CN106947820A - Purposes of the VCAN in adenocarcinoma of colon diagnosis and treatment - Google Patents

Purposes of the VCAN in adenocarcinoma of colon diagnosis and treatment Download PDF

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CN106947820A
CN106947820A CN201710232967.6A CN201710232967A CN106947820A CN 106947820 A CN106947820 A CN 106947820A CN 201710232967 A CN201710232967 A CN 201710232967A CN 106947820 A CN106947820 A CN 106947820A
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vcan
colon
adenocarcinoma
medicine
product
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CN106947820B (en
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杨承刚
董东
杜海威
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Qingdao Yangshen Biomedical Co Ltd
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Beijing Medintell Bioinformatic Technology Co Ltd
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Abstract

The invention discloses the molecular marker that VCAN genes can be diagnosed as adenocarcinoma of colon, the experiment proves that：Compared with normal structure, VCAN genes expression quantity in adenocarcinoma of colon tissue is low.It can be used for the medicine for preparing treatment adenocarcinoma of colon the invention also discloses VCAN genes.The achievement in research of the present invention provides a kind of new adenocarcinoma of colon methods for clinical diagnosis, while there is provided a kind of medicine new target drone for treating adenocarcinoma of colon.

Description

Purposes of the VCAN in adenocarcinoma of colon diagnosis and treatment
Technical field
The present invention relates to diagnosing tumor, treatment, prediction prognosis field, more particularly it relates to detect that VCAN is different Often it is the diagnosing tumor of means, predicts method of prognosis；And the tumor therapeutic agent of activation VCAN genes or protein.
Background technology
Adenocarcinoma of colon is one of most common malignant tumour of the mankind, and in recent years, its incidence of disease gradually rises, and accounts for all pernicious The second of tumour.
The generation of adenocarcinoma of colon, development is a complex process for being related to polygenes change.Its pathogenic process and cell point Split, break up, the interaction of the spatial and temporal expression exception of gene group and its protein it is relevant.Identification is related to adenocarcinoma of colon morbidity Oncogene or tumor suppressor gene, to research adenocarcinoma of colon Carcinogenesis Mechanism and its diagnosis, treat and prevent it is significant.
The content of the invention
An object of the present invention is to provide one kind to diagnose colon by detecting VCAN genes or protein expression difference The method of gland cancer.
The second object of the present invention is to provide one kind to predict colon by detecting VCAN genes or protein expression difference The method of gland cancer prognosis.
The third object of the present invention is to provide one kind to treat adenocarcinoma of colon by activating VCAN genes or VCAN albumen Method.
The fourth object of the present invention is to provide a kind of method for being used to screen the medicine for the treatment of adenocarcinoma of colon.
The fifth object of the present invention is to provide a kind of medicine for being used to treat adenocarcinoma of colon.
To achieve these goals, present invention employs following technical scheme：
The invention provides application of the detection VCAN product in adenocarcinoma of colon diagnostic tool is prepared.
Further, the product of the detection VCAN includes the product of detection VCAN gene expression amounts.
Further, the product of the detection VCAN includes that the product of VCAN gene mRNAs can be quantified, and/or can determine Measure the product of VCAN albumen.
The product of the quantitative VCAN gene mRNAs of the present invention can be based on playing its work(using the known method of nucleic acid molecules Energy：Such as PCR, such as Southern hybridization, Northern hybridization, dot blot, FISH (FISH), DNA microarray, ASO Method, high-flux sequence platform etc..It can qualitatively, quantitatively or semi-quantitatively implement analysis using the product.
Nucleic acid included in the said goods can be obtained by chemical synthesis, or be contained by being prepared from biomaterial Expect the gene of nucleic acid, then expand it to obtain using the primer for expecting nucleic acid designed for amplification.
Further, the PCR method is known method, for example, ARMS (Amplification Refractory Abruptly-changing system is not answered in Mutation System, amplification) method, RT-PCR (reverse transcriptase-PCR) method, nesting PCR methods etc..Amplification Nucleic acid can be by using dot blotting hybridization method, Surface Plasmon Resonance (SPR methods), PCR-RFLP methods, original position RT-PCR Method, PCR-SSO (sequence specific oligonucleotide) method, PCR-SSP methods, AMPFLP (amplifiable fragment length polymorphism) method, MVR-PCR methods and PCR-SSCP (single-strand conformation polymorphism) methods are detected.
The product that VCAN gene mRNAs can be quantified includes the specific amplified VCAN genes used in real-time quantitative PCR Primer, the primer sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.
The primer that product includes can by being prepared by chemical synthesis, by using those skilled in the art will know that Method be suitably designed with reference to Given information, and prepared by chemical synthesis.
Nucleic acid recited above may also include probe, and the probe can be prepared by chemical synthesis, by using this The method that art personnel know appropriately is designed with reference to Given information, and is prepared by chemical synthesis, or can be led to Cross and prepared from biomaterial containing the gene for expecting nucleotide sequence, and expanded using the primer that nucleotide sequence is expected designed for amplification Increase it to prepare.
The product of the quantitative VCAN albumen of the present invention can be based on playing its function using the known method of antibody：For example, ELISA, radioimmunoassay, immunohistochemical method, western blot etc. can be included.
The product of the quantitative VCAN albumen of the present invention includes the antibody or its fragment of specific binding VCAN albumen.It can make With the antibody or its fragment of any structure, size, immunoglobulin class, origin etc., as long as it combines target protein.This The antibody or its fragment that the detection product of invention includes can be monoclonals or polyclonal.Antibody fragment refers to reservation antibody Antibody a part of (Partial Fragment) to the binding activity of antigen or the peptide containing an antibody part.Antibody fragment can include F (ab′)2, Fab ', Fab, scFv (scFv), the Fv (dsFv) of disulphide bonding or its polymer, dimerization V areas it is (dual anti- Body) or peptide containing CDR.The product of the quantitative VCAN albumen of the present invention can include encoding antibody or Encoding Antibody Fragment The nucleic acid of the separation of amino acid sequence, the carrier comprising the nucleic acid, and carry the cell of the carrier.
Antibody can be by obtaining well known to a person skilled in the art method.Retain target all or in part for example, preparing The mammalian cell expression vector that the polypeptide of protein or integration encode their polynucleotides is used as antigen.Exempted from using antigen After epidemic disease animal, from by immune animal adaptive immune cell and merging myeloma cell to obtain hybridoma.Then from hybridization Knurl culture collects antibody.The antibody of acquisition can finally be implemented by using VCAN albumen for being used as antigen or part thereof Antigentic specificity purifies to obtain the monoclonal antibody for VCAN albumen.Polyclonal antibody can be prepared as follows：With with above Identical antigen-immunized animal, collects blood sample from by immune animal, serum is isolated from blood, then using upper State antigen and antigentic specificity purifying is implemented to serum.The antibody or the antibody by using acquisition that can be obtained by using ferment treatment Sequence information obtain antibody fragment.
The combination of label and antibody or its fragment can be implemented by method as commonly known in the art.For example, can With following fluorescent marker protein or peptide：Clean protein or peptide with phosphate buffer, addition DMSO, buffer, etc. standard Standby dyestuff, then mixed solution, is placed 10 minutes then at room temperature.In addition, the labelling kit of commercialization can be used in mark, it is all Such as biotin labeling reagent box, such as biotin labeling reagent box-NH2, biotin labeling reagent box-SH (DojindoLaboratories)；Alkali phosphatase enzyme mark kit such as alkali phosphatase enzyme mark kit-NH2, alkaline phosphorus Sour enzyme labelling kit-SH (Dojindo Laboratories)；Peroxidase labelling kit such as peroxidase mark Remember kit-NH2, peroxidase labelling kit-NH2 (Dojindo Laboratories)；Phycobniliprotein labelled reagent Box such as phycobniliprotein labelling kit-NH2, phycobniliprotein labelling kit-SH, B- phycoerythrin labelling kit-NH2, B- phycoerythrin labelling kits-SH, R-PE labelling kit-NH2, R-PE labelling kit SH (DojindoLaboratories)；Fluorescent labeling reagent box such as fluorescein labelling kit-NH2, HiLyte Fluor (TM) 555 labelling kit-NH2, the labelling kit-NH2 of HiLyte Fluor (TM) 647 (Dojindo Laboratories)；And DyLight 547 and DyLight647 (Techno Chemical Corp.), Zenon (TM), Alexa Fluor (TM) antibody Labelling kit, Qdot (TM) antibody labeling kit (Invitrogen Corporation) and EZ- label protein marks Remember kit (Funakoshi Corporation).For correct labeling, suitable instrument can be used to detect by mark Antibody or its fragment.
As the sample of the detection product according to the present invention, the tissue sample for example obtained from biopsy subject can be used Or fluid.Sample is not particularly limited, as long as it is suitable to the measure of the present invention；For example, it can include tissue, blood, blood plasma, Serum, lymph, urine, serous cavity liquid, spinal fluid, synovia, aqueous humor, tear, saliva or its fraction or treated material Material.
In specific embodiments of the present invention, tissue of the sample from subject.
Further, the product of the quantitative VCAN genes or VCAN albumen can be detection VCAN genes or VCAN albumen Reagent, it can also be kit, chip, test paper etc. comprising the reagent or be measured using the high pass of the reagent Sequence platform.
Present invention also offers a kind of instrument for diagnosing adenocarcinoma of colon, the instrument can detect VCAN gene expression amounts.
Further, the instrument includes that the reagent of VCAN gene mRNAs can be quantified, and/or can quantify VCAN eggs White reagent.
Further, the reagent that can quantify VCAN gene mRNAs is the specific amplified used in real-time quantitative PCR The primer of VCAN genes, the primer sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.
Further, the instrument of the diagnosis adenocarcinoma of colon includes but is not limited to chip, kit, test paper or high pass measurement Sequence platform；High-flux sequence platform is a kind of instrument of special diagnosis adenocarcinoma of colon, with the development of high throughput sequencing technologies, The structure of the gene expression profile of one people will be turned into and very easily worked.By the base for contrasting Disease and normal population Because of express spectra, the exception for easily analyzing which gene is related to disease.Therefore, VCAN genes are known in high-flux sequence The abnormal purposes for falling within VCAN related to adenocarcinoma of colon, equally within protection scope of the present invention.
The number for the amino acid that the detection product of the present invention, the anti-VCAN antibody or its fragment that use in diagnostic tool are recognized Mesh is not particularly limited, as long as antibody can combine VCAN.
Present invention also offers a kind of method for diagnosing adenocarcinoma of colon, methods described comprises the following steps：
(1) sample of subject is obtained；
(2) expression of VCAN genes or albumen in Samples subjects is detected；
(3) the VCAN genes measured or the expression of albumen are associated with the whether ill of subject.
(4) compared with the control, the expression reduction of VCAN genes or albumen, then the subject is judged with colon gland Cancer is judged as recurrence or adenocarcinoma of colon patient's quilt with the risk with adenocarcinoma of colon or adenocarcinoma of colon patient It is judged as prognosis mala.
Present invention also offers a kind for the treatment of method of adenocarcinoma of colon, methods described includes activation VCAN genes or VCAN eggs In vain.
Further, methods described includes promoting the expression of VCAN genes, or promotes expression or the enhancing VCAN of VCAN albumen The activity of albumen.
, can be by after testing drug be added to cancer cell present invention also offers a kind of screening technique of tumour medicine Or some period after testing drug is applied to tumor model animal measures the expression of VCAN genes or VCAN albumen Improve the effect of tumor prognosis to determine tumour medicine.More specifically, when VCAN genes or the expression of VCAN albumen When being raised after addition or administration testing drug or when recovering normal level, the medicine may be selected and is used as improvement tumor prognosis Medicine.
Present invention also offers a kind of medicine for treating adenocarcinoma of colon, the medicine includes VCAN activator.
The VCAN of invention activator is unrestricted, as long as the activator can promote or strengthen VCAN or be related to VCAN The expression of the material of upstream or downstream pathway or activity, and for the treatment effective medicine of tumour.
Present invention also offers application of the above-mentioned activator in the medicine for preparing treatment adenocarcinoma of colon.
Further, the activator include VCAN genes, VCAN albumen, promoted type miRNA, promoted type transcriptional control because Son or promoted type targeting micromolecular compound.
On the one hand the activator of the present invention can be used for the missing or deficiency for supplementing endogenic VCAN albumen, by improving The expression of VCAN albumen, so as to treat the adenocarcinoma of colon caused by VCAN hypoproteinosis.On the other hand it can be used for strengthening VCAN The activity of albumen, so as to treat adenocarcinoma of colon.
The medicine of the present invention can be administered alone as medicine or be applied together with other medicines.Can be with medicine of the invention The other medicines that thing is applied together are unrestricted, as long as it does not damage therapeutic or preventive medicine the effect of the present invention i.e. Can, it is preferred that the medicine for treating or preventing tumour can include such as alkylating agent, such as ifosfamide, ring phosphinylidyne Amine, Dacarbazine, Temozolomide, Nimustine, busulfan, procarbazine, melphalan and Ranimustine；Antimetabolite, such as Enocitabine, capecitabine, Carmofur, Cladribine, gemcitabine, cytarabine, cytarabine octadecyl phosphate (cytarabine ocfosfate), Tegafur, UFT, Tegafur gimeracil oteracil potassium, deoxidation fluorine urine Glycosides, hydroxycarbamide, fluorouracil, fludarabine, pemetrexed, Pentostatin, mercaptopurine and methotrexate (MTX)；Plant alkaloid, it is all As Irinotecan, Etoposide, Sobuzoxane, docetaxel, nogitecan, Palmer altruism, vinorelbine, eldisine and Vincaleukoblastinum；Antitumor antibiotic, such as actinomycin D, Aclarubicin, Amrubicin, idarubicin, epirubicin, Zinostatin Stimalamer, daunorubicin, Doxorubicin, THP, bleomycin, Peplomycin, mitomycin C and mitoxantrone； Medicine based on platinum, such as oxaliplatin, carboplatin, cis-platinum and Nedaplatin；Hormonal medicaments, such as Anastrozole, Exemestane, Estramustine, ethinyloestradiol, chlormadinone, Goserelin, TAM, dexamethasone, Toremifene, Bicalutamide, Flutamide, Prednisolone, Fosfestrol, mitotane, methyltestosterone, Medroxyprogesterone, Mepitiostane, Leuprorelin and Letrozole；Biological respinse is modified Agent, such as interferon-' alpha ', interferon beta, interferon gamma, interleukin, ubenimex, dry BCG and lentinan；With molecular targeted medicine Thing, such as Imatinib (imatinib), Gefitinib (gefitinib), gemtuzumab, ozogamicin, Tamibarotene, song Appropriate monoclonal antibody, Tretinoin, bortezomib (bortezomib) and Rituximab etc..
The medicine of the present invention can be prepared into various formulations as needed.Including but not limited to, percutaneous, mucous membrane, nose, buccal, Tablet, solution, granule, patch, paste, capsule, aerosol or suppository sublingual or orally use.
The route of administration of the medicine of the present invention is unrestricted, as long as it can play desired therapeutic effect or preventive effect i.e. Can, including but not limited to intravenous, intraperitoneal, intraocular, intra-arterial, intrapulmonary, orally, and in vesicle, intramuscular, tracheal strips, subcutaneously , local by pleura by skin, suction, by mucous membrane, skin, stomach is intra-articular, intra-ventricle, rectum, vagina, In skull, in urethra, in liver, in knurl.In some cases, can systematically it be administered.It is to be partly administered in some cases.
The dosage of the medicine of the present invention is unrestricted, can as long as obtaining desired therapeutic effect or preventive effect To carry out appropriate determination according to symptom, sex, age etc..The medicine of the present invention or the dosage of prophylactic agent can be with use examples Determined such as to the therapeutic effect or preventive effect of disease as index.
In the context of the present invention, " diagnosis adenocarcinoma of colon " include judge subject whether suffered from adenocarcinoma of colon, Judge that subject whether there is the risk with adenocarcinoma of colon, judge whether adenocarcinoma of colon patient has been recurred and shifted, judged Prognosis situation reactive or that judge adenocarcinoma of colon patient of the adenocarcinoma of colon patient to drug therapy.
" treatment " used herein is covered treatment-related in such as mankind of the mammal with relevant disease or illness Disease or morbid state, and including：
(1) prevention disease or morbid state occur in mammal, especially when the mammal is susceptible in the disease Diseased state, but when being not yet diagnosed with this morbid state；
(2) suppress disease or morbid state, that is, prevent it from occurring；Or
(3) disease or morbid state are alleviated, even if disease or morbid state disappear.
Term " treatment " is usually directed to the treatment mankind or animal (for example, being applied by animal doctor), wherein can reach some pre- The therapeutic effect of phase, for example, suppressing the development (including reduce development speed, stop development) of illness, improving illness and healing Illness.Also include the treatment as precautionary measures (such as preventing).Pair illness is not developed into also but develop into illness danger The purposes of the patient of danger, is also included within term " treatment ".
The advantages of the present invention：
The present invention's is found that a kind of molecular marker for diagnosing adenocarcinoma of colon, can be in colon using the molecular marker The early stage that gland cancer occurs can be used as judging the survival rate there is provided patient.
The medicine of the activator for including VCAN genes or albumen of the present invention can be used as the treatment of new adenocarcinoma of colon Medicine.
Brief description of the drawings
Fig. 1 displays detect differential expression of the VCAN genes in adenocarcinoma of colon tissue and normal structure using QPCR；
Difference of Fig. 2 displays using Western blot experiment detection VCAN genes in adenocarcinoma of colon tissue and normal structure Different expression；
Fig. 3 displays detect the overexpression efficiency of VCAN genes using Western blot experiments；
Fig. 4 displays detect the influence that VCAN gene expressions are bred to colon adenocarcinoma cell using mtt assay；
Influence of Fig. 5 displays using flow cytomery VCAN gene expressions to colon adenocarcinoma cell apoptosis.
Specific embodiment
The present invention is further detailed explanation with reference to the accompanying drawings and examples.Following examples are merely to illustrate this Invention rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in embodiment, generally according to conventional strip Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) described in condition, or according to the condition proposed by manufacturer.
Embodiment 1 screens difference expression gene
1st, experiment material
The lesion resection tissue specimen of hospital's row operative treatment and 10 complete colorectal cancer patients of medical history data is selected, oneself Through being diagnosed as adenocarcinoma of colon, male 5, female 5 through pathology department；Formulated according to by american cancer joint committee (AJCC, 2009) Stages of colon and rectal cancer standard：I-II phases 6, III-IV phases 4.Histological grade:Differentiated 3, middle differentiation 4 is low Differentiation 3.Preoperative equal footline radiotherapy, chemotherapy and biological agent treatment.Normal group is woven to normal distal colon mucosa tissue 10, it is all from healthy colonoscopy examinee.
2nd, RNA is extracted, cDNA is synthesized
, then will according to Reverse Transcriptase kit specification according to Trizol specifications operation extracting histocyte total serum IgE MRNA reverse transcriptions are cDNA.
3rd, biotin labeling cRNA hybridizes
Using cDNA as template, using MessageAmpTM II-Biotin Arna Amplification Kit (Ambion Company) in-vitro transcription synthesizing biotinylated mark cRNA, after purification by the eucaryote express spectra single-wheel core of Affymetrix companies Piece amplification program adds 5 × fragmentation buffer and obtains fragmentation cRNA of the size distribution in 35~200nt；Prepared by target completes Afterwards, using eucaryote Hybridization Control Kit (Affymetrix companies) preparing hybrid liquid, hybridization solution is injected Chip balance be positioned in hybrid heater, 45 DEG C, 60r/min rotation hybridization 16h (Hybridization Oven 640, Affymetrix companies), then Affymetrix companies provide washing work station in (Fluidics Station 450, Affymetrix companies) complete chip cleaning dyeing；
4th, scan and analyze
UsingScanner 3000 (Affymetrix companies) scanner scanning image.Scan image is first WithOperating Software Version1.4 (GCOS 1.4, Affymetrix company) software is schemed Conversion as arriving signal value, is converted into raw data file.Then the invariant set in software dChip 2006 are used Normalization methods and Model-base ExpressionIndex models are further counted to GCOS output results According to analysis.According to the P values detected in each chip, as detected value Call values (the Absolute Call, Abs of data set When Call) in the absence of A (Absent) or critical value M (Marginal), it is considered as not expressing, only Abs Call values are to deposit Just it is used for further analysis in P (present) data set.
5th, the screening of histological difference expressing gene
The screening of difference expression gene utilizes Significant Analysis of Microarray Software (SAM) algorithm is carried out.
6th, result
Chip filters out 485 difference expression genes altogether.Compared with normal structure, up-regulated expression in adenocarcinoma of colon tissue Gene is 289, and the gene that expression is lowered is 196.
Checking of the difference expression gene of embodiment 2 in large sample
1st, research object
The lesion resection tissue specimen of hospital's row operative treatment and 50 complete colorectal cancer patients of medical history data is selected, oneself Through being diagnosed as adenocarcinoma of colon, male 20, female 30 through pathology department；Made according to by american cancer joint committee (AJCC, 2009) Fixed stages of colon and rectal cancer standard：I-II phases 15, III-IV phases 35.Histological grade：Differentiated 17, middle differentiation 15 Example, low differentiation 18.Preoperative equal footline radiotherapy, chemotherapy and biological agent treatment.Normal group is woven to normal lower distal colon and sticked Membrane tissue 40, is all from healthy colonoscopy examinee.
2nd, RNA is extracted and cDNA synthesis
Method according to embodiment 1 carries out RNA and extracted and cDNA synthesis.
3、QPCR
Primer is designed by primer-design software Primer 5.0, and Dalian treasured biotech firm synthesizes with Shanghai Ying Jun companies. VCAN genes and reference gene the primer sequence are as follows：
VCAN gene primer sequences
5 '-GGAGAAGACTGTGTTGTA-3 ' (SEQ ID NO.1),
5’-CGTATAGGTGAGATGGTAAT-3’(SEQ ID NO.2)；
GAPDH gene primer sequences
5 '-AAGGTCGGAGTCAACGGATTTG-3 ' (SEQ ID NO.3),
5’-CCATGGGTGGAATCATATTGGAA-3’(SEQ ID NO.4)。
QPCR reactions are carried out using cDNA2.0 μ l.Amplification program is：95 DEG C of 5min, (95 DEG C of 5s, 60 DEG C of 60s) * 45 are followed Ring.Using SYBR Green as fluorescent marker, in the enterprising performing PCR reaction of Light Cycler fluorescence real-time quantitative PCR instrument, Δ Δ CT methods carry out relative quantification.
4th, Western blot are detected
Extract total protein of cell.The total protein of extraction is quantified using BCA protein concentrations kit.Each sample takes After 50 μ g total proteins, 12%SDS-PAGE electrophoresis 1.5h, transferring film.5% skimmed milk power is closed, 4 DEG C of overnight incubations of primary antibody, secondary antibody 37 DEG C be incubated 2h.The luminous ECL luminescent solutions colour developing of electrochemical process, gel imaging system exposure colour developing；
5th, statistical procedures
The gray value of protein band is analyzed using Image J softwares, using β-actin as internal reference, by VCAN albumen The gray value of band is normalized.Result data is represented in the way of mean+SD, is used SPSS13.0 statistical softwares carry out statistical analysis, and difference between the two is examined using t, it is believed that work as P<There is system when 0.05 Meter learns meaning.
6th, result
(1) QPCR results
As shown in figure 1, compared with normal structure, VCAN gene mRNA levels are significantly reduced in adenocarcinoma of colon tissue, difference With statistical significance (P<0.05).
(2) Western blot results
As shown in Fig. 2 compared with normal structure, VCAN protein levels are significantly reduced in adenocarcinoma of colon tissue, and difference has Statistical significance (P<0.05).
Influence of the expression of embodiment 3VCAN genes to colon adenocarcinoma cell multiplication capacity
1st, VCAN gene recombination plasmids are built
(1) coded sequence of VCAN genes is expanded；
(2) amplimer is designed；
(3) the VCAN genes after amplification are connected in expression vector pcDNA3.0, build pcDNA3.0-VCAN restructuring tables Up to carrier.
The culture and transfection of 1.2 colon adenocarcinoma cells
HT-29 cells are cultivated in the RPMI-1640 culture mediums containing 10% hyclone, culture parameters：37 DEG C, volume integral Number is 5%CO2Saturated humidity.Liposome Lipofectamine2000 is used for transfection reagent.2 groups of experiment point：Negative control group (transfection pcDNA3.0)；Experimental group (transfection pcDNA3.0-VCAN).Take the logarithm the HT-29 cells in growth period, be inoculated in 6 holes thin Born of the same parents' culture plate.Tissue Culture Plate coverage rate is about 70%-80% after 24h.Rotaring transfecting mode is with reference to Lipofectamine2000 explanations Book is carried out.
1.3Western blot experiment detections pcDNA3.0-VCAN overexpression efficiency
Step be the same as Example 2.
1.4 results
As shown in figure 3, compared with negative control group (transfection pcDNA3.0), experimental group (transfection pcDNA3.0-VCAN) is thin VCAN expressing quantities are significantly raised in born of the same parents, and difference has statistical significance (P<0.05).
2nd, MTT experiment analysis cell-proliferation activity
The each group cell of exponential phase is collected, adjustment cell density is 1*104/ ml, is inoculated in 96 orifice plates, per hole 100 μ L, be placed in 37 DEG C, volume fraction be 5%CO2, saturated humidity incubator in cultivate.Respectively at progress after 24,48,72h MTT is detected：Add the μ l (5%) of MTT solution 10 per hole, be placed in 37 DEG C, volume fraction be 5%CO2, saturated humidity incubator in train Support.Cultivate after 3h, carefully remove supernatant, to add per hole and place 30min on 150 μ L DMSO, horizontal shaker.ELIASA determines wavelength A values under 490nm.
3rd, experimental result
As a result as shown in figure 4, compared with negative control group (transfection pcDNA3.0) cell, experimental group (transfects pcDNA3.0- VCAN) cell propagation is slow, and difference has statistical significance (P<0.05).It is above-mentioned test result indicate that, VCAN gene expressions suppression The propagation of colon adenocarcinoma cell is made.
Influence of the expression of embodiment 4VCAN genes to colon adenocarcinoma cell apoptosis
1st, step：Collect each group cell after transfection 24h, glacial phosphoric acid w salt buffer washes 2 times, 1xBinding Buffer It is 10 to adjust cell concentration4/ ml, after the AnnexinV dilutions mixing for adding 5 μ L, adds 2.5 μ L propidium iodide, mixes It is even.Lucifuge stands 10min on ice chest, adds the μ L of 1xBinding Buffer 400, upper machine testing apoptosis ratio.
2nd, experimental result
As a result as shown in figure 5, compared with negative control group (transfection pcDNA3.0) cell, experimental group (transfects pcDNA3.0- VCAN) apoptosis rate is raised, and difference has statistical significance (P<0.05).It is above-mentioned test result indicate that, VCAN gene expressions Promote the apoptosis of colon adenocarcinoma cell.
The explanation of above-described embodiment is only intended to understand the method for the present invention and its core concept.It should be pointed out that for this For the those of ordinary skill in field, under the premise without departing from the principles of the invention, some improve can also be carried out to the present invention And modification, these are improved and modification will be also fallen into the protection domain of the claims in the present invention.
SEQUENCE LISTING
<110>Beijing Yang Shen biology information technologies Co., Ltd
<120>Purposes of the VCAN in adenocarcinoma of colon diagnosis and treatment
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213>Artificial sequence
<400> 1
ggagaagact gtgttgta 18
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
cgtataggtg agatggtaat 20
<210> 3
<211> 22
<212> DNA
<213>Artificial sequence
<400> 3
aaggtcggag tcaacggatt tg 22
<210> 4
<211> 23
<212> DNA
<213>Artificial sequence
<400> 4
ccatgggtgg aatcatattg gaa 23

Claims (10)

1. detect application of the VCAN product in diagnosis adenocarcinoma of colon instrument is prepared.
2. application according to claim 1, it is characterised in that the product of the detection VCAN includes detection VCAN gene tables Up to the product of amount.
3. application according to claim 1 or 2, it is characterised in that the product of the detection VCAN includes quantifying The product of VCAN gene mRNAs, and/or the product of VCAN albumen can be quantified.
4. application according to claim 3, it is characterised in that the reagent that can quantify VCAN gene mRNAs includes real When quantitative PCR in the primer of specific amplified VCAN genes that uses, the primer sequence such as SEQ ID NO.1 and SEQ ID Shown in NO.2；The reagent that VCAN albumen can be quantified includes the antibody of specific binding VCAN albumen.
5. a kind of instrument for diagnosing adenocarcinoma of colon, it is characterised in that the instrument includes that VCAN gene expression amounts can be detected Instrument.
6. instrument according to claim 5, it is characterised in that the instrument includes that VCAN gene mRNAs can be quantified Reagent, and/or the reagent of VCAN albumen can be quantified.
7. instrument according to claim 6, it is characterised in that the reagent that can quantify VCAN gene mRNAs is real-time The primer of the specific amplified VCAN genes used in quantitative PCR, the primer sequence such as SEQ ID NO.1 and SEQ ID NO.2 It is shown.
8. a kind of medicine for treating adenocarcinoma of colon, it is characterised in that the medicine includes VCAN activator.
9. medicine according to claim 8, it is characterised in that the activator can promote or strengthen VCAN or be related to The expression of the material of VCAN upstreams or downstream pathway or activity.
10. application of the activator in the medicine for preparing treatment adenocarcinoma of colon described in claim 8 or 9.
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