Summary of the invention
One of the objects of the present invention is to provide one kind to be diagnosed down by detection INCENP gene or protein expression difference
The method of pharynx cancer.
The second object of the present invention is to provide one kind by detection INCENP gene or protein expression difference to predict down
The method of pharynx cancer prognosis.
The third object of the present invention is that providing a kind of treat by activation INCENP gene or INCENP albumen swallows
The method of cancer.
The fourth object of the present invention is to provide a kind of method for screening the drug for the treatment of hypopharyngeal cancer.
The fifth object of the present invention is to provide a kind of for treating the drug of hypopharyngeal cancer.
To achieve the goals above, present invention employs following technical solutions:
The present invention provides the products of detection INCENP gene or INCENP albumen in preparing hypopharyngeal cancer diagnostic tool
Purposes.
The present invention also provides the products of detection INCENP gene or INCENP albumen to predict hypopharyngeal cancer prognosis work in preparation
Purposes in tool.
Further, the product of the detection INCENP gene or INCENP albumen includes detection INCENP gene or INCENP
The product of the expression of albumen.The product includes that can combine the nucleic acid of INCENP gene or can combine INCENP egg
White substance (such as antibody).The nucleic acid is able to detect the expression of INCENP gene;The substance is able to detect
The expression of INCENP albumen.
The product of detection INCENP gene of the invention can play its function based on the known method of nucleic acid molecules is used:
As PCR, such as Southern hybridization, Northern hybridization, dot blot, fluorescence in situ hybridization (FISH), DNA microarray, ASO method,
High-flux sequence platform etc..It can qualitatively, quantitatively or semi-quantitatively implement analysis using the product.
Include that nucleic acid in the said goods can be obtained by chemical synthesis, or by containing from biomaterial preparation
It is expected that the gene of nucleic acid, then using primer amplification designed for amplification expectation nucleic acid, it is obtained.
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 method etc..Amplification
Nucleic acid can be by using dot blotting hybridization method, Surface Plasmon Resonance (SPR method), PCR-RFLP method, original position RT-PCR
Method, PCR-SSO (sequence specific oligonucleotide) method, PCR-SSP method, AMPFLP (amplifiable fragment length polymorphism) method,
MVR-PCR method and PCR-SSCP (single-strand conformation polymorphism) method detect.
Nucleic acid recited above includes the primer for expanding INCENP gene, and the primer for including in product can be by passing through
Synthesis is learned to prepare, by using those skilled in the art will know that method be suitably designed with reference to Given information, and pass through
It is prepared by chemical synthesis.
In specific embodiments of the present invention, the nucleic acid is amplimer used in QPCR experiment, the primer
Sequence such as SEQ ID NO.1 (positive sequence) and SEQ ID NO.2 (reverse sequence) shown in.
Nucleic acid recited above may also include probe, and the probe can be prepared by chemical synthesis, by using this
The method that field technical staff knows appropriately is designed with reference to Given information, and is prepared by chemical synthesis, or can lead to
The gene for containing desired nucleic acid sequence from biomaterial preparation is crossed, and is expanded using the primer designed for amplification expectation nucleic acid sequence
Increase it to prepare.
The product of detection INCENP albumen of the invention can play its function: example based on the known method of antibody is used
It such as, may include ELISA, radioimmunoassay, immunohistochemical method, western blot etc..
The product of detection INCENP albumen of the invention includes the antibody or its segment for specifically binding INCENP albumen.It can
To use the antibody or its segment of any structure and size, immunoglobulin class, origin etc., as long as it is in conjunction with target protein
It can.The antibody or its segment for including in testing product of the invention can be monoclonal or polyclonal.Antibody fragment refers to guarantor
Stay peptide of the antibody to the active antibody of the combination of antigen a part of (Partial Fragment) or containing antibody a part.Antibody fragment can be with
Including F (ab ')2, Fab ', Fab, scFv (scFv), disulphide bonding Fv (dsFv) or its polymer, the area dimerization V
(double antibody) or peptide containing CDR.The product of detection INCENP albumen of the invention may include encoding antibody or encoding antibody
The isolated nucleic acid of the amino acid sequence of segment, the carrier comprising the nucleic acid, and carry the cell of the carrier.
Antibody can be obtained by the way that well known to a person skilled in the art methods.For example, preparation retains target all or in part
The mammalian cell expression vector of their polynucleotides of the polypeptide or integration coding of protein is as antigen.Exempted from using antigen
After epidemic disease animal, from the immune animal adaptive immune cell of process and myeloma cell is merged to obtain hybridoma.Then from hybridization
Tumor culture collects antibody.It finally can be real by using antibody of the INCENP albumen for being used as antigen or part thereof to acquisition
Antigentic specificity purifying is applied to obtain the monoclonal antibody for INCENP albumen.Polyclonal antibody can be prepared as follows: with
Identical antigen-immunized animal above collects blood sample from by immune animal, serum is isolated from blood, is then made
Implement antigentic specificity to serum with above-mentioned antigen to purify.It can be by the antibody that is obtained with enzymatic treatment or by using acquisition
The sequence information of antibody obtains antibody fragment.
The combination of marker and antibody or its segment 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. standards
Standby dyestuff, then mixed solution, then at being placed at room temperature for 10 minutes.In addition, the labelling kit of commercialization can be used in label, 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 kit-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-NH2,647 labelling kit-NH2 of HiLyte Fluor (TM) (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- marker protein mark
Remember kit (Funakoshi Corporation).For correct labeling, suitable instrument can be used to detect by label
Antibody or its segment.
As the sample according to testing product of the 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 for measurement of the invention;For example, it may 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.
In the present invention, " prognosis " refers to mistake of the tumor patient after inhibiting by surgical procedure etc. or alleviating tumour growth
Journey or result.In the present specification, prognosis can be by surgical procedure inhibit or alleviate tumour growth after 1,2,3,4,5,6,
7,8,9,10,15,20 years or more long when life state.Prognosis can be by checking biomarker, that is, INCENP albumen or volume
The gene of code INCENP albumen is predicted.Prognosis prediction can be performed such that according to biomarker with or without or increasing
Or reduce, determine that the prognosis of patient is good or bad, or determine the probability of good prognosis or poor prognosis.
In the present invention, " prognosis bona " refer to inhibit or alleviate for patient by surgical procedure etc. tumour growth it
Afterwards, patient's long-term (such as 3,5,6,7,8,9,10,15,20 years or longer) does not have critical condition.Alternatively, good prognosis can anticipate
Refer to and survives in such long-time, sent out again without transfer, without recurrence or nothing.For example, prognosis bona can mean at least 3 years or outstanding
It is to survive at least 5 years, preferably without transfer or recurrence.The most preferred state of prognosis bona is survival for a long time without disease.Such as
Used herein, " prognosis bona " can also include any such state, wherein it can be found that disease such as shifts, still
It is pernicious low and do not severely impact survival ability.
In the present invention, " prognosis mala " refers to that patient is short after inhibiting or alleviating tumour growth by surgical procedure etc.
Fatal condition occurs in period (such as 1,2,3,4,5 year or shorter).Alternatively, poor prognosis refers in such short-term extremely
It dies, shift, recur or sends out again.For example, poor prognosis can mean Preventive or dead at least 3 years or especially at least 5 years
It dies.
Prediction prognosis is referred to the process of prediction status of patient or as a result, is not meant to be predicted with 100% accuracy
The process or result of status of patient.Prediction prognosis refers to whether a possibility that determining certain processes or result increases, and simultaneously unexpectedly
Taste by determining a possibility that certain processes or result occurs or not compared with certain processes or result.Such as this
For invention, in the present invention in the horizontal patient reduced of INCENP gene or INCENP albumen, with the trouble for not showing this feature
Person compares, and more likely observes particular procedure or result.
Further, it is described detection INCENP gene or INCENP albumen product can be detection INCENP gene or
The reagent of INCENP albumen is also possible to include kit, chip, test paper of the reagent etc., is also possible to using the examination
The high-flux sequence platform of agent.
The present invention also provides a kind of tool for diagnosing hypopharyngeal cancer, the tool is able to detect INCENP gene or INCENP
The expression of albumen.The tool includes the nucleic acid that can combine INCENP gene or the object that can combine INCENP albumen
Matter (such as antibody).The nucleic acid is able to detect the expression of INCENP gene;The substance is able to detect INCENP albumen
Expression.
Further, the property of the nucleic acid and the substance is the same as noted earlier.
Further, the tool of the diagnosis hypopharyngeal cancer includes but is not limited to chip, kit, test paper or high-flux sequence
Platform;High-flux sequence platform is a kind of tool of special diagnosis hypopharyngeal cancer, with the development of high throughput sequencing technologies, to one
The building of personal gene expression profile will become very easily work.By the gene table for comparing Disease and normal population
Up to spectrum, the exception for being easy to analyze which gene is related to disease.Therefore, the different of INCENP gene is known in high-flux sequence
The often purposes for also belonging to INCENP gene related to hypopharyngeal cancer, equally within protection scope of the present invention.
The present invention also provides a kind of tools for predicting hypopharyngeal cancer prognosis, and the prediction hypopharyngeal cancer prognostic tool includes can
In conjunction with INCENP gene nucleic acid or can in conjunction with INCENP albumen substance (such as antibody).The nucleic acid is able to detect
The mRNA level in-site of INCENP gene;The substance is able to detect the expression of INCENP albumen.
Further, the property of the nucleic acid and the substance is the same as noted earlier.
Further, the tool of the prediction hypopharyngeal cancer prognosis includes but is not limited to chip, kit, test paper or high throughput
Microarray dataset;High-flux sequence platform is a kind of tool of special diagnosis hypopharyngeal cancer, with the development of high throughput sequencing technologies,
Very easily work will be become to the building of the gene expression profile of a people.By the base for comparing Disease and normal population
Because of express spectra, the exception for being easy to analyze which gene is related to disease.Therefore, INCENP gene is known in high-flux sequence
The exception purposes for also belonging to INCENP gene related to hypopharyngeal cancer, equally within protection scope of the present invention.
The amino acid that anti-INCENP antibody used in testing product of the invention, diagnostic tool or its segment are identified
Number is not particularly limited, as long as antibody can combine INCENP.
The present invention also provides a kind of diagnosis hypopharyngeal cancer or the methods for predicting hypopharyngeal cancer prognosis, and the method includes walking as follows
It is rapid:
(1) sample of subject is obtained;
(2) expression of INCENP gene or albumen in Samples subjects is detected;
(3) it associates whether by the expression of the INCENP gene or albumen that measure with the illness of subject.
(4) compared with the control, the expression of INCENP gene or albumen reduces, then the subject is diagnosed as swallowing
Cancer or the subject are confirmed as prognosis mala.
The present invention also provides a kind for the treatment of methods of hypopharyngeal cancer, and the method includes activation INCENP gene or INCENP
Albumen.
Further, the method includes promoting the expression of INCENP gene, or the expression or enhancing of promotion INCENP albumen
The activity of INCENP albumen.
The present invention also provides a kind of screening techniques of tumour medicine, can be by after adding testing drug to cancer cell
Or the expression of some period measurement INCENP gene or INCENP albumen after applying testing drug to tumor model animal
Level improves the effect of tumor prognosis to measure tumour medicine.More specifically, when INCENP gene or INCENP albumen
It is swollen as improving that the drug may be selected when increasing after adding or applying testing drug or when restoring normal level in expression
The therapeutic agent of tumor prognosis.
The present invention also provides a kind of drugs of activator containing INCENP gene or INCENP albumen.
The present invention also provides application of the above-mentioned activator in the drug of preparation treatment hypopharyngeal cancer.
The activator of INCENP gene or INCENP albumen of the invention is unrestricted, as long as can promote or enhance
INCENP or be related to the upstream INCENP or downstream pathway substance expression or activity, and for treatment the effective drug of tumour be
It can.
Further, the activator includes INCENP gene, INCENP albumen, promoted type miRNA, promoted type transcriptional control
The factor or promoted type target small molecule compound.
The activator further includes carrier or host cell comprising carrying INCENP gene.
On the one hand activator of the invention can be used for supplementing the missing or deficiency of endogenic INCENP albumen, by mentioning
The expression of high INCENP albumen, thus hypopharyngeal cancer caused by treating because of INCENP hypoproteinosis.On the other hand can be used for enhancing
The activity of INCENP albumen, to treat hypopharyngeal cancer.
Drug of the invention can be used as medicine and be administered alone or apply together with other medicines.It can be with medicine of the invention
The other medicines that object is applied together are unrestricted, as long as it does not damage therapeutic or preventive medicine effect of the invention i.e.
It can, it is preferred that the drug for treating or preventing tumour may 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, tegafur-Uracil, 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, pirarubicin, bleomycin, Peplomycin, mitomycin C and mitoxantrone;
Drug based on platinum, such as oxaliplatin, carboplatin, cis-platinum and Nedaplatin;Hormonal medicaments, such as Anastrozole, Exemestane,
Estramustine, ethinyloestradiol, chlormadinone, Goserelin, tamoxifen, dexamethasone, Toremifene, Bicalutamide, Flutamide,
Prednisolone, Fosfestrol, mitotane, methyltestosterone, Medroxyprogesterone, Mepitiostane, Leuprorelin and Letrozole;Biological respinse modification
Agent, such as interferon-' alpha ', interferon beta, interferon gamma, interleukin, ubenimex, dry BCG and lentinan;With molecular targeted medicine
Object, such as Imatinib (imatinib), Gefitinib (gefitinib), gemtuzumab, ozogamicin, Tamibarotene, song
Appropriate monoclonal antibody, Tretinoin, bortezomib (bortezomib) and Rituximab etc..
Drug of the invention can be prepared into various dosage forms 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 administration method of drug of the invention is unrestricted, as long as it can play desired therapeutic effect or preventive effect i.e.
Can, including but not limited to intravenously, in peritonaeum, intraocularly, intra-arterial, intrapulmonary is taken orally, in vesicle, intramuscular, intratracheally, subcutaneously
, local by pleura by skin, sucking, by mucous membrane, skin, stomach is intra-articular, intra-ventricle, rectum, vagina,
In skull, in urethra, in liver, in tumor.In some cases, it can systematically be administered.It is locally to be administered in some cases.
The dosage of drug of the invention is unrestricted, can as long as obtaining desired therapeutic effect or preventive effect
To carry out appropriate determination according to symptom, gender, age etc..Example can be used in the dosage of therapeutic agent or prophylactic agent of the invention
Such as the therapeutic effect of disease or preventive effect are determined as index.
In the context of the present invention, " diagnosis hypopharyngeal cancer " both included judge subject whether suffered from hypopharyngeal cancer or
Including judging that subject whether there is the risk with hypopharyngeal cancer.
" treatment " used herein is covered treatment-related in such as mankind of the mammal with related disease or illness
Disease or morbid state, and include:
(1) prevent disease or morbid state occurs in mammals, especially when the mammal is susceptible in the disease
Diseased state, but when being not yet diagnosed with this morbid state;
(2) inhibit disease or morbid state, that is, prevent its generation;Or
(3) alleviate disease or morbid state, even if disease or morbid state subside.
Term " treatment " is usually directed to treatment mankind or animal (for example, being applied by animal doctor), wherein can reach certain pre-
The therapeutic effect of phase, for example, inhibiting the development (including reduce development speed, stop development) of illness, improving illness and healing
Illness.It further include the treatment as precautionary measures (such as prevention).To not yet development be illness but have development be the illness endanger
The purposes of the patient of danger, is also included in term " treatment ".
The advantages of the present invention:
Of the invention has found a kind of molecular marker for diagnosing hypopharyngeal cancer, can be in hypopharyngeal cancer using the molecular marker
The early stage of generation can be used as judging, provide the survival rate of patient.
In addition, the present invention is capable of providing significant information to determine treatment side for patient by the prognosis of prediction patient
Case strategy.
Of the invention includes that the therapeutic agent of the activator of INCENP gene or albumen can be used as the treatment of new hypopharyngeal cancer
Drug.