CN106947821A - Biomarker for diagnosis and treatment adenocarcinoma of colon - Google Patents
Biomarker for diagnosis and treatment adenocarcinoma of colon Download PDFInfo
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Abstract
The invention discloses the molecular marker that COL18A1 genes can be early diagnosed as adenocarcinoma of colon.The experiment proves that, compared with normal structure, the COL18A1 gene expressions in adenocarcinoma of colon tissue are significantly reduced, and prove that COL18A1 can influence colon adenocarcinoma cell to breed and apoptosis by being overexpressed experiment.According to the achievement in research of the present invention, can research and develop can promote the medicine of COL18A1 gene expressions, so as to realize prevention and treatment clinically for adenocarcinoma of colon.
Description
Technical field
The present invention relates to diagnosing tumor, treatment, prediction prognosis field, more particularly it relates to detect COL18A1
Abnormal is diagnosing tumor, the prediction method of prognosis of means；And the tumor therapeutic agent of activation COL18A1 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 knot by detecting COL18A1 genes or protein expression difference
The method of enteraden cancer.
The second object of the present invention is to provide one kind to predict knot by detecting COL18A1 genes or protein expression difference
The method of enteraden cancer prognosis.
The third object of the present invention is to provide one kind to treat knot by activating COL18A1 genes or COL18A1 albumen
The method of enteraden cancer.
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 COL18A1 product in adenocarcinoma of colon diagnostic tool is prepared.
Further, the product of the detection COL18A1 includes the product of detection COL18A1 gene expression amounts.
Further, the product of the detection COL18A1 includes that the product of COL18A1 gene mRNAs can be quantified, and/or
The product of COL18A1 albumen can be quantified.
The product of the quantitative COL18A1 gene mRNAs of the present invention can be based on playing it using the known method of nucleic acid molecules
Function：As PCR, such as Southern hybridization, Northern hybridization, dot blot, FISH (FISH), DNA microarray,
ASO methods, 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 COL18A1 gene mRNAs can be quantified includes the specific amplified used in real-time quantitative PCR
The primer of COL18A1 genes, 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 COL18A1 albumen of the present invention can be based on playing its function using the known method of antibody：Example
Such as, ELISA, radioimmunoassay, immunohistochemical method, western blot etc. can be included.
The product of the quantitative COL18A1 albumen of the present invention includes the antibody or its fragment of specific binding COL18A1 albumen.
The antibody or its fragment of any structure, size, immunoglobulin class, origin etc. can be used, as long as it combines target protein
.The antibody or its fragment that the detection product of the present invention includes can be monoclonals or polyclonal.Antibody fragment refers to
Retain peptide of the antibody to the antibody a part of (Partial Fragment) of the binding activity of antigen or containing an antibody part.Antibody fragment can
With including F (ab ')2, Fab ', Fab, scFv (scFv), disulphide bonding Fv (dsFv) or its polymer, dimerization V
Area's (double antibody) or the peptide containing CDR.The product of the quantitative COL18A1 albumen of the present invention can include encoding antibody or coding
The nucleic acid of the separation of the amino acid sequence of antibody fragment, 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.Finally can be by using antibody of the COL18A1 albumen for being used as antigen or part thereof to acquisition
Implement antigentic specificity purifying to obtain the monoclonal antibody for COL18A1 albumen.Polyclonal antibody can be prepared as follows：With
Antigen-immunized animal same as above, collects blood sample from by immune animal, serum is isolated from blood, then
Antigentic specificity purifying is implemented to serum using above-mentioned antigen.The antibody that can be obtained by using ferment treatment or by using acquisition
The sequence information of antibody 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 COL18A1 genes or COL18A1 albumen can be detection COL18A1 genes or
The reagent of COL18A1 albumen, it can also be kit, chip, test paper etc. comprising the reagent or use the examination
The high-flux sequence platform of agent.
Present invention also offers a kind of instrument for diagnosing adenocarcinoma of colon, the instrument can detect COL18A1 gene expressions
Amount.
Further, the instrument includes that the reagent of COL18A1 gene mRNAs can be quantified, and/or can quantify
The reagent of COL18A1 albumen.
Further, the reagent that can quantify COL18A1 gene mRNAs is the special expansion used in real-time quantitative PCR
Increase the primer of COL18A1 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, COL18A1 bases are known in high-flux sequence
The exception of the cause purposes for falling within COL18A1 related to adenocarcinoma of colon, equally within protection scope of the present invention.
The amino acid that the detection product of the present invention, the anti-COL18A1 antibody or its fragment that use in diagnostic tool are recognized
Number be not particularly limited, as long as antibody can combine COL18A1.
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 COL18A1 genes or albumen in Samples subjects is detected；
(3) the COL18A1 genes measured or the expression of albumen are associated with the whether ill of subject.
(4) compared with the control, the expression reduction of COL18A1 genes or albumen, then the subject, which is judged, suffers from knot
Enteraden cancer is judged as recurrence or adenocarcinoma of colon trouble with the risk with adenocarcinoma of colon or adenocarcinoma of colon patient
Person is judged as prognosis mala.
Present invention also offers a kind for the treatment of method of adenocarcinoma of colon, methods described include activation COL18A1 genes or
COL18A1 albumen.
Further, methods described includes promoting the expression of COL18A1 genes, or promotes the expression or increasing of COL18A1 albumen
The activity of strong COL18A1 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 table of COL18A1 genes or COL18A1 albumen
Tumour medicine is determined up to level improves the effect of tumor prognosis.More specifically, when COL18A1 genes or COL18A1 eggs
The medicine may be selected as changing when being raised after adding or applying testing drug or when recovering normal level in white expression
The medicine of kind tumor prognosis.
Present invention also offers a kind of medicine for treating adenocarcinoma of colon, the medicine includes COL18A1 activator.
The COL18A1 of invention activator is unrestricted, as long as the activator can promote or strengthen COL18A1 or relate to
And expression or the activity of the material of COL18A1 upstreams or downstream pathway, 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 includes COL18A1 genes, COL18A1 albumen, promoted type miRNA, promoted type transcription tune
Control the factor 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 COL18A1 albumen, pass through
The expression of COL18A1 albumen is improved, so as to treat the adenocarcinoma of colon caused by COL18A1 hypoproteinosis.On the other hand it can use
In the activity of enhancing COL18A1 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 COL18A1 genes or albumen of the present invention can be used as controlling for new adenocarcinoma of colon
Treat medicine.
Brief description of the drawings
Fig. 1 displays detect differential expression of the COL18A1 genes in adenocarcinoma of colon tissue and normal structure using QPCR；
Fig. 2 displays are using Western blot experiment detection COL18A1 genes in adenocarcinoma of colon tissue and normal structure
Differential expression；
Fig. 3 displays detect the overexpression efficiency of COL18A1 genes using Western blot experiments；
Fig. 4 displays detect the influence that COL18A1 gene expressions are bred to colon adenocarcinoma cell using mtt assay；
Influence of Fig. 5 displays using flow cytomery COL18A1 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；It is prepared by target
Cheng Hou, using eucaryote Hybridization Control Kit (Affymetrix companies) preparing hybrid liquid, injection hybridization
The chip balance of liquid is 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.
COL18A1 genes and reference gene the primer sequence are as follows：
COL18A1 gene primer sequences
5 '-AAGATTCCAGAAGTGAAGAAGT-3 ' (SEQ ID NO.1),
5’-ATCTGAGCCAGGAAGTGT-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 10s, 60 DEG C of 65s) * 42
Circulation.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 COL18A1 eggs
The gray value of informal voucher 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, COL18A1 gene mRNA levels are significantly reduced in adenocarcinoma of colon tissue, poor
It is different that there is statistical significance (P<0.05).
(2) Western blot results
As shown in Fig. 2 compared with normal structure, COL18A1 protein levels are significantly reduced in adenocarcinoma of colon tissue, difference tool
Statistically significant (P<0.05).
Influence of the expression of the COL18A1 genes of embodiment 3 to colon adenocarcinoma cell multiplication capacity
1st, COL18A1 gene recombination plasmids are built
(1) coded sequence of COL18A1 genes is expanded；
(2) amplimer is designed；
(3) the COL18A1 genes after amplification are connected in expression vector pcDNA3.0, build pcDNA3.0-COL18A1
Recombinant expression 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-COL18A1).Take the logarithm the HT-29 cells in growth period, be inoculated in 6 holes
Tissue Culture Plate.Tissue Culture Plate coverage rate is about 70%-80% after 24h.Rotaring transfecting mode is said with reference to Lipofectamine2000
Bright book is carried out.
1.3 Western blot experiment detections pcDNA3.0-COL18A1 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-COL18A1)
COL18A1 expressing quantities are significantly raised in cell, 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-
COL18A1) cell propagation is slow, and difference has statistical significance (P<0.05).It is above-mentioned test result indicate that, COL18A1 genes
The expression inhibiting propagation of colon adenocarcinoma cell.
Influence of the expression of the COL18A1 genes of embodiment 4 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-
COL18A1) apoptosis rate is raised, and difference has statistical significance (P<0.05).It is above-mentioned test result indicate that, COL18A1 bases
Because expression promotes to finish the apoptosis of enteraden cancer 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>Biomarker for diagnosis and treatment adenocarcinoma of colon
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213>Artificial sequence
<400> 1
aagattccag aagtgaagaa gt 22
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence
<400> 2
atctgagcca ggaagtgt 18
<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 COL18A1 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 COL18A1 includes detection COL18A1
The product of gene expression amount.
3. application according to claim 1 or 2, it is characterised in that the product of the detection COL18A1 includes quantifying
The product of COL18A1 gene mRNAs, and/or the product of COL18A1 albumen can be quantified.
4. application according to claim 3, it is characterised in that described to quantify the reagent bag of COL18A1 gene mRNAs
Include the primer of the specific amplified COL18A1 genes used in real-time quantitative PCR, the primer sequence such as SEQ ID NO.1 and SEQ
Shown in ID NO.2；The reagent that COL18A1 albumen can be quantified includes the antibody of specific binding COL18A1 albumen.
5. a kind of instrument for diagnosing adenocarcinoma of colon, it is characterised in that the instrument includes that COL18A1 gene expression amounts can be detected
Instrument.
6. instrument according to claim 5, it is characterised in that the instrument includes that COL18A1 genes can be quantified
MRNA reagent, and/or the reagent of COL18A1 albumen can be quantified.
7. instrument according to claim 6, it is characterised in that it is described can quantify COL18A1 gene mRNAs reagent be
The primer of the specific amplified COL18A1 genes used in real-time quantitative PCR, the primer sequence such as SEQ ID NO.1 and SEQ
Shown in ID NO.2.
8. a kind of medicine for treating adenocarcinoma of colon, it is characterised in that the medicine includes COL18A1 activator.
9. medicine according to claim 8, it is characterised in that the activator can promote or strengthen COL18A1 or relate to
And expression or the activity of the material of COL18A1 upstreams or downstream pathway.
10. application of the activator in the medicine for preparing treatment adenocarcinoma of colon described in claim 8 or 9.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101594908A (en) * | 2007-01-03 | 2009-12-02 | 加利福尼亚大学董事会 | Transfection is used for the treatment of the apparatus and method of gene, siRNA, shRNA carrier and the other biological medical diagnosis and the medicine of large animal and people's arthritis and other plastic surgery's diseases in the body born of the same parents |
| CN101772511A (en) * | 2007-02-16 | 2010-07-07 | 太平洋艾瑞有限公司 | Blocking the migration or metastasis of cancer cells by affecting adhesion proteins and the uses of new compounds thereof |
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2017
- 2017-04-11 CN CN201710232968.0A patent/CN106947821B/en active Active
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|---|---|---|---|---|
| CN101594908A (en) * | 2007-01-03 | 2009-12-02 | 加利福尼亚大学董事会 | Transfection is used for the treatment of the apparatus and method of gene, siRNA, shRNA carrier and the other biological medical diagnosis and the medicine of large animal and people's arthritis and other plastic surgery's diseases in the body born of the same parents |
| CN101772511A (en) * | 2007-02-16 | 2010-07-07 | 太平洋艾瑞有限公司 | Blocking the migration or metastasis of cancer cells by affecting adhesion proteins and the uses of new compounds thereof |
| CN106659765A (en) * | 2014-04-04 | 2017-05-10 | 德玛医药 | Use of dianhydrogalactitol and analogs or derivatives thereof to treat non-small-cell carcinoma of the lung and ovarian cancer |
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