remove bubble removing, clip electricity transfer folder.Electricity transfer liquid, insertion electricity transfer folder, by electricity are filled it up with electrophoresis tank
Swimming groove is put in refrigerator (will be put into pre-cooling in refrigerator before electricity transfer liquid), connects electrode, and turn-on current transfers the NC films of folder
The positive pole of reply electrophoresis tank.
3rd, close：Rinsed once with 1xTBS.Add containing 5% alipoidic milk power TBS Block buffers, be placed in shaken cultivation
Closed in case；
4th, an anti-hybridization：Confining liquid is abandoned, is added and is resisted (Anti-C2CD4D antibody with the one of an anti-diluted
(ab139105)) hybridization solution, is placed in 4 DEG C of hybridized overnights, is hybridized within second day in shaken cultivation case；
5th, an anti-hybridization solution is reclaimed, film 3 times is washed with TBST；
6th, TBST, two anti-(Goat Anti-Rabbit IgG, the HRP that addition Block buffer dilutes are abandoned
Conjugated (CW0103)) hybridization solution, it is placed in shaken cultivation case and is hybridized；
7th, two corresponding anti-solution is abandoned, and film 3 times is washed with TBST；
8th, ECL chemiluminescences and image acquisition and analysis：According to high sensitivity chemistry luminescence detection kit, (health is century
Article No. CW0049B), concrete steps are with reference to description.
9th, data normalization is carried out as internal reference using β-Actin, the C2CD4D using in cancer beside organism is calculated as sample for reference
The relative expression levels of C2CD4D albumen in experimental group.
5th, experimental result
A1 groups and C1 groups are put to death within the 4th week after modeling respectively, put to death within the 6th week A2 groups and C2 groups, are put to death A3 groups and C3 within the 8th week
The sample detection result that group is obtained shows that experimental group A1, A2, A3 group is compared with matched group C1, C2 with C3 groups, C2CD4D albumen tables
Up to substantially reduction (P<0.01), in matched group C1, C2 and C3 groups C2CD4D protein expressions without significant difference, but experimental group A1, A2,
In A3 groups, C2CD4D protein expression differences are notable, wherein A1>A2>A3(P<0.01).Referring specifically to shown in Fig. 1.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Ye Weiliang
<120>A kind of molecular marked compound of detection carcinoma of prostate and its application
<130> 1642
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence
<400> 1
actctgcctt ggagctattg g 21
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
ccacttcttt ggtgctcggt 20
<210> 3
<211> 21
<212> DNA
<213>Artificial sequence
<400> 3
ggagcgagat ccctccaaaa t 21
<210> 4
<211> 23
<212> DNA
<213>Artificial sequence
<400> 4
ggctgttgtc atacttctca tgg 23
<210> 5
<211> 1713
<212> DNA
<213>Artificial sequence
<400> 5
agaataatgc agacaactga tcactgcaag tcactctgcc ttggagctat tggaatggct 60
ccagaacagg gcactggggg ccaaggcagg atttcaacca ggagggtttt acagaagatg 120
gacttcacct gtgagggctg gggttggaac cgagcaccaa agaagtggcc actgtccact 180
gatggccgtc tccctcttct ctgtagccct cccccacccc taagcagccc agcttcagca 240
gaggaggcct gggcacagga atggattgtt gctagaaacc tggcttcagc tggaggaagt 300
cagaagaccg ggctgatggc cgcgcagaca gagctgtgtt tatccaggaa aagagccccc 360
cagactgcgc cgccctttct ttttctgctc aaataggggt ccgcgctccc agtgggtccg 420
gagaccgggg cgcggtggct gaccgcccgg tgtacgccag catcactcac ctctgactcc 480
tctgcgctcc tcgcccagcc ccgctccgcc gagcactccc atctgagacg ggatttggac 540
cctctcggcc ctgcggaatt cggaattcca ttaccccacc caccgcatgt ggctcttgga 600
aaaagctggc tataaggtgg gggccgcgga gcctgcggcc cgttgggcgc cttccggcct 660
gttctccaag cgtcgcgccc cgggcccgcc cacaagcgcc tgccccaacg tcctcacccc 720
ggatcgcatc ccgcagttct tcatcccgcc tcggctcccg gacccgggcg gcgcagtgcc 780
cgcggcccgg cggcacgtgg cggggcgcgg cctccccgcg acctgctcgc tgcctcacct 840
ggcgggccgc gaaggctggg ccttcctgcc cgagagcccg cacacgcgcc ggcgggaatc 900
cctgttccac gggccgccac ctgccccggc cgggggactc cccgcggcgc agtcccggct 960
gcacgtctcc gccccggacc tgcgcctctg ccgggccccc gacagcgaca cggcctcgtc 1020
gccggactcg tcgcccttcg gctccccgcg gccaggcctg ggccggcgcc gggtgtccag 1080
gcctcactct ctgtccccag aaaaagcgag ctcggccgat accagcccgc actcgccgcg 1140
ccgcgccggg ccgcccacgc cgccgctctt ccacctggac ttcctgtgct gccagctgcg 1200
gcccacgcgc gagagcgtgc tgcgcctggg gccccgcggc gggcagctgc ggctctccac 1260
cgaatatcag gccgggcccg ggcggctgcg gctgcgccta gtgagcgccg agggcctgcc 1320
ccggccgcgg tcccgccccg ggagcggcgg cggcggctgc tgtgtggtgc tgaggctgcg 1380
gccccgcgtc cggccgcggg agcagcagag ccgcgtggtc aagtgcagcg ccaaccccat 1440
cttcaacgag gatttctttt tcgacgggct cggccccccg gacctggccg cccgcagcct 1500
gagagccaag gtgctagaca ggggcgcggg acttcgcagg gatgtgctgc tgggggagtg 1560
cgagacgccc ctcattgcgc tgctgccccc gctgggtggg ggactaggtc ccgggtcatc 1620
cctggcgccc acccatctca gcctgtagcc tgagcccctg gcttcctcag gacgtctcca 1680
ctgtgtctgc agtccacatt ctttccaccc tgc 1713
Claims (9)
1. it is a kind of detection carcinoma of prostate molecular marked compound, it is characterised in that the molecular marked compound be the C2CD4D or mRNA
Expressing protein and its fragment, analogs and derivatives.
2. molecular marked compound as claimed in claim 1, it is characterised in that the expression product of the described C2CD4D or mRNA exists
Expression in prostate cancer is lowered.
3. a kind of method that whether detection carcinoma of prostate correlation mRNA expressions are lowered, it is characterised in which includes following step
Suddenly:
(1) expression of carcinoma of prostate correlation mRNA in testing sample is detected, wherein carcinoma of prostate correlation mRNA is
C2CD4D；
(2) expression of carcinoma of prostate correlation mRNA in cell to be measured is compared with a control, so that it is determined that carcinoma of prostate table
Whether lower up to level.
4. method as claimed in claim 3, it is characterised in that the testing sample is cell tissue sample.
5. method as claimed in claim 3, it is characterised in that the detection is by nucleic acid chip detection method, fluorescent quantitation
PCR, protein chip detection method, immunization method or Ag-Ab detection method are carried out.
6. a kind of prostatic cancer diagnostic reagent kit, it is characterised in that the test kit includes that specific amplification carcinoma of prostate is related
The primer and description of mRNA, the carcinoma of prostate correlation mRNA is C2CD4D.
7. test kit as claimed in claim 6, it is characterised in that the primer has SEQ ID NO:1 and SEQ ID NO:2
Shown primer.
8. a kind of prostatic cancer diagnostic reagent kit, it is characterised in that the test kit include detecting relating to prostate cancers because
Chip and description, the chip are selected from the group：
(1) there is the point sample DNA in the substrate and point sample point sample area on the substrate, the point sample DNA to include for DNA chip, the chip
Relating to prostate cancers is specifically bound to because of the probe of transcript, the relating to prostate cancers is because being C2CD4D；
(2) protein chip, the chip have the deposited protein in the substrate and point sample point sample area on the substrate, the deposited protein
Including relating to prostate cancers is specifically bound to because of the antibody of expression product, the relating to prostate cancers is because being C2CD4D.
9. a kind of preparation for treating carcinoma of prostate, it is characterised in that in the preparation containing the reagent for promoting C2CD4D expression and
Promote the reagent of C2CD4D expression products.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610962167.5A CN106544430A (en) | 2016-11-04 | 2016-11-04 | A kind of molecular marked compound of detection carcinoma of prostate and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610962167.5A CN106544430A (en) | 2016-11-04 | 2016-11-04 | A kind of molecular marked compound of detection carcinoma of prostate and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106544430A true CN106544430A (en) | 2017-03-29 |
Family
ID=58395154
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610962167.5A Withdrawn CN106544430A (en) | 2016-11-04 | 2016-11-04 | A kind of molecular marked compound of detection carcinoma of prostate and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106544430A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020045683A1 (en) * | 2018-09-01 | 2020-03-05 | 国立大学法人北海道大学 | Medicine for preventing and/or treating stress load-related disease |
| US11733232B2 (en) | 2017-03-01 | 2023-08-22 | National University Corporation Hokkaido University | Method for producing disease modeling non-human animal, disease modeling non-human animal, and method for screening drug and method for determining risk of disease using the same |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102549167A (en) * | 2009-05-12 | 2012-07-04 | 皇家飞利浦电子股份有限公司 | Phosphodiesterase 4d7 as prostate cancer marker |
| CN105316340A (en) * | 2015-09-16 | 2016-02-10 | 复旦大学 | MIR27A-3P serving as prostate cancer molecular marker and application of same to diagnostic reagent kit |
| CN105316341A (en) * | 2015-12-08 | 2016-02-10 | 浙江理工大学 | LncRNA and application thereof as prostatic cancer detection marker or prostatic cancer prognosis recurrence marker |
| CN105658810A (en) * | 2013-06-13 | 2016-06-08 | 南澳大学 | Methods for detecting prostate cancer |
-
2016
- 2016-11-04 CN CN201610962167.5A patent/CN106544430A/en not_active Withdrawn
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102549167A (en) * | 2009-05-12 | 2012-07-04 | 皇家飞利浦电子股份有限公司 | Phosphodiesterase 4d7 as prostate cancer marker |
| CN105658810A (en) * | 2013-06-13 | 2016-06-08 | 南澳大学 | Methods for detecting prostate cancer |
| CN105316340A (en) * | 2015-09-16 | 2016-02-10 | 复旦大学 | MIR27A-3P serving as prostate cancer molecular marker and application of same to diagnostic reagent kit |
| CN105316341A (en) * | 2015-12-08 | 2016-02-10 | 浙江理工大学 | LncRNA and application thereof as prostatic cancer detection marker or prostatic cancer prognosis recurrence marker |
Non-Patent Citations (1)
| Title |
|---|
| 刘陶迪: "大鼠青春期前期精原干细胞自我更新和分化相关基因表达的初步研究", 《内蒙古大学-学位论文》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11733232B2 (en) | 2017-03-01 | 2023-08-22 | National University Corporation Hokkaido University | Method for producing disease modeling non-human animal, disease modeling non-human animal, and method for screening drug and method for determining risk of disease using the same |
| WO2020045683A1 (en) * | 2018-09-01 | 2020-03-05 | 国立大学法人北海道大学 | Medicine for preventing and/or treating stress load-related disease |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106755344A (en) | Molecular marked compound and its application for the diagnosis of cancer of pancreas clinical prognosis | |
| CN104711341B (en) | DLK1 gene is preparing the application in gastrointestinal stromal tumor diagnostic reagent | |
| CN109402252A (en) | Acute myeloid leukemia risk assessment gene marker and its application | |
| CN106544430A (en) | A kind of molecular marked compound of detection carcinoma of prostate and its application | |
| CN109022583A (en) | Hsa_circ_0021977 is preparing the application on Diagnosis of Breast cancer product | |
| CN101768214A (en) | Human tumor marker-Tim17 polypeptide and application thereof | |
| CN106755309A (en) | Application of the molecular marked compound in cancer of pancreas prognosis evaluation product is prepared | |
| CN109182528A (en) | A kind of glioblastoma auxiliary diagnosis based on ITGB5 gene, prognostic evaluation kit and its application method | |
| CN106148562A (en) | For detecting the label of carcinoma of prostate | |
| CN106367526A (en) | Product for diagnosing prostatic cancer and application thereof | |
| CN106282366A (en) | A kind of molecular marked compound relevant to carcinoma of prostate and application thereof | |
| CN108034713A (en) | Postmenopausal Osteoporosis diagnosis and treatment target spot and its application | |
| CN106148554A (en) | A kind of mark for carcinoma of prostate detection and the application in test kit thereof | |
| CN106148556A (en) | Application in prostate cancer diagnosis mark for the C22orf41 | |
| CN106498062A (en) | A kind of product of diagnosis of prostate cancer and its application | |
| CN106498051B (en) | Application of the ZNF800 gene in preparation osteoporosis early screening product | |
| CN106399509A (en) | Product for diagnosing prostate cancer and use thereof | |
| CN107881240B (en) | The diagnosis and treatment marker of osteosarcoma | |
| CN108085386B (en) | The identification of the reference gene of osteosarcoma miRNA detection | |
| CN106148563A (en) | The biomarker of carcinoma of prostate | |
| CN105950714B (en) | It is a kind of diagnose osteoarthritis product and its application | |
| CN105886628B (en) | Application of the SPRR1A gene in preparation osteoarthritis diagnostic products | |
| CN106282374A (en) | FAM13AOS is as molecular marked compound and the application thereof detecting carcinoma of prostate | |
| CN109439760A (en) | Application of the ARRDC2 in assessment oral squamous cell carcinomas development process | |
| CN106148561A (en) | The diagnosis and treatment label of carcinoma of prostate |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20170329 |
|
| WW01 | Invention patent application withdrawn after publication |