CN102803968A - Esophageal cancer marker - Google Patents
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- CN102803968A CN102803968A CN2011800067190A CN201180006719A CN102803968A CN 102803968 A CN102803968 A CN 102803968A CN 2011800067190 A CN2011800067190 A CN 2011800067190A CN 201180006719 A CN201180006719 A CN 201180006719A CN 102803968 A CN102803968 A CN 102803968A
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Abstract
As a result of immunohistochemical staining of human cancer pathological tissue sections and normal tissue sections with various marker candidates, the antibody against the SLC38A4 protein, out of 80 spontaneously developed types of antigen-specific antibodies against a transporter, was found to react specifically to esophageal cancer tissue and in particular to show high reactivity in esophageal cancer tissue in early well-differentiated stages.
Description
Technical field
The present invention relates to: (another name: SANT4) protein expression is the inspection method of the cancer of the esophagus of index with SLC38A4; The cancer of the esophagus that comprises anti-SLC38A4 protein antibody detects uses composition; This cancer of the esophagus detects the manufacturing approach with composition; The cancer of the esophagus that comprises anti-SLC38A4 protein antibody detects uses kit, and the screening technique that utilizes the cancer of the esophagus curative of SLC38A4 protein.
Background technology
Cancer of the esophagus is the tumour that betides the epithelium property source of esophagus.On histology, mainly be categorized as the dermoid cancer of esophageal mucosa membrane carcinogenesis and the gland cancer of esophageal gland canceration.In Japan, dermoid cancer accounts for 90% of cancer of the esophagus integral body, and 5% of remainder is a gland cancer.From then on can know that Japanese cancer of the esophagus is dermoid cancer basically.And, being mainly in more than 60 year old the male sex, men and women's ratio is 3～5:1.Because do not have adventitia (serous coat) as the morphologic characteristic of esophagus, the cancer cell of propagation soaks into easily towards periphery, also transfers to lymph node easily, so the progress of canceration is fast.Add owing to the shortage subjective symptom, thereby early detection postpones easily.The prognosis of cancer of the esophagus is extreme difference in comprising the digestive system cancer of cancer of the stomach, colorectal cancer, and 5 whole annual survival rates of cancer of the esophagus are about 14%.Though carcinogen is not clear, ban on opium-smoking and the opium trade can reduce the incidence of disease of esophagus dermoid cancer, and it is relevant with the esophagus dermoid cancer to enlighten smoking thus.
The diagnosis of cancer of the esophagus utilizes health check-up discovery, imaging diagnosis and tumor markers.Health check-up is found to exist hardly in the cancer in early days.Sometimes find the enlargement of lymph nodes on right side or left collarbone top late in the cancer.Among the patient after being diagnosed as cancer of the esophagus, 74% is carved with the dysphagia that comprises that esophagus is uncomfortable when making a definite diagnosis, and 14% swallows pain.
In imaging diagnosis, can utilize the barium sulphate X ray to take pictures to observe easily narrowing down and/or being out of shape of esophagus.But this inspection method is difficult to detect early carcinoma.Scope the most usefully in the discovery of cancer in early days.Yet, in common endoscopic views, also usually be difficult to find to rest on the shallow table cancer on the mucosal surface.For this reason, utilize Lugol's solution (Lugol solution) dye (dye endoscopy).Lugol's solution is with the staining for glycogen that is rich in the normal mucous membrane scaly epithelium.If because the cancerization of mucous membrane and atypical epitheliumization and the glycogen amount significantly reduces, then diseased region is by Lugol's solution dyeing and form the not colored zone of white.But Lugol's solution not colored zone is not specific to cancer, is positive at esophagitis, atrophy position yet.Also can use the blue dyeing of iodine, in this case, normal mucosa forms not colored zone and carninomatosis becomes and dyed blueness.In this dyeing, it is conditio sune qua non that carninomatosis becomes exposing surface, and it is positive that the intracutaneous cancer does not then show.Which kind of situation no matter, the pathological diagnosis of the biopsy that utilizing combines with scope carries out becomes making a definite diagnosis of cancer of the esophagus.Confirming as under the situation of cancer of the esophagus,, implementing ultrasoni fiberscope inspection and/or CT (computer tomography) in order to judge its invasive depth (progressive stage).Thereby whether can detect, thereby the progressive stage of diagnosing esophageal cancer to the infiltration of the surrounding tissue of cancer of the esophagus and/or to the transfer of lymph node, far-end organ.The evaluation that PET inspection is considered to being difficult to utilize the MET that CT judges is useful.
For the diagnosis that utilizes tumor markers; As stated; Because cancer of the esophagus more than 90% is a dermoid cancer in Japan, so more often be utilized as the SCC (dermoid cancer related antigen) of the index of the dermoid cancer of cervix, esophagus.The diagnosis rate before operation of utilizing SCC separately is about 30%.As the tumor markers except that SCC, use CEA, CYFRA21-1 etc.But, using under the situation of these labels, positive rate is higher along with the development of the progression rates of cancer, but in early days under the situation of cancer diagnosis low.About autoantibody, though p53 antibody has the higher characteristic of positive rate in more early stage case, positive rate still has only about 20-30%.
On the other hand, the enhancing of the unconventionality expression of transport protein is pointed out to become one of index of canceration (patent documentation 1～3, non-patent literature 1).Characteristic as cancerous tumor cell can be enumerated significant propagation and/or transfer, but needs a large amount of nutrient sources because realize these propagation and/or transfer, so cancer cell increases the expression of glucose, amino acid whose transport protein sometimes.For example, known L type amino acid transporter LAT1 high expressed (non-patent literature 2) in people's various cancerous tissues (prostate cancer, colorectal cancer, carcinoma of urinary bladder, Barrett esophageal adenocarcinoma, oral cavity squamous cell carcinoma, liver cancer).Yet, comprise all expression on a small quantity in normal cell of transport protein that LAT1 has reported so far, be the specific transport protein of cancer (non-patent literature 3 and 4) hardly.
As stated, up to the present, do not find to show the mark of high positive rate to the high mark of cancer of the esophagus specificity or to early esophageal cancer.Cancer of the esophagus not only lacks subjective symptom, and have after morbidity easily shift, the characteristic of poor prognosis.Therefore, need to identify high and can realize the tumor markers of the early detection of cancer of the esophagus to the cancer of the esophagus specificity.
The prior art document
Patent documentation
Patent documentation 1: the flat 11-248546 communique of the special hope of Japan
Patent documentation 2: Japan special hope 2004-76282 communique
Patent documentation 3: the flat 10-126648 communique of the special hope of Japan
Patent documentation: No. 2008/096416 pamphlet of International Publication
Non-patent literature
Non-patent literature 1:Imai H, et al.Histopathology.2009 Jun; 54 (7): 804-13.
Non-patent literature 2:Kondoh N, et al.Int J Oncol.2007 Jul; 31 (1): 81-7.
Non-patent literature 3:del Amo EM, et al.Eur J Pharm Sci.2008 Oct 2; 35 (3): 161-74.Epub 2008 Jul 5.
Non-patent literature 4:Mackenzie B, Erickson JD.Pflugers Arch.2004 Feb; 447 (5): 784-95.Epub 2003 Jul 4.Review.
Non-patent literature 5:Sugawara M, et al.Biochim Biophys Acta.2000 Dec 20; 1509 (1-2): 7-13.
Non-patent literature 6:Hatanaka T, et al.Biochim Biophys Acta.2001 Feb 9; 1510 (1-2): 10-7.
Non-patent literature 7:Gu S, et al.Genomics.2001 Jun 15; 74 (3): 262-72.
Non-patent literature 8:Sundberg BE, et al.J Mol Neurosci.2008 Jun; 35 (2): 179-93.Epub 2008 Apr 17.
Non-patent literature 9:Desforges M, et al.J Physiol.2009 Jan 15; 587 (Pt 1): 61-72.Epub, 2008 Nov 17.
Non-patent literature 10:Gao H, et al.Biol Reprod.2009 Jun; 80 (6): 1196-208.Epub 2009 Jan 28.
Non-patent literature 11:Desforges M, et al.Am J Physiol Cell Physiol.2006Jan; 290 (1): C305-12.Epub 2005 Sep 7.
Non-patent literature 12:Song B, et al.World J Gastroenterol.2005 Mar 14; 11 (10): 1463-72.
Summary of the invention
Invent problem to be solved
The present invention makes in view of as above situation, and its objective is provides high and can realize the tumor markers of the early detection of cancer of the esophagus to the cancer of the esophagus specificity.And then the purpose of this invention is to provide: be the inspection method of the cancer of the esophagus (especially preferably early esophageal cancer) of target with this tumor markers; The cancer of the esophagus that comprises the molecule that is used to detect this tumor markers detects uses composition; The manufacturing approach of said composition; The cancer of the esophagus that comprises said composition detects uses kit, and the screening technique that has utilized the cancer of the esophagus curative of this tumor markers.
Be used to solve the method for problem
SLC38A4 protein (is called: the amino acid transporter (non-patent literature 4) that SNAT4) belongs to sodion dependent form; Its gene at first 2000 from the muscle of rat by cloning, for the people calendar year 2001 origin come from the cultured cell of liver by cloning (non-patent literature 5～7).SLC38A4 protein structurally is classified as glutamine transport protein superfamily, but in fact the compatibility to glutamine is low, and its turn-over capacity is far below other glutamine transport proteins.On the other hand, mainly carry L-alanine and asparagine (non-patent literature 4).
About the location of SLC38A4 in adult, from the muscle of rat and liver, detect liver content the highest (non-patent literature 6～8) in people's tissue through the RNA blotting in the mRNA level.From the skeletal muscle except that liver, brain, lung, heart, small intestine, kidney, pancreas, placenta, uterus, amplify gene (non-patent literature 9 and 10) through the RT-PCR method.Locating information about the protein level of SLC38A4 detects in liver and placenta through immunoblotting, in placenta, detects (non-patent literature 9 and 11) through immuning tissue's dyeing.About with the relation of cancer, in the cell line JHH4 of hepatocellular carcinoma, confirmed the expression of mRNA, but with normal liver tissue be par, express to strengthen not being identified (patent documentation 2) experimentally.In addition; In mouse; To the utilization of height metastatic strain hepatocellular carcinoma Hca-F and low metastatic hepatocellular carcinoma Hca-P the result of gene expression analysis of genetic chip; Reported the SLC38A4 gene in Hca-F than high expressed, but with the cause-effect relationship of hepatoma Metastasis not clear (non-patent literature 12).Like this, about the expression of SLC38A4 and the relation of esophagus or cancer of the esophagus, unknown so far.
Present inventors are in view of above-mentioned background; In order to explore cancer specificity marker thing; For various mark candidates, carried out immuning tissue's dyeing to section of people's carninomatosis reason and normal tissues section, the result finds; In the independently developed antigen-specific antibodies that is directed against 80 kinds of transport proteins, to the antibody of SLC38A4 protein to cancer of the esophagus tissue (particularly early stage cancer of the esophagus tissue) specificity and reaction consumingly.And; Present inventors based on SLC38A4 protein in cancer of the esophagus high expressed, in normal tissues, almost detect less than such knowledge; Discovery can be that index is checked cancer of the esophagus with the SLC38A4 protein expression, and utilizing the immunological method of anti-SLC38A4 protein antibody is useful in the inspection of cancer of the esophagus.And then present inventors find, can utilize SLC38A4 protein to carry out the screening of cancer of the esophagus curative.
Promptly; The present invention relates to: be the method for index inspection cancer of the esophagus (being preferably early stage cancer of the esophagus especially) with the SLC38A4 protein expression; The cancer of the esophagus that comprises anti-SLC38A4 protein antibody detects uses composition; The manufacturing approach of said composition, the cancer of the esophagus that comprises anti-SLC38A4 protein antibody detects uses kit, and the screening technique that utilizes the cancer of the esophagus curative of SLC38A4 protein.More specifically, the present invention provides following (1)～(14).
(1) inspection method of cancer of the esophagus wherein, comprising: the operation that detects the SLC38A4 protein expression from the isolated cell or tissue of subject.
(2), wherein, use antibody to detect the SLC38A4 protein expression according to (1) described method.
(3) according to (2) described method, wherein, antibody is the antibody that identification comprises the zone of the amino acid sequence that the sequence number 2 in the SLC38A4 protein put down in writing.
(4) according to each said method of (1)～(3), wherein, cancer of the esophagus is early stage cancer of the esophagus.
(5) cancer of the esophagus detects and uses composition, wherein, comprises anti-SLC38A4 protein antibody.
(6) according to (5) described composition, wherein, antibody is the antibody that identification comprises the zone of the amino acid sequence that the sequence number 2 in the SLC38A4 protein put down in writing.
(7) according to (5) or (6) described composition, wherein, cancer of the esophagus is early stage cancer of the esophagus.
(8) manufacturing approach of (5) described composition wherein, comprises following operation:
(a) perhaps it has the operation of an immunogenic part to immune SLC38A4 protein, and
(b) operation of the antibody of separation and/or purifying and SLC38A4 protein bound.
(9) manufacturing approach of (6) said composition wherein, comprises following operation:
(a) immunity comprises the operation of the peptide of the amino acid sequence that the sequence number 2 in the SLC38A4 protein put down in writing, and
(b) operation of the antibody that combines of the zone of the amino acid sequence put down in writing of separation and/or purifying and the sequence number 2 in comprising SLC38A4 protein.
(10) according to (9) or (10) described manufacturing approach, wherein, composition is that composition is used in the detection of early stage cancer of the esophagus.
(11) cancer of the esophagus detects and uses kit, wherein, comprises (5)～(7) described composition.
(12) screening technique of cancer of the esophagus curative wherein, comprises following operation:
(a) SLC38A4 protein or its a part of operation are provided,
(b) operation that candidate compound is contacted with SLC38A4 protein or its part, and
(c) select operation with SLC38A4 protein or its a part of compound that combines.
(13) screening technique of cancer of the esophagus curative wherein, comprises following operation:
The operation of (a) cancer of the esophagus animal pattern (except that the people) being granted candidate compound or contrasting,
(b) gather the operation of tissue of the esophageal of this animal pattern, and
(c) the SLC38A4 protein expression in the tissue of detect gathering and is compared, and selects to reduce the operation of the compound of SLC38A4 protein expression.
(14) according to (12) or (13) described screening technique, wherein, curative is the curative of early stage cancer of the esophagus.
The effect of invention
In the present invention, distinguished that the SLC38A4 protein expression is detected specifically, particularly is detected with high positive rate in cancer of the esophagus in the cancer of the esophagus that is in early stage high differential period.Therefore, according to being the inspection method of the cancer of the esophagus of the present invention of index, can detect cancer of the esophagus in early days with high precision with the SLC38A4 protein expression.Thus, can carry out the treatment of cancer of the esophagus, can make bigger contribution the improvement of patient's treatment and patient's prognosis at the commitment of the progress of cancer of the esophagus.In addition, according to the screening technique of the cancer of the esophagus curative of the present invention that utilizes SLC38A4 protein, can identify the curative candidate compound effectively.The cancer of the esophagus of the present invention that comprises anti-SLC38A4 protein antibody detects in the screening of curative of inspection with composition, the cancer of the esophagus of cancer of the esophagus detection kit in the invention described above, cancer of the esophagus extremely useful.
Description of drawings
Fig. 1 shows that the amino acid full length sequence of the SLC38A4 protein derive from the people is used as the figure of immunogenic amino acid sequence (in the square frame: the 29th～the 47th) with synthetic.
Fig. 2 shows the figure that will analyze the result who gets to the antibody titer of the antiserum, antigen compatibility antibody purification and the normal rabbit serum that obtain after the rabbit immunity through antigen solid phase ELISA.The X axle is represented antigen concentration (μ g/ml), and the Y axle is represented absorbance (OD450).
Fig. 3 is the cell that show to use forced expression SLC38A4, estimates result's the figure of the performance of anti-SLC38A4 protein antibody through flow cytometer.The X axle is represented the fluorescence volume as the egfp Azami-Green of the index of target gene expression.The Y axle is represented the reactivity that two of PE mark resists.
Fig. 4 is the cell that show to use forced expression SLC38A4, estimates result's the microphotograph of the performance of anti-SLC38A4 protein antibody through cellular immunity dyeing.Left figure shows the reactivity to the 293T cell of not expressing SLC38A4 protein, and right figure shows the reactivity to the 293T cell of transient expression SLC38A4 protein.
Fig. 5 shows to use cancer of the esophagus patient sample, confirms the chromatic result's of anti-SLC38A4 protein antibody microphotograph through immuning tissue's dyeing.Use typical 2 examples.Left figure is to use the result at patient's normal position, and right figure is to use the result of patient's the tissue that comprises cancer portion.
Fig. 6 is the figure of positive rate of each grade that show to use the cancer of the esophagus sample of anti-SLC38A4 protein antibody.
Fig. 7 shows the I level of use WHO pathological classification and the cancer of the esophagus sample of I-II level, utilizes anti-SLC38A4 protein antibody to carry out the result's of immuning tissue's dyeing microphotograph.Typical 4 examples that show the I level among the figure.
Fig. 8 shows the II level of use WHO pathological classification and the cancer of the esophagus sample of II-III level, utilizes anti-SLC38A4 protein antibody to carry out the result's of immuning tissue's dyeing microphotograph.Typical 4 examples that show the II level among the figure.
Fig. 9 is the cancer of the esophagus sample that shows the III level of using the WHO pathological classification, utilizes anti-SLC38A4 protein antibody to carry out the result's of immuning tissue's dyeing microphotograph.Typical 4 examples that show the III level among the figure.
Figure 10 is the figure of score of tissue staining that shows each grade of the cancer of the esophagus sample utilize anti-SLC38A4 protein antibody.
Figure 11 shows to use anti-SLC38A4 protein antibody digestive system cancer sample 6 examples (sdenocarcinoma of stomach, large intestine gland cancer, the carcinoma of the rectum, cancer of pancreas, hepatocellular carcinoma, kidney) except that cancer of the esophagus to be carried out the result's of immuning tissue's dyeing microphotograph.The typical case that shows the tissue staining of each cancer sample among the figure.
Embodiment
< inspection method of cancer of the esophagus >
The present invention provides the inspection method of cancer of the esophagus, wherein, comprising: the operation that detects the SLC38A4 protein expression from the isolated cell or tissue of subject.
In the present invention, " cell or tissue " is meant the cell or tissue that becomes sample (object) when in inspection method of the present invention, detecting the SLC38A4 protein expression.Cell or tissue to from the isolated state of biosome is used the present invention." isolate " and be meant through from biosome extraction cell or tissue from subject, thus the state that the biosome in this cell or tissue and its source is isolated fully.Subject as the extraction cell or tissue is not limited to the cancer patient, can be object with strong ordinary person's (comprise and possibly suffer from cancer person) also.Will be in biopsy (Biopsy) part of internal organs that gather, subject and/or tissue supply with the inspection method of being invented.
Pathological tissue usually the state in being present in biosome, promptly with on every side cell bonding state under (as tissue) preparation; Use in the method for the invention, but also can after pathological tissue is isolated from cell on every side, use in the method for the invention.
Be used for testing result under the situation that is diagnosed as purpose of cancer of the esophagus the SLC38A4 protein expression; As pathological tissue, preferred use through other diagnosises be judged as be cancer tissue, to be judged as be the high tissue of the probability of cancer, or might be the tissue of cancer.Employed tissue be preferably through other diagnosises be judged as be cancer tissue, or to be judged as be the high tissue of probability of cancer.Here,, for example can enumerate, use the diagnosis of x-ray imaging inspection, endoscopy, ultrasonic inspection, CT examination, MRI inspection, PET inspection, tumor markers etc. as other diagnosises.Usually, use through in the above diagnosis more than one to suspect it is the tissue of cancer.
" the SLC38A4 protein " of detect expressing in the present invention, typically is the protein that comprises the amino acid sequence that sequence number 1 put down in writing.But the amino acid sequence of protein maybe be because sudden change of its encoding gene etc. and suddenling change at occurring in nature (that is inhuman building site).Therefore, in the present invention, the natural mutation of such SLC38A4 protein also can become detected object.
In the present invention, " detect the SLC38A4 protein expression " and comprise the detection that has or not of SLC38A4 protein expression and detection two aspects of the degree of expression.SLC38A4 protein expression amount can be used as absolute magnitude or relative quantity is grasped.Under the situation of grasping relative quantity, for example, can relatively judge with the SLC38A4 protein expression amount of the standard model of preparing." standard model " is the sample of having confirmed whether to express SLC38A4 protein in advance.For example, can be with the pathological tissue of confirming as the position that has cancer of the esophagus as standard model of the present invention.In addition, the tissue (normal tissues) of not suffering from cancer also can be used as standard model of the present invention.
The detection of the SLC38A4 protein expression among the present invention is preferably carried out through immunological method.As immunological method, for example can enumerate immunohistochemistry staining method, ELISA method, radiommunoassay, FCM, immuno-precipitation, Western blotting etc.In immunological method, use anti-SLC38A4 protein antibody, this antibody is contacted with SLC38A4 protein, be index with this antibody to SLC38A4 combination of proteins property (binding capacity), detect SLC38A4 protein.Here " contact " be meant this antibody of physiological condition held and the SLC38A4 protein that can discern SLC38A4 protein at anti-SLC38A4 protein antibody.For example; Using this antibody to carry out under the situation of dyeing of the SLC38A4 protein on the cell surface; Be meant and soak into the solution that contains antibody from the isolated cell or tissue of subject; The solution that perhaps will contain antibody fully drips or is sprayed in this cell or tissue, is placed on this antibody and can discerns under the physiological condition that is present in the SLC38A4 in the cell or tissue.According to immunological detection, can be rapidly and sensitivity detect well, operate also easy.Detection method of the present invention to the burden of patient body little aspect also be favourable.
As the anti-SLC38A4 protein antibody that uses in the detection method of the present invention, as long as the specificity that has SLC38A4 protein combines, to the not special restriction of its kind and/or source.Be preferably the antibody of the 29th～the 47th amino acid sequence " GIGNSEKAAMSSQFANEDT " (sequence number 2) of identification SLC38A4 protein (sequence number 1).
If as anti-SLC38A4 protein antibody, use the antibody be combined with mark substance, then can directly measure and the antibody amount of SLC38A4 protein bound through detecting this mark, be easy.But then, also there is the antibody trouble of preparing to be combined with mark substance in this method, and the general low such problem of detection sensitivity.Therefore in the present invention, preferably utilize the two anti-methods that are combined with mark substance, and/or utilized for two anti-combinations and the Indirect Detecting Method such as method of the polymkeric substance that gets with mark substance.Here " two is anti-" is the antibody that shows specific bond property to anti-SLC38A4 protein antibody.For example, preparing as rabbit antibody under the situation of anti-SLC38A4 protein antibody, resisting as two and can use anti-rabbit igg antibody.To the antibody that derives from various biological species such as rabbit, goat, mouse, commercially available have can use two anti-, can select suitable two anti-ly according to the source biological species difference of anti-SLC38A4 protein antibody, uses in the present invention.Can also use the albumin A, the Protein G that are combined with mark substance to wait and replace two to resist.
The material that serves as a mark can be enumerated peroxidase, beta-D-galactosidase, microperoxisome, horseradish peroxidase (HRP), fluorescein isothiocynate (FITC), rhodamine isothiocyanate (RITC), alkaline phosphatase, biotin and radiomaterial etc.In the present invention,, make itself and avidin peroxidase reaction if use the biotin material that serves as a mark, then can be with the antibody of high-sensitivity detection and SLC38A4 protein bound.
The immunohistochemical staining of bio-tissue generally carries out through following steps (1)～(10).Wherein, about the immunohistochemistry staining method of bio-tissue can with reference to various documents with become book (for example, " ferment antibody act, change the third edition " crosses limit Gui one, and middle root one fringe is compiled, and learns border enterprise picture).
(1) fixing FFPE
To fix with formalin or paraformaldehyde, absolute ethyl alcohol etc. through the tissue samples that surgery is gathered from biosome.Use FFPE then.Generally behind dehydration of alcohols, handle, use FFPE at last with xylene.Sample behind the FFPE is cut into the slice of required thickness (for example, 3～5 μ m are thick), it is stretched on microslide.In addition, also can use sometimes pure fixed preparation, dry sealing sample, freeze sample etc. and replace the FFPE sample.
(2) take off paraffin
Generally handle successively with xylene, alcohol and purified water.
(3) pre-treatment (antigen retrieval)
Carry out enzyme processing, heat treated and/or pressurized treatments etc. as required for antigen retrieval.
(4) endogenous peroxydase is removed
Use under the situation of peroxidase at mark substance as when dyeing, in advance with the hydrogen peroxide water treatment to remove endogenous peroxidase activity.
(5) inhibition of nonspecific reaction
To cut into slices and handle a few minutes～dozens of minutes to suppress nonspecific reaction with bovine serum albumin solution (for example, 1% solution).In addition, if use the antibody-solutions that contains bovine serum albumin(BSA) to carry out ensuing one anti-reaction, then can omit this operation.
(6) antibody response
The antibody that is diluted to debita spissitudo is added drop-wise in the section on the microslide, reacts dozens of minutes～several hours then.After reaction finishes, with suitable buffer solution for cleaning such as phosphate buffers.
(7) interpolation of labelled reagent
The material that serves as a mark often uses peroxidase.In above-mentioned antibody response; Also can use the anti-SLC38A4 protein antibody that is combined with peroxidase; Under the situation of the anti-SLC38A4 protein antibody of mark not, can also use to be combined with two of peroxidase and to resist, be combined with Protein G, albumin A of peroxidase etc.For example, using under the two anti-situation,, react dozens of minutes～several hours then with two anti-being added drop-wise in the section on the microslide that are combined with peroxidase.After reaction finishes, with suitable buffer solution for cleaning such as phosphate buffers.
(8) chromogenic reaction
In the Tris damping fluid, dissolve DAB (3,3 '-diaminobenzidine).Add aquae hydrogenii dioxidi then.Make the colour developing of such modulation soak into a few minutes (for example 5 minutes) of cutting into slices, make its colour developing with solution.After the colour developing, will cut into slices and fully clean, to remove DAB with tap water.
(9) nuclear staining
The Mayer haematine is reacted carry out nuclear staining several seconds～tens seconds.Clean and develop the color (a few minutes usually) with flowing water.
(10) dehydration, sharpening, sealing
After with dehydration of alcohols, carry out sharpening with xylene and handle, at last with sealings such as synthetic resin or glycerine, mountant.
Cell or tissue is the probability of cancer and/or the cancerization position in the tissue, and the ratio of the cell that is colored with the staining power of immunostaining and/or through immunostaining has strong correlativity.Therefore, in the present invention, be evaluation and/or the cancerization position the tissue specific of the probability of cancer through detecting that the SLC38A4 protein expression can carry out from the isolated cell or tissue of subject.Thus, can visually confirm position that cancer of the esophagus exists and situation (comprise the situation of metastasis site and/or transfer, to the existing way of the cancer cell of new tissue infiltration etc.).Particularly in the cancer of the esophagus that is in early stage high differential period, be detected with high positive rate owing to the SLC38A4 protein expression, thereby according to the method for the invention, the stage of decidable pathological tissue (high differential period).Compare with the late cancer of grade development; The cancer cell of high differential period is more with normal squamous cell similarity on cellular morphology; Be difficult to realize cancer and normal differentiation; But utilized the tissue staining of the present invention of anti-SLC38A4 protein antibody, extremely useful in detection through visual and normal nondescript early carcinoma.In the present invention, " early stage cancer of the esophagus " specifically is the cancer of the esophagus that does not reach the stage of III level, is preferably the cancer of the esophagus of I level or II level.With the cancer patient is that object is implemented the information that said method obtains, can be in utilization in the evaluation of the evaluation of this patient's the state of an illness or grasp, result of treatment etc.For example, if with treat the method for embodiment of the present invention concurrently, can be the basic evaluation result of treatment with the information that as a result of obtains then.Specifically, can be through studying the chromatic variation in the pathological tissue granting after the medicament method of embodiment of the present invention, judge result of treatment by the variation of the increase and decrease of dyeing part.Can utilize the monitor of method of the present invention like this as result of treatment.On the other hand, with the people except the patient, promptly do not regard as the information that the artificial object of cancer obtains, can be in utilization in the judgement evaluation of suffering from that has or not of cancer of the esophagus etc.If based on method of the present invention, then can carry out the diagnosis of cancer based on the so excellent index of objectivity of dyeability.
The diagnosis of the cancer in the subject normally (comprises the people who receives doctor's indication by the doctor.Down together.) carry out, obtain through method of the present invention, the diagnosis to the doctor plays a role about the data of the SLC38A4 protein expression amount in the pathological tissue.Therefore, method of the present invention also can be described as the method for the data that collection, prompting play a role to doctor's diagnosis.
< cancer of the esophagus detects with composition and manufacturing approach thereof >
The present invention provides cancer of the esophagus to detect and uses composition, wherein, comprises anti-SLC38A4 protein antibody.As long as anti-SLC38A4 protein antibody has the specific bond property to SLC38A4 protein, to the not special restriction of its kind and/or source.The antibody that in cancer of the esophagus of the present invention detects with composition, uses can be monoclonal antibody, also can be polyclonal antibody.In addition, the antibody that in cancer of the esophagus of the present invention detects with composition, uses can be the polymeric form (for example, dimer, tripolymer, the tetramer, polymer) of the functional fragment and/or the functional fragment of antibody.As such functional fragment and/or its polymer, for example can enumerate Fab, Fab ', F (ab ') 2, Fv, scFv, sc (Fv) 2, dsFv and bivalent antibody (diabody) etc.Here " Fab " is meant the Fab of monovalence of the immunoglobulin (Ig) of a part that comprises a light chain and heavy chain.Can obtain through the papain digestion of antibody and through recombination method." Fab ' " comprises or 2 above halfcystines of the hinge area of antibody, owing to added a small amount of residue and different with Fab at the carboxyl terminal of heavy chain CH1 domain." F (ab ') 2 " is the Fab of divalence of immunoglobulin (Ig) that comprises the part of two light chains and two heavy chains." Fv " is the antibody fragment with minimum of complete antigen recognizing and binding site.Fv is the dimer that is formed by connecting securely through non-covalent bond variable region of heavy chain and variable region of light chain." scFv " comprises the variable region of heavy chain and the variable region of light chain of antibody, and these zones are present in the single polypeptied chain." sc (Fv) 2 " is that two variable region of heavy chain and two variable region of light chain are combined into strand through joint (linker) and get." dsFv " is the Fv of disulfide bond stabilization." bivalent antibody " is the little antibody fragment with two antigen binding sites, and this fragment packet is contained in the variable region of heavy chain that combines with variable region of light chain in the same polypeptied chain, and each zone forms pairing with the complementary region of other chains.
As anti-SLC38A4 protein antibody, be preferably the antibody of the 29th～the 47th amino acid sequence " GIGNSEKAAMSSQFANEDT " (sequence number 2) of identification SLC38A4 protein (sequence number 1).
The manufacturing approach that cancer of the esophagus of the present invention detects with composition comprises: immune SLC38A4 protein or its have the operation of an immunogenic part, and the operation of the antibody of separation and/or purifying and SLC38A4 protein bound.Further can comprise the operation of mixing as other compositions of composition permission.
It is known in this area that cancer of the esophagus of the present invention detects with the preparation method for antibody that uses in the composition; For example be recorded in Harlow and Lane; Antibodies:A Laboratory Manual (New York:Cold Spring Harbor Laboratory Press, 1988).Antibody can utilize common immunological method and phage display method to prepare.
Polyclonal Antibody Preparation can be carried out according to following steps.At first prepare antigen (for example, the total length of the SLC38A4 protein that sequence number 1 is put down in writing or its part), use this antigen that animals such as rabbit are carried out immunity then.SLC38A4 protein as antigen can prepare through separation and purification from the biosome sample.In addition, also can use SLC38A4 protein as recombinant protein preparation as antigen.Recombinant protein is prepared as follows: the gene of the SLC38A4 protein of will encoding or its part are inserted in the carrier with the state that can express, and this carrier is imported suitable host, again expressed protein in the host are carried out separation and purification.The peptide zone of can also be with SLC38A4 protein, being preferably the amino acid sequence " GIGNSEKAAMSSQFANEDT " (sequence number 2) that comprises the 29th～the 47th is prepared into the fused protein with GST, beta galactosidase, maltose-binding protein matter, histidine (His) label etc., uses this fused protein as antigen.Such fused protein can pass through method in common separation and purification easily.
Repeat immunity as required,, obtain serum through centrifugal treating etc. in the moment blood sampling that tiring of antibody fully risen.The serum that obtains can be processed IgG level branch through the affinity chromatography of utilizing Protein G, albumin A etc.Can be through utilizing SLC38A4 protein or its a part of affinity purification, from antiserum, IgG level are divided further separation and purification with as immunogenic SLC38A4 protein or its a part of antibody that combines.
The polyclonal antibody that in cancer of the esophagus of the present invention detects with composition, uses is preferably the peptide that immunity comprises the 29th～the 47th amino acid sequence " GIGNSEKAAMSSQFANEDT " (sequence number 2) of SLC38A4 protein especially, as the antibody separation and purification that combines with this peptide and must polyclonal antibody.
On the other hand, monoclonal antibody can be according to following step preparation.At first according to implementing immune operation with above-mentioned same step.Repeat immunity as required, win antibody produced cell from immune animal in the moment that antibody titer fully rises.Then the antibody produced cell and the myeloma cell of gained are merged and the acquisition hybridoma.Then select to produce the clone who target protein is had the antibody of high specific.Can after with the hybridoma monoclonalization,, obtain target antibody through the selected clone's of purifying nutrient solution.On the other hand, also can make hybridoma propagation, make it in the ascites internal breeding its intraperitoneal of being transplanted to animal (for example mouse) then, then ascites carried out purifying, thereby obtain target antibody to more than the required number.In the purifying of the purifying of above-mentioned nutrient solution or ascites, the preferred affinity chromatography of having utilized Protein G, albumin A of using.Also can use the antigen immobilization in addition affinity chromatography.Can also use methods such as ion-exchange chromatography, gel permeation chromatography, ammonium sulfate classification and centrifuging in addition.These methods can be used separately or combination in any is used.
Antibody or its gene with such acquisition are the basis; Functional fragment and/or its polymer (for example, dimer, tripolymer, the tetramer and polymer) that can prepare antibody such as Fab, Fab ', F (ab ') 2, Fv, scFv, sv (Fv) 2, dsFv and bivalent antibody.
As stated, under situation about directly detecting with the antibody amount of SLC38A4 protein bound, the anti-SLC38A4 protein antibody of gained can directly pass through marks uses such as enzyme, fluorescence.On the other hand, implement to utilize two anti-when waiting Indirect Detecting Method that detects with the antibody amount of SLC38A4 protein bound, the also anti-SLC38A4 protein antibody of mark (is anti-) not, and to discerning the two anti-marks that carry out of this antibody.
Use composition as cancer of the esophagus detection of the present invention, except containing anti-SLC38A4 protein antibody, can also contain other compositions that allow as composition.As other such compositions, for example can enumerate, support body, excipient, disintegrant, buffering agent, emulsifying agent, suspending agent, stabilizing agent, preservative agent, antiseptic, physiology salt, tagged compound, two anti-etc.As excipient, can use lactose, starch, D-sorbite, D-mannitol, white sugar etc.As disintegrant, can use starch, CMC, lime carbonate etc.As buffering agent, can use phosphate, citrate, acetate etc.As emulsifying agent, can use gum arabic, sodium alginate, bassora gum etc.As suspending agent, can use glyceryl monostearate, aluminum monostearate, methylcellulose, CMC, CMC, lauryl sodium sulfate etc.As stabilizing agent, can use propylene glycol, diethylin sulphite, ascorbic acid etc.As preservative agent, can use phenol, BZK, phenmethylol, methaform, methyl p-hydroxybenzoate etc.As antiseptic, can use BZK, P-hydroxybenzoic acid, methaform etc.
< cancer of the esophagus detects and uses kit >
The present invention also provides and comprises above-mentioned cancer of the esophagus and detect with the cancer of the esophagus detection of composition and use kit.In the kit of the present invention, except cancer of the esophagus detects with the composition (antibody standard substance), required substrate, positive control and/or the negative control of detection that can composite marking, or the damping fluid of use in the dilution of reagent and/or washing etc.Be under the situation of antibody standard substance with unlabelled antibody, kit of the present invention can make up will be with (for example, two anti-, Protein G, the albumin A etc.) marking of the material of this antibodies material.And kit of the present invention can comprise the operation instructions of this kit.Kit of the present invention is useful in the diagnosis of cancer of the esophagus for example.
[screening of cancer of the esophagus curative]
The present invention also provides the screening technique of cancer of the esophagus curative.Its a kind of mode is to be the screening technique of target with SLC38A4 protein; Be the method that comprises following operation: SLC38A4 protein or its a part of operation are provided; The operation that candidate compound and SLC38A4 protein or its part contacts, and the operation of selection and SLC38A4 protein or its a part of compound that combines.Through this method, can obtain the compound with the SLC38A4 protein bound, carry out the screening of cancer of the esophagus curative.As candidate compound, can use chemosynthesis or natural low molecular compound, natural or synthetic protein, peptide, antibody (comprising antibody of the present invention), cell extract, culture supernatant etc.Using as candidate compound under the situation of antibody, can wait the combination of measuring candidate compound active through EIA, ELISA.The compound that use is screened with the synthetic of protein bound through the high flux method of combinatorial chemistry technique also is to well known to a person skilled in the art technology (Verdine GL.Nature (ENGLAND) 1996 Nov 7; 384:11-13, Hogan JC Jr.Nature (ENGLAND) 1996Nov 7; 384:17-19).The compound that obtains so also can use in the target screening of cancer of the esophagus.
Other type is to be the screening technique of index with the SLC38A4 protein expression; It is the method that comprises following operation: the operation of cancer of the esophagus animal pattern (except that the people) being granted candidate compound or contrast; The operation of tissue of separating the esophageal of this animal pattern; And the SLC38A4 protein expression in the detection isolated tissue, and compare, select to reduce the operation of the compound of SLC38A4 protein expression.This method can with above-mentioned be that the screening technique of target combines to implement with SLC38A4 protein, also can independently implement.
In the method, at first prepare cancer of the esophagus animal model, for example, can bring out dermoid cancer, obtain the animal model (for example, with reference to TOHKEMY 2005-110601 communique) of cancer of the esophagus at the subcutaneous injection ethanol or the acetaldehyde of waiting of ALDH2 knock-out mice.Also can make the cancer of the esophagus animal model in the immunodeficient mouse in addition through people's esophageal cancer cell is implanted into.
Then, this animal pattern is granted candidate compound or contrast.As candidate compound, can use chemosynthesis or natural low molecular compound, natural or synthetic protein, peptide, antibody (comprising antibody of the present invention), cell extract, culture supernatant etc.As contrast, can use positive control and/or negative control.Here, as negative control, use physiological saline etc. usually.It also can be the material of having distinguished readily good therapeutic effect.As positive control, use and to have distinguished material that result of treatment is arranged etc.The method of granting is well known to a person skilled in the art, can from per os, intravenous injection, intracutaneous or subcutaneous etc. suitable the selection.
Then, the tissue of the esophageal of disjunctive model animal.Separated being organized under the situation of supplying with immunostaining, as stated, fixedly processing such as FFPE.
At last, the compound of comparing reduction SLC38A4 protein expression is selected and contrasted to the SLC38A4 protein expression in the detection isolated tissue.Detecting through immunostaining under the situation of SLC38A4 protein expression; Grant the colored graph picture of group and the colored graph picture that candidate compound is granted group through comparing positive control; Select dyeing that candidate compound grants group for more negative (for example; A little less than the staining power, the ratio of the cancer cell of perhaps strong dyeing is few) compound.Colored graph picture as contrast; Also can prepare positive control and grant the colored graph picture of group (the colored graph picture of granting group that the material of result of treatment is arranged: standard colored graph picture) replace negative control to grant the colored graph picture of group, select to bring on an equal basis the compound of negative colored graph picture abovely.
Embodiment
Below illustrate in greater detail the present invention through embodiment, but the invention is not restricted to these embodiment.
(1) making of antibody
Amino acid sequence information according to SLC38A4 protein; Carry out secondary structure prediction through computing machine; Expose on contact rate, flexibility, the surface that combines itself and solvent, whole factors of antigenicity, water wettability and polarity, and the result judges the position antigenicity the highest (Fig. 1) of the 29th～the 47th amino acid sequence " GIGNSEKAAMSSQFANEDT " (sequence number 2).The peptide that synthetic comprises the 29th～the 47th amino acid sequence makes it combine with KLH, mixes with Freund's complete adjuvant then, 6 times the rabbit more than two is carried out immunity week about.Be purified into the IgG of antigentic specificity the antiserum that uses immobilization to have the sepharose 4B post of antigenic peptides after immunity, to obtain.For IgG purification, use immobilization to have 96 orifice plates of antigenic peptides to carry out ELISA, its tire (absorbance OD450) is under the situation more than 1.0 when antigen concentration 0.4 μ g/ml, is used for later experiment.Tiring that the IgG level of the antigentic specificity that obtains in the present embodiment is divided is 1.2 (Fig. 2).
(2) confirming performance of antibody
The antibody of gained preferably shows reactive in to the cell dyeing of the 293T cultured cell of the total length of forced expression SLC38A4 protein and flow cytometer (FCM).Expression vector has used pcDNA3.1 (invitrogen company).Load the gene that loads egfp Azami-Green (Amalgam company) as the IRES sequence of RES and in the tight downstream of this IRES sequence in the downstream of SLC38A4 gene.This expression vector is utilized in the instantaneous importing 293T cell of lipofectamine (invitrogen company), and the cell of Azami-Green fluorescence radiation is judged as the cell that has imported the SLC38A4 gene.
At first, for the 293T cell (1 * 10 after importing at gene
5Individual cell) perforate on the cell membrane, the paraformaldehyde/phosphate buffer with 4% (PBS) is fixed 15 minutes at 4 ℃, and the TritonX-100/PBS with 0.1% handled 15 minutes under the room temperature then.Then; At room temperature make antibody-solutions (in the PBS that contains 1% BSA and 0.1%TritonX-100 of 100 μ l, diluting) and the cell effect 1 hour of 1 μ g/ml; Then as two anti-anti-rabbit igg antibody (BECKMANCOULTER corporate system, 200 times of dilutions that make the PE mark.Dilution is identical with an anti-dilution) reaction.When antibody and antigen presentation cell effect, because of the luminous cell of Azami-Green (green) simultaneously because of PE (ruddiness) luminous (that is, in the FCM data, loose point move) to upper right.Staining cell is launched with scatter diagram; The result looses point to upper right mobile under the situation of using the rabbit igg that uses as negative control; On the other hand; (the MBL system: under situation PL14) and use under the situation of anti-SLC38A4 protein antibody, the point that looses is to upper right move (Fig. 3) using myc tag antibody as positive control.In addition; Will be as the importing of contrast the simulation gene 293T cell and the 293T cell that imported the SLC38A4/SNAT4 gene with above-mentioned likewise handle after, use UV fluorescent microscope (the OLYMPUS IX71 of OLYMPUS company fluorescence microscope system) is observed.Consequently, in control cells, almost can't see fluorescence (redness), and in the 293T cell that has imported the SLC38A4/SNAT4 gene, detect fluorescence (redness) (Fig. 4).Judge the antibody specificity ground identification SLC38A4 protein of preparation from the result of Fig. 2～4.
(3) utilize the histotomy (FFPE) of antibody to dye
In order to make the sample unification derive from the Asian, all histotomies (having carried out FFPE) are bought from the Outdo company (China) in Shanghai.At first, use each 2 example of normal portion of esophagus (left side) and esophagus dermoid cancer portion (right side) to carry out chromatic affirmation.By this dyeability; Staging (Pathology and Genetics of Tumours of the Digestive System Edited by Stanley R.Hamilton Lauri A.Aaltonen IARCPress Lyon based on WHO; 2000, p16) estimated the progression rates of esophagus dermoid cancer.In this is estimated; With " high differentiation (well differentiaed) " in the staging of WHO as the I level; " middle differentiation (moderately differentiaed) " as the II level, and " low differentiation (poorly differentiaed) " is as III level (below identical).Patient Com01-D7 and Com01-D8 are the esophagus dermoid cancer of WHO pathological classification I level.For histotomy is taken off Treating Cuttings with Paraffin Wax, will be in xylene 5 minutes processing carry out 3 times, will be in 100% ethanol 5 minutes processing carry out 2 times, will be in 95% ethanol 5 minutes processing carry out 1 time, will be in 90% ethanol 5 minutes processing carry out 1 time, will be in 80% ethanol 5 minutes processing carry out 1 time, will be in 70% ethanol 5 minutes processing carry out 1 time, then will be in PBS 5 minutes processing carry out (all processing are all at room temperature carried out) 3 times.Then, in order to carry out antigen retrieval, histotomy is immersed in the 10mM citrate buffer solution (pH value 6) that contains 0.05% Tween20, handled 5 minutes at 125 ℃ with autoclave.In order to eliminate endogenic peroxidase activity, in containing the PBS of 3% aquae hydrogenii dioxidi, at room temperature handled 10 minutes, the PBS (lock solution) with the BSA that contains 5% normal goats serum and 0.5% at room temperature handled 30 minutes then.The excessive solution that uses cloth to wipe with adds an amount of being diluted to the anti-SLC38A4 protein antibody (fully soaking the degree of histotomy) of 1 μ g/ml with lock solution, at room temperature reacts 2 hours.PBS with containing 0.05% Tween20 at room temperature washs 3 times, each 5 minutes, as two anti-stostes (fully soaking the degree of histotomy) of adding an amount of Histostar (Ms+Rb) (MBL company), at room temperature reacts 60 minutes then.PBS with containing 0.05% Tween20 at room temperature washs 3 times, and each 5 minutes, then with DAB substrate solution (MBL company) reaction 10 minutes.Through stop reaction with the water washing histotomy.Behind the brazilwood extract dyeing, carry out processed, process sample with preparation of specimen's liquid (loose unrestrained nitre) with ethanol and xylene.Carry out microscopy and record with (OLYMPUS IX71) under the bright-field microscope, results verification anti-SLC38A4 protein antibody with the reaction of normal esophageal, and with esophagus dermoid cancer reaction (Fig. 5).
Then; Through using 96 routine cancer of the esophagus patient's samples of record in table 1, the table 2 with above-mentioned same method (is I level 27 examples of high differentiated in the pathological classification of WHO; I-II level 4 examples between height differentiation and middle differentiation; In II level 36 examples of differentiation, II-III level 3 examples between middle differentiation and low differentiation, PD III level 26 examples) carry out tissue staining.
Table 1
Table 2
Wherein, the degree of dyeing is divided into 4 stages according to its staining power and distribution.Promptly; The situation that seized cancerous tissue integral body dyes very doughtily is a strong positive (++); The situation of seized cancerous tissue bulk dyeing positive (+); Dyeing of seized cancerous tissue part of the whole or whole situation about dyeing are the weak positive (+-) weakly, the complete achromophil situation of seized in addition cancerous tissue negative (-).Consequently, in the seized tissue samples of investigated biopsy from esophagus dermoid cancer patient, strong positive is 10 examples; The positive is 24 examples, and the weak positive is 18 examples, and feminine gender is 44 examples; With strong positive to the weak positive all add with and positive example add up to 52 examples, be equivalent to all 54%.In addition; According to WHO pathological classification rank the result is classified; Then judge 26 examples positive (positive rate 83.9%) (Fig. 6 and Fig. 7) in 31 examples that are classified as I level and I-II level; Be classified as 22 examples positive (positive rate 56.4%) (Fig. 6 and Fig. 8) in 39 examples of II level and II-III level, be classified as 4 examples positive (positive rate 15.4%) (Fig. 6 and Fig. 9) in 26 examples of III level.Distinguish by this result, in the cancer of the esophagus that is in more early stage high differential period, utilize the positive rate high (Fig. 6) of anti-SLC38A4 protein antibody.In addition, according to the staining power classification, then in the I level, strong positive is 25.8% with the positive example in each grade; The positive is 32.3%, and the weak positive is 25.8%, and in the II level, strong positive is 5.1%; The positive is 30.8%, and the weak positive is 20.5%, and in the III level, strong positive is 0% in addition; The positive is 7.7%, and the weak positive is 7.7%, has found the high more tendency (Figure 10) of staining power in the low more then tissue staining of grade.Think that thus the cancer of the esophagus tissue staining that utilizes anti-SLC38A4 protein antibody is extremely useful to the detection that visually is difficult to the normal early carcinoma of distinguishing.
In addition, in order to estimate the dyeability of in other digestive system cancers, utilizing anti-SLC38A4 protein antibody, sdenocarcinoma of stomach, large intestine gland cancer, rectal adenocarcinoma, cancer of pancreas, hepatocellular carcinoma and kidney are carried out tissue staining (each 4 example of each cancer kind) according to the method for above-mentioned Fig. 5.These samples are all available from Shanghai OutDo company.Consequently in these 6 kinds of cancers, do not confirm dyeability (Figure 11).Show that utilizing the dyeing of anti-SLC38A4 protein antibody is to the specific reaction of squamous cell carcinoma of esophagus.Utilizability on the industry
As discussed above, according to the present invention, can be the index high precision and detect cancer of the esophagus in early days with the SLC38A4 protein expression.Thus, can carry out the treatment of cancer of the esophagus, improve patient's treatment success ratio, and can seek the improvement of patient's prognosis at the commitment of the development of cancer of the esophagus.In addition, according to the present invention, can be target, and be index, carry out the screening of the curative of cancer of the esophagus effectively with the SLC38A4 protein expression with SLC38A4 protein.Like this, the present invention can make bigger contribution to exploitation of diagnosis, treatment and the curative of cancer of the esophagus etc.
Claims (14)
1. the inspection method of cancer of the esophagus wherein, comprising: the operation that detects the SLC38A4 protein expression from the isolated cell or tissue of subject.
2. method according to claim 1 wherein, uses antibody to detect the SLC38A4 protein expression.
3. method according to claim 2, wherein, antibody is the antibody that identification comprises the zone of the amino acid sequence that the sequence number 2 in the SLC38A4 protein put down in writing.
4. according to each said method of claim 1～3, wherein, cancer of the esophagus is early stage cancer of the esophagus.
5. cancer of the esophagus detects and uses composition, wherein, comprises anti-SLC38A4 protein antibody.
6. composition according to claim 5, wherein, antibody is the antibody that identification comprises the zone of the amino acid sequence that the sequence number 2 in the SLC38A4 protein put down in writing.
7. according to claim 5 or 6 described compositions, wherein, cancer of the esophagus is early stage cancer of the esophagus.
8. the manufacturing approach of the described composition of claim 5 wherein, comprises following operation:
(a) perhaps it has the operation of an immunogenic part to immune SLC38A4 protein, and
(b) operation of the antibody of separation and/or purifying and SLC38A4 protein bound.
9. the manufacturing approach of the said composition of claim 6 wherein, comprises following operation:
(a) immunity comprises the operation of the peptide of the amino acid sequence that the sequence number 2 in the SLC38A4 protein put down in writing, and
(b) operation of the antibody that combines of the zone of the amino acid sequence put down in writing of separation and/or purifying and the sequence number 2 in comprising SLC38A4 protein.
10. according to claim 9 or 10 described manufacturing approaches, wherein, composition is that composition is used in the detection of early stage cancer of the esophagus.
11. cancer of the esophagus detects and uses kit, wherein, comprises the described composition of claim 5～7.
12. the screening technique of cancer of the esophagus curative wherein, comprises following operation:
(a) SLC38A4 protein or its a part of operation are provided,
(b) operation that candidate compound is contacted with SLC38A4 protein or its part, and
(c) select operation with SLC38A4 protein or its a part of compound that combines.
13. the screening technique of cancer of the esophagus curative wherein, comprises following operation:
The operation of (a) cancer of the esophagus animal pattern (except that the people) being granted candidate compound or contrasting,
(b) gather the operation of tissue of the esophageal of this animal pattern, and
(c) the SLC38A4 protein expression in the tissue of detect gathering and is compared, and selects to reduce the operation of the compound of SLC38A4 protein expression.
14. according to claim 12 or 13 described screening techniques, wherein, curative is the curative of early stage cancer of the esophagus.
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| CN106811548A (en) * | 2017-04-11 | 2017-06-09 | 北京泱深生物信息技术有限公司 | SLC38A4 as adenocarcinoma of colon diagnosis and treatment target |
| CN109142755A (en) * | 2018-10-09 | 2019-01-04 | 福建省立医院 | It is a kind of diagnose early stage esophageal squamous cell carcinoma four kinds of autoantibody combined detection kits and application |
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| US20140296079A1 (en) * | 2012-12-03 | 2014-10-02 | Neogenomics Laboratories | Methods for early detection of esophageal cancer |
| CN108828229B (en) * | 2018-06-15 | 2021-03-16 | 深圳华大生命科学研究院 | Esophageal cancer tumor marker combination and application thereof |
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| CN103424553A (en) * | 2013-07-18 | 2013-12-04 | 上海交通大学医学院附属瑞金医院 | HbA1c level detection reagent used as NAFLD detection reagent and used for screening medicine for treating NAFLD |
| CN106811548A (en) * | 2017-04-11 | 2017-06-09 | 北京泱深生物信息技术有限公司 | SLC38A4 as adenocarcinoma of colon diagnosis and treatment target |
| CN109142755A (en) * | 2018-10-09 | 2019-01-04 | 福建省立医院 | It is a kind of diagnose early stage esophageal squamous cell carcinoma four kinds of autoantibody combined detection kits and application |
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| Publication number | Publication date |
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| CN102803968B (en) | 2014-09-24 |
| WO2011090166A1 (en) | 2011-07-28 |
| JP2011149859A (en) | 2011-08-04 |
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