Summary of the invention
An object of the present invention is that providing a kind of diagnoses uterus by detection TNS11 gene or protein expression difference
The method of endometrial carcinomas.
The two of the purpose of the present invention are that providing a kind of predicts uterus by detection TNS11 gene or protein expression difference
The method of endometrial carcinomas prognosis.
The three of the purpose of the present invention are that providing a kind of treats intrauterine by activation TNS11 gene or TNS11 albumen
The method of film cancer.
A kind of method that the four of the purpose of the present invention are to provide medicine for screening treatment carcinoma of endometrium.
The five of the purpose of the present invention are to provide a kind of medicine for treating carcinoma of endometrium.
To achieve these goals, present invention employs following technical scheme:
The invention provides the product of detection TNS11 gene or TNS11 albumen in preparation carcinoma of endometrium diagnostic tool
Purposes.
Present invention also offers the product of detection TNS11 gene or TNS11 albumen in preparation prediction carcinoma of endometrium prognosis
Purposes in instrument.
Further, the product of described detection TNS11 gene or TNS11 albumen includes detecting TNS11 gene or TNS11 albumen
The product of expression.Described product includes can be in conjunction with the nucleic acid of TNS11 gene or can be in conjunction with the thing of TNS11 albumen
Matter (such as antibody).Described nucleic acid can detect the expression of TNS11 gene;Described material can detect TNS11 albumen
Expression.
The product of the detection TNS11 gene of the present invention can play its function based on the known method using nucleic acid molecules:
As PCR, as Southern hybridization, Northern hybridization, dot blot, fluorescence in situ hybridization (FISH), DNA microarray, ASO method,
High-flux sequence platform etc..This product is used can qualitatively, quantitatively or semi-quantitatively to implement to analyze.
The nucleic acid being included in the said goods can be obtained by chemosynthesis, or by containing from biomaterial preparation
Expect the gene of nucleic acid, then use be designed for amplification expectation nucleic acid primer amplification it obtain.
Further, described PCR method is known method, such as, and ARMS (Amplification Refractory
Mutation System, abruptly-changing system is not answered in amplification) method, RT-PCR (reverse transcriptase-PCR) method, nested PCR method etc..Amplification
Nucleic acid can by use dot blotting hybridization method, Surface Plasmon Resonance (SPR method), PCR-RFLP method, in situ 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 expanding TNS11 gene, and the primer that product includes can be by by changing
Learning synthesis to prepare, the method that be those skilled in the art will know that by use is suitably designed with reference to Given information, and passes through
Prepared by chemosynthesis.
In specific embodiments of the present invention, described nucleic acid is the amplimer used in QPCR experiment, described primer
Sequence such as SEQ ID NO.1 (forward sequence) and SEQ ID NO.2 (reverse sequence) shown in.
Nucleic acid recited above may also include probe, and described probe can be prepared by chemosynthesis, by using this
The method that skilled person knows appropriately designs with reference to Given information, and is prepared by chemosynthesis, or can lead to
Cross the gene containing expectation nucleotide sequence from biomaterial preparation, and use the primer expansion being designed for amplification expectation nucleotide sequence
Increase it to prepare.
The product of the detection TNS11 albumen of the present invention can play its function based on the known method using antibody: such as,
ELISA, radioimmunoassay, immunohistochemical method, western blot etc. can be included.
The product of the detection TNS11 albumen of the present invention includes antibody or its fragment of specific binding TNS11 albumen.Permissible
Use antibody or its fragment of any structure, size, immunoglobulin class, origin etc., as long as it combines target protein.
Antibody or its fragment that the detection product of the present invention includes can be monoclonal or polyclonal.Antibody fragment refers to that reservation is anti-
Body is to an antibody part (Partial Fragment) of the combination 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 district (dual anti-
Body) or containing the peptide of CDR.The product of the detection TNS11 albumen of the present invention can include encoding antibody or Encoding Antibody Fragment
The nucleic acid of the separation of aminoacid sequence, comprises the carrier of this nucleic acid, and carries the cell of this carrier.
Antibody can be by well known to a person skilled in the art that method obtains.Such as, preparation retains target all or in part
The polypeptide of protein or the mammalian cell expression vector of integration their polynucleotide of coding are as antigen.Use antigen is exempted from
After epidemic disease animal, from passing through immune animal adaptive immune cell fused bone myeloma cells to obtain hybridoma.Then from hybridization
Antibody collected by tumor culture.TNS11 albumen or its part antibody reality to obtaining of antigen finally can be used as by use
Execute antigenic specificity purification and obtain the monoclonal antibody for TNS11 albumen.Polyclonal antibody can be prepared as follows: with upper
The antigen-immunized animal that literary composition is identical, collects blood sample from the animal through immunity, isolates serum, then use from blood
Serum is implemented antigenic specificity purification by above-mentioned antigen.Can be by the antibody obtained with ferment treatment or by resisting that use obtains
The sequence information of body obtains antibody fragment.
The combination of label and antibody or its fragment can be implemented by method as commonly known in the art.Such as, may be used
With following fluorescent marker protein or peptide: clean protein or peptide with phosphate buffer, add with DMSO, buffer agent, etc. standard
Standby dyestuff, then mixed solution, place 10 minutes then at room temperature.It addition, labelling can the labelling kit of commodity in use, all
Such as biotin labeling reagent box, such as biotin labeling reagent box-NH2, biotin labeling reagent box-SH
(DojindoLaboratories);Alkali phosphatase enzyme mark test kit such as alkali phosphatase enzyme mark test kit-NH2, alkalescence phosphorus
Acid enzyme labelling test kit-SH (Dojindo Laboratories);Peroxidase labelling test kit such as peroxidase mark
Note test kit-NH2, peroxidase labelling test 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, HiLyte Fluor (TM) 647 labelling kit-NH2 (Dojindo Laboratories);And
DyLight 547 and DyLight647 (Techno Chemical Corp.), Zenon (TM), Alexa Fluor (TM) antibody
Labelling kit, Qdot (TM) antibody labeling test kit (Invitrogen Corporation) and EZ-label protein mark
Note test kit (Funakoshi Corporation).For correct labeling, it is possible to use suitable instrument detects through labelling
Antibody or its fragment.
Sample as the detection product according to the present invention, it is possible to use the tissue sample such as obtained from biopsy experimenter
Or fluid.Sample is not particularly limited, as long as it is suitable to the mensuration of the present invention;Such as, it can include tissue, blood, blood plasma,
Serum, lymph fluid, urine, serous cavity liquid, spinal fluid, synovial fluid, aqueous humor, tear, saliva or its fraction or treated material
Material.
In specific embodiments of the present invention, described sample is from the tissue of experimenter.
In the present invention, " prognosis " refer to that cancer patient is by the suppression such as surgical procedure or the mistake after alleviating tumor growth
Journey or result.In this manual, prognosis can be suppressed or alleviate after tumor growth 1 by surgical procedure, 2,3,4,5,6,
7,8,9,10,15,20 years or more long time life state.Prognosis can be by checking biomarker i.e. TNS11 albumen or volume
The gene of code TNS11 albumen is predicted.Prognosis prediction can be performed such that according to biomarker with or without, or raise
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 suppressed for patient by surgical procedure etc. or alleviate tumor growth it
After, patient (such as 3,5,6,7,8,9,10,15,20 years or longer) over a long time does not has critical condition.Or, good prognosis can be anticipated
Refer to survive in the most long-time, without transfer, without recurrence or without sending out again.Such as, prognosis bona can mean at least 3 years or outstanding
It is survival at least 5 years, preferably without transfer or recurrence.The most preferred state of prognosis bona is the long-term survival without disease.As
Used herein, " prognosis bona " can also include any such state, wherein it appeared that disease is as shifted, but
Pernicious low and do not severely impact survival ability.
In the present invention, " prognosis mala " refers to that patient is by the suppression such as surgical procedure or short after alleviating tumor growth
Fatal condition is there is in period (such as 1,2,3,4,5 years or shorter).Or, poor prognosis refers in such short-term dead
Die, shift, recur or send out again.Such as, poor prognosis can mean at least 3 years or Preventive or dead in especially at least 5 years
Die.
Prediction prognosis refers to predict process or the result of status of patient, is not meant to predict with the accuracy of 100%
The process of status of patient or result.Prediction prognosis refers to whether the probability determining some process or result increases, and the most unexpectedly
Taste to be compared by situation about not occurring with some process or result and is determined some process of generation or the probability of result.Such as this
For invention, in the patient that in the present invention, the level of TNS11 gene or TNS11 albumen reduces, with the patient not showing this feature
Compare, more likely observe particular procedure or result.
Further, the product of described detection TNS11 gene or TNS11 albumen can be detection TNS11 gene or TNS11 egg
White reagent, can also be to comprise the test kit of described reagent, chip, reagent paper etc., it is also possible to be the high pass using described reagent
Measure sequence platform.
Present invention also offers the instrument of a kind of diagnosis of endometrial carcinoma, described instrument can detect TNS11 gene or
The expression of TNS11 albumen.Described instrument includes can be in conjunction with the nucleic acid of TNS11 gene or can be in conjunction with TNS11 albumen
Material (such as antibody).Described nucleic acid can detect the expression of TNS11 gene;Described material can detect TNS11 egg
White expression.
Further, the character of described nucleic acid and described material is with noted earlier.
Further, the instrument of described diagnosis of endometrial carcinoma includes but not limited to chip, test kit, reagent paper or high flux
Order-checking platform;High-flux sequence platform is the instrument of a kind of special diagnosis of endometrial carcinoma, along with high throughput sequencing technologies
Development, will become the structure of the gene expression profile of a people and work the most easily.By contrast Disease and normal person
The gene expression profile of group, the exception easily analyzing which gene is relevant to disease.Therefore, know in high-flux sequence
The exception of the TNS11 gene purposes that fall within TNS11 gene relevant to carcinoma of endometrium, equally protection scope of the present invention it
In.
Present invention also offers a kind of instrument predicting carcinoma of endometrium prognosis, described prediction carcinoma of endometrium prognostic tool
Including can be in conjunction with the nucleic acid of TNS11 gene or can be in conjunction with the material (such as antibody) of TNS11 albumen.Described nucleic acid can
The mRNA level in-site of detection TNS11 gene;Described material can detect the expression of TNS11 albumen.
Further, the character of described nucleic acid and described material is with noted earlier.
Further, the instrument of described prediction carcinoma of endometrium prognosis includes but not limited to chip, test kit, reagent paper or height
Flux order-checking platform;High-flux sequence platform is the instrument of a kind of special diagnosis of endometrial carcinoma, along with high-flux sequence skill
The development of art, will become the structure of the gene expression profile of a people and work the most easily.By contrast Disease and just
The gene expression profile of ordinary person group, the exception easily analyzing which gene is relevant to disease.Therefore, know in high-flux sequence
The exception of the TNS11 gene purposes that fall within TNS11 gene relevant to carcinoma of endometrium, equally protection scope of the present invention it
In.
It is amino acid whose that the anti-TNS11 antibody used in detection product, the diagnostic tool of the present invention or its fragment are identified
Number is not particularly limited, as long as antibody can be in conjunction with TNS11.
Present invention also offers a kind of diagnosis of endometrial carcinoma or the method for prediction carcinoma of endometrium prognosis, described method bag
Include following steps:
(1) sample of experimenter is obtained;
(2) TNS11 gene or the expression of albumen in detection Samples subjects;
(3) the TNS11 gene recorded or the expression of albumen are associated with the whether ill of experimenter.
(4) compared with the control, the expression of TNS11 gene or albumen reduces, then this experimenter is diagnosed as intrauterine
Film cancer, or this experimenter is confirmed as prognosis mala.
Present invention also offers the Therapeutic Method of a kind of carcinoma of endometrium, described method include activate TNS11 gene or
TNS11 albumen.
Further, described method includes the expression promoting TNS11 gene, or promotes expression or the enhancing of TNS11 albumen
The activity of TNS11 albumen.
Present invention also offers the screening technique of a kind of cancer drug, can be by after cancerous cell be added testing drug
Or the expression water measuring TNS11 gene or TNS11 albumen certain period after cancer model animal is used testing drug
Put down and measure cancer drug and improve the effect of cancer prognosis.More specifically, when TNS11 gene or the expression of TNS11 albumen
When level raises after adding or using testing drug or when recovering normal level, this medicine optional is pre-as improving cancer
After medicine.
Present invention also offers a kind of containing TNS11 gene or the medicine of the activator of TNS11 albumen.
Present invention also offers the application in the medicine of preparation treatment carcinoma of endometrium of the above-mentioned activator.
The TNS11 gene of the present invention or the activator of TNS11 albumen are unrestricted, as long as can promote or strengthen
TNS11 or relate to the expression of material or the activity of TNS11 upstream or downstream pathway, and for the treatment effective medicine of cancer i.e.
Can.
Further, described activator include TNS11 gene, TNS11 albumen, promoted type miRNA, promoted type transcriptional control because of
Son or promoted type targeting micromolecular compound.
Described activator also includes comprising carrier or the host cell carrying TNS11 gene.
On the one hand the activator of the present invention may be used for supplementing disappearance or the deficiency of endogenic TNS11 albumen, by carrying
The expression of high TNS11 albumen, thus the carcinoma of endometrium that treatment causes because of TNS11 hypoproteinosis.On the other hand may be used for increasing
The activity of strong TNS11 albumen, thus treat carcinoma of endometrium.
The medicine of the present invention can be administered alone as medicine or use together with other medicines.Can be with the medicine of the present invention
The other medicines that thing is used together are unrestricted, as long as it does not damage the effect of the therapeutic of the present invention or preventive medicine i.e.
Can, it is preferred that the medicine for treatment or prophylaxis of cancer 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, cytosine arabinoside, cytosine arabinoside octadecyl phosphate
(cytarabine ocfosfate), ftorafur, UFT, ftorafur gimeracil oteracil potassium, deoxidation fluorine urine
Glycosides, hydroxyurea, fluorouracil, fludarabine, pemetrexed, pentostatin, mercaptopurine and methotrexate;Plant alkaloid, all
As irinotecan, etoposide, sobuzoxane, docetaxel, nogitecan, Pa Litasai, vinorelbine, vindesine and
Vinblastine;Antitumor antibiotic, such as actinomycin D, aclarubicin, amrubicin, idarubicin, epirubicin, zinostatin
Stimalamer, daunorubicin, doxorubicin, pirarubicin, bleomycin, peplomycin, ametycin and mitoxantrone;
Medicine based on platinum, such as oxaliplatin, carboplatin, cisplatin and nedaplatin;Hormonal medicaments, such as Anastrozole, exemestane,
Estramustine, ethinylestradiol, chlormadinone, goserelin, tamoxifen, dexamethasone, toremifene, bicalutamide, flutamide,
Prednisolone, fostestrol, 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 as various dosage form as required.Include but not limited to, percutaneous, mucosa, nose, buccal,
Tablet, solution, granule, patch, unguentum, capsule, aerosol or the suppository that Sublingual or per os 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 is
Can, include but not limited to intravenous, intraperitoneal, ophthalmic, intra-arterial, in lung, be administered orally, in vesicle, intramuscular, tracheal strips, subcutaneous
, by skin, by pleura, local, suck, by mucosa, skin, the intestines and stomach, intraarticular, in ventricle, rectum, vagina,
In skull, in urethra, in liver, in tumor.In some cases, can systematically be administered.It is to be administered partly in some cases.
The dosage of the medicine of the present invention is unrestricted, as long as obtaining desired therapeutic effect or preventive effect, and can
To carry out appropriate determination according to symptom, sex, age etc..The medicine of the present invention or the dosage of prophylactic agent can make use-case
As the therapeutic effect of disease or preventive effect are determined as index.
In the context of the present invention, " diagnosis of endometrial carcinoma " both includes judging that experimenter has suffered from intrauterine
Film cancer, also include judging whether experimenter exists the risk suffering from carcinoma of endometrium.
" treatment " used herein contains what treatment in the mammal such as mankind suffering from relevant disease or disease was correlated with
Disease or morbid state, and include:
(1) prevention disease or morbid state occur in mammal, especially susceptible in described disease when this mammal
Diseased state, but not yet it is diagnosed when suffering from this morbid state;
(2) suppression disease or morbid state, i.e. stop it to occur;Or
(3) disease or morbid state are alleviated, even if disease or morbid state disappear.
Term " is treated " and is usually directed to treat the mankind or animal (such as, applied by veterinary), wherein can reach some pre-
The therapeutic effect of phase, such as, suppresses the development (including reducing development speed, making development stop) of disease, improves disease and healing
Disease.Also include the treatment as preventive measure (such as prevention).This disease danger is developed into the most not developing into disease
The purposes of patient of danger, is also included within during term " treats ".
Advantages of the present invention and beneficial effect:
The molecular marker being found that a kind of diagnosis of endometrial carcinoma of the present invention, uses this molecular marker can be at son
The early stage that endometrial carcinoma occurs can be used as judging, it is provided that the survival rate of patient.
It addition, by the prognosis predicting patient, the present invention can provide significant information to determine treatment side for patient
Case strategy.
The medicine of the activator including TNS11 gene or albumen of the present invention can be used as controlling of new carcinoma of endometrium
Treat medicine.
The difference expression gene filtered out verified by embodiment 2 large sample
Consider prior art yet there are no the gene carrying out studying about this gene and carcinoma of endometrium dependency as time
Select gene, consider the result of gene sequencing simultaneously, select TNS11 gene (it is expressed in endometrial tissues and lowers) to carry out
Checking.
1, sample collection
Endometrial 50 example, normal endometrial tissue 60 example is collected according to the method for embodiment 1.
2, verify in mRNA level in-site
2.1 extract tissue RNA
Step is with embodiment 1.
2.2 reverse transcription
Use Reverse Transcriptase kit, with RT Buffer, l μ g total serum IgE is carried out converse record and synthesize cDNA.Use 25 μ l
Reaction system, each sample takes 1 μ g total serum IgE as template ribonucleic acid, is separately added into following components in PCR pipe: DEPC water, 5 × inverse
Transcription buffer, 10mmol/l dNTP, 0.1mmol/l DTT, 30 μm mol/l Oligo dT, 200U/ μ l MMLVRT, template
RNA.Hatch 1 hour for 42 DEG C, 72 DEG C 10 minutes, of short duration centrifugal.
2.3PCR
Primer-design software Primer Premier 5.0 is used to design mRNA fluorescent quantitation upstream and downstream PCR primer, synthesis
Primer sequence, uses the operation of SYBR Green PCR Master Mix test kit, and concrete steps by specification operates, and adopts
By 25 μ l reaction systems, each sample arranges 3 parallel pipes, and all amplified reactions are above can with ensure result
By property.Preparing following reaction system (as shown in table 1), operations is all carried out on ice:
The each component of table 1 quantitative fluorescent PCR and respective volume
With GAPDH as internal reference, using SYBR Green I as fluorescent marker, at Light Cycler quantitative fluorescent PCR
The enterprising performing PCR of instrument reacts, and determines that purpose band, Δ Δ CT method carry out relative quantification by melt curve analysis analysis and electrophoresis.
TNS11 gene primer sequence is as follows:
Forward primer: 5 '-CACTACAGCAACATTTCT-3 ' (SEQ ID NO.1);
Downstream primer: 5 '-GCACAATCTTATCCTCATA-3 ' (SEQ ID NO.2).
GAPDH gene primer sequence is as follows:
Forward primer: 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ' (SEQ ID NO.3);
Downstream primer: 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO.4)
2.4 result
Result shows, compared with normal endometrial tissue, in endometrial, the mRNA level in-site of TNS11 gene is bright
Aobvious downward, relative expression quantity is 0.19 ± 0.011, and difference has statistical significance (P < 0.05).
3, verify on protein level
3.1 extract tissue total protein
The operation of protein extraction is carried out according to the description of EpiQuik tissue/cell total protein extraction test kit.
3.2Western blot detects
The protein quantification of extraction is carried out SDS-PAGE electrophoresis, carry out afterwards transferring film, closing, one anti-hatch, two anti-hatch,
Colour developing.
3.3 statistical procedures
Image J software is used to be analyzed, with β-actin as internal reference, by TNS11 albumen the gray value of protein band
The gray value of band is normalized.Result data is all to represent in the way of mean+SD, uses
SPSS13.0 statistical software carries out statistical analysis, and difference between the two uses t inspection, it is believed that when P < has system when 0.05
Meaning learned by meter.
3.4 result
Result is as it is shown in figure 1, compared with normal endometrial tissue, in endometrial, TNS11 protein level shows
Writing and reduce, difference has statistical significance (P < 0.05).