CN102803968B - Esophageal cancer marker - Google Patents
Esophageal cancer marker Download PDFInfo
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- CN102803968B CN102803968B CN201180006719.0A CN201180006719A CN102803968B CN 102803968 B CN102803968 B CN 102803968B CN 201180006719 A CN201180006719 A CN 201180006719A CN 102803968 B CN102803968 B CN 102803968B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5082—Supracellular entities, e.g. tissue, organisms
- G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
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: taking SLC38A4 (another name: SANT4) protein expression is as the inspection method of the cancer of the esophagus of index, the cancer of the esophagus detection composition that comprises anti-SLC38A4 protein antibody, this cancer of the esophagus detects the manufacture method with composition, the cancer of the esophagus detection kit that comprises anti-SLC38A4 protein antibody, and utilize the screening technique of the cancer of the esophagus curative of SLC38A4 protein.
Background technology
Cancer of the esophagus is the tumour that betides the epithelium source of esophagus.In histology, classification is the dermoid cancer of epithelium canceration and the gland cancer of esophageal gland canceration.In Japan, dermoid cancer accounts for 90% of cancer of the esophagus entirety, and 5% of remainder is gland cancer.From then on known, Japanese cancer of the esophagus is dermoid cancer substantially.And, being mainly in the male sex of more than 60 year old, men and women's ratio is 3～5:1.Because there is no adventitia (serous coat) as the morphologic feature of esophagus, the cancer cell of propagation easily infiltrates towards periphery, also easily transfers to lymph node, so the progress of canceration is fast.Add owing to lacking subjective symptom, thereby early detection easily postpones.The prognosis of cancer of the esophagus is extreme difference in the Alimentary System including cancer of the stomach, colorectal cancer, and 5 annual survival rates of cancer of the esophagus entirety are 14% left and right.Although carcinogen is not clear, ban on opium-smoking and the opium trade can reduce the incidence of disease of esophagus dermoid cancer, enlightens thus smoking relevant with esophagus dermoid cancer.
The diagnosis of cancer of the esophagus utilizes health check-up discovery, imaging diagnosis and tumor markers.Health check-up is found in cancer, to exist hardly in early days.In cancer, sometimes find late the enlargement of lymph nodes on right side or left collarbone top.In the patient who is diagnosed as after cancer of the esophagus, 74% is carved with the dysphagia including esophagus is uncomfortable in the time making a definite diagnosis, and 14% swallows pain.
In imaging diagnosis, can utilize barium sulphate X ray to take pictures and observe easily narrowing 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.But, in common endoscopic views, be also usually difficult to find to rest on the shallow table cancer on mucosal surface.For this reason, utilize Lugol's solution (Lugol solution) dye (dye endoscopy).Lugol's solution is by the staining for glycogen being rich in normal mucous membrane scaly epithelium.If due to the cancerization of mucous membrane and atypical epithelium and glycogen amount significantly reduces, diseased region is not dyeed by Lugol's solution and forms 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 is dyed to blueness.In this dyeing, it is conditio sune qua non that carninomatosis becomes exposing surface, and intracutaneous cancer is not aobvious positive.No matter which kind of situation, the pathological diagnosis of the biopsy that utilizing combines with scope carries out becomes making a definite diagnosis of cancer of the esophagus.In the situation that being defined as cancer of the esophagus, in order to judge its invasive depth (progressive stage), implement ultrasoni fiberscope inspection and/or CT (computer tomography).Thereby can not be tested with not to the infiltration of the surrounding tissue of cancer of the esophagus and/or to the transfer of lymph node, remote organ, thereby the progressive stage of diagnosing esophageal cancer.PET checks that the evaluation being considered to being difficult to the metastasis of utilizing CT judgement is useful.
For the diagnosis that utilizes tumor markers, as mentioned above, because be dermoid cancer in more than 90% cancer of the esophagus of Japan, so be more often 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 separately SCC is 30% left and right.As the tumor markers except SCC, use CEA, CYFRA21-1 etc.But, using these labels in the situation that, along with the development of the progression rates of cancer, positive rate is higher, but in early days in the situation of cancer diagnosis low.About autoantibody, although p53 antibody has the higher feature of positive rate in more early stage case, positive rate still only has 20-30% left and right.
On the other hand, the unconventionality expression of transport protein strengthens to be noted can become one of index of canceration (patent documentation 1～3, non-patent literature 1).Feature as cancerous tumor cell can be enumerated significant propagation and/or transfer, but needs a large amount of nutrient sources because of realizing these propagation and/or shifting, 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 squamous cell carcinoma, liver cancer).But the transport protein of having reported so far including LAT1 is all expressed on a small quantity in normal cell, is the specific transport protein of cancer (non-patent literature 3 and 4) hardly.
As mentioned above, up to the present, do not find the high mark of cancer of the esophagus specificity or early esophageal cancer shown to the mark of high positive rate.Cancer of the esophagus not only lacks subjective symptom, and have in morbidity easily shift afterwards, the feature of poor prognosis.Therefore, need qualification high and can realize the tumor markers of the early detection of cancer of the esophagus to cancer of the esophagus specificity.
Prior art document
Patent documentation
Patent documentation 1: the flat 11-248546 communique of Japanese Patent Application
Patent documentation 2: Japanese Patent Application 2004-76282 communique
Patent documentation 3: the flat 10-126648 communique of Japanese Patent Application
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 in view of as above situation and make, its objective is the tumor markers that provides high to cancer of the esophagus specificity and can realize the early detection of cancer of the esophagus.And then the object of this invention is to provide: the inspection method of the cancer of the esophagus (being particularly preferably early esophageal cancer) taking this tumor markers as target, comprise the cancer of the esophagus detection composition for detection of the molecule of this tumor markers, the manufacture method of said composition, the cancer of the esophagus detection kit that comprises said composition, and utilized the screening technique of the cancer of the esophagus curative of this tumor markers.
For solving the method for problem
SLC38A4 protein (another name: the amino acid transporter (non-patent literature 4) that SNAT4) belongs to sodion dependent form, its gene is first being cloned from the muscle of rat in 2000, for people's being cloned (non-patent literature 5～7) in calendar year 2001, origin came from the cultured cell of liver.SLC38A4 protein is structurally classified as glutamine transport protein superfamily, but in fact low to the compatibility of glutamine, and its turn-over capacity is far below other glutamine transport proteins.On the other hand, mainly carry ALANINE and asparagine (non-patent literature 4).
Location about SLC38A4 in adult detects by RNA blotting in mRNA level from the muscle of rat and liver, liver content the highest (non-patent literature 6～8) in people's tissue.From the skeletal muscle except liver, brain, lung, heart, small intestine, kidney, pancreas, placenta, uterus, amplify gene (non-patent literature 9 and 10) by RT-PCR method.About the locating information of the protein level of SLC38A4, detect in liver and placenta by immunoblotting, dye by immuning tissue and in placenta, detect (non-patent literature 9 and 11).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 experimentally (patent documentation 2).In addition, in mouse, for 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 SLC38A4 gene in Hca-F compared with 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.
The present inventors are in view of above-mentioned background, in order to explore cancer Specific marker, for various mark candidates, carry out the immuning tissue's dyeing for people's cancer pathological section and normal structure section, found that, in the independently developed antigen-specific antibodies for 80 kinds of transport proteins, for the antibody of SLC38A4 protein to cancer of the esophagus tissue (particularly early stage cancer of the esophagus tissue) specificity and reaction consumingly.And, the present inventors based on SLC38A4 protein in cancer of the esophagus high expressed, in normal structure, almost can't detect such knowledge, discovery can check cancer of the esophagus taking SLC38A4 protein expression as index, and utilize the immunological method of anti-SLC38A4 protein antibody is useful in the inspection of cancer of the esophagus.And then the present inventors find, can utilize SLC38A4 protein to carry out the screening of cancer of the esophagus curative.
; the present invention relates to: the method that (is particularly preferably early stage cancer of the esophagus) taking SLC38A4 protein expression as index checking cancer of the esophagus; the cancer of the esophagus detection composition that comprises anti-SLC38A4 protein antibody; the manufacture method of said composition; the cancer of the esophagus detection kit that comprises anti-SLC38A4 protein antibody, and utilize the screening technique of the cancer of the esophagus curative of SLC38A4 protein.More specifically, the 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) method according to (1), wherein, detects SLC38A4 protein expression with antibody.
(3) method according to (2), wherein, antibody is the antibody in the identification region that comprises the amino acid sequence that the sequence number 2 in SLC38A4 protein records.
(4) according to method described in any one of (1)～(3), wherein, cancer of the esophagus is early stage cancer of the esophagus.
(5) cancer of the esophagus detection composition, wherein, comprises anti-SLC38A4 protein antibody.
(6) composition according to (5), wherein, antibody is the antibody in the identification region that comprises the amino acid sequence that the sequence number 2 in SLC38A4 protein records.
(7) according to the composition (5) or (6) described, wherein, cancer of the esophagus is early stage cancer of the esophagus.
(8) manufacture method of the composition described in (5), wherein, comprises following operation:
(a) immune SLC38A4 protein or its have the operation of an immunogenic part, and
(b) operation of the antibody of separation and/or purifying and SLC38A4 protein bound.
(9) manufacture method of (6) described composition, wherein, comprises following operation:
(a) operation of the peptide that immunity comprises the amino acid sequence that the sequence number 2 in SLC38A4 protein records, and
(b) operation of the antibody of being combined in the region of the amino acid sequence that separation and/or the purifying sequence number 2 in comprising SLC38A4 protein is recorded.
(10) manufacture method according to (9) or (10), wherein, composition is the detection composition of early stage cancer of the esophagus.
(11) cancer of the esophagus detection kit, wherein, comprises (5)～(7) described composition.
(12) screening technique of cancer of the esophagus curative, wherein, comprises following operation:
(a) provide SLC38A4 protein or its a part of operation,
(b) operation that candidate compound is contacted with SLC38A4 protein or its part, and
(c) operation of the compound of selection and SLC38A4 protein or its a part of combination.
(13) screening technique of cancer of the esophagus curative, wherein, comprises following operation:
(a) cancer of the esophagus animal pattern (except people) is granted to the operation of candidate compound or contrast,
(b) gather the operation of the tissue of the esophageal of this animal pattern, and
(c) detect the SLC38A4 protein expression in the tissue gathering, and compare, select the operation of the compound that reduces SLC38A4 protein expression.
(14) screening technique according to (12) or (13), wherein, curative is the curative of early stage cancer of the esophagus.
The effect of invention
In the present invention, distinguish that SLC38A4 protein expression is detected specifically in cancer of the esophagus, particularly in the cancer of the esophagus in the early stage differentiated stage, be detected with high positive rate.Therefore,, according to the inspection method of the cancer of the esophagus of the present invention taking SLC38A4 protein expression as index, can detect in early days cancer of the esophagus with high precision.Thus, can carry out at the commitment of the progress of cancer of the esophagus the treatment of cancer of the esophagus, can make larger contribution to the improvement of patient's treatment and patient's prognosis.In addition,, according to the screening technique of cancer of the esophagus curative of the present invention that utilizes SLC38A4 protein, can effectively identify curative candidate compound.The cancer of the esophagus of the present invention that comprises anti-SLC38A4 protein antibody detects in the screening with the curative of the inspection of composition, the cancer of the esophagus of cancer of the esophagus detection kit in the invention described above, cancer of the esophagus extremely useful.
Brief description of the drawings
Fig. 1 shows that the amino acid full length sequence of the SLC38A4 protein that derives from people is used as the figure of immunogenic amino acid sequence (in square frame: the 29th～the 47th) with artificial synthesizing.
Fig. 2 shows the antibody titer of the antiserum, antigen compatibility antibody purification and the normal rabbit serum that obtain after rabbit immunity to be analyzed to the figure of the result obtaining by antigen solid phase ELISA.X-axis represents antigen concentration (μ g/ml), and Y-axis represents absorbance (OD450).
Fig. 3 shows the cell that uses forced expression SLC38A4, evaluates the figure of the result of the performance of anti-SLC38A4 protein antibody by flow cytometer.X-axis represents the fluorescence volume of the egfp Azami-Green of the index of expressing as target gene.Y-axis represents two anti-reactivities of PE mark.
Fig. 4 shows the cell that uses forced expression SLC38A4, by the dye microphotograph of result of performance of the anti-SLC38A4 protein antibody of evaluation of cellular immunity.Left figure shows the reactivity of the 293T cell to not expressing SLC38A4 protein, and right figure shows the reactivity of the 293T cell to transient expression SLC38A4 protein.
Fig. 5 shows to use esophageal cancer patients sample, by the dye microphotograph of chromatic result of the anti-SLC38A4 protein antibody of confirmation of immuning tissue.Use typical 2 examples.Left figure is the result that uses patient's normal position, and right figure is the result that uses patient's the tissue that comprises cancer portion.
Fig. 6 is the figure that shows the positive rate of each grade of the cancer of the esophagus sample that uses anti-SLC38A4 protein antibody.
Fig. 7 shows to use the I level of WHO pathological classification and the cancer of the esophagus sample of I-II level, utilizes anti-SLC38A4 protein antibody to carry out the microphotograph of the result of immuning tissue's dyeing.In figure, show typical 4 examples of I level.
Fig. 8 shows to use the II level of WHO pathological classification and the cancer of the esophagus sample of II-III level, utilizes anti-SLC38A4 protein antibody to carry out the microphotograph of the result of immuning tissue's dyeing.In figure, show typical 4 examples of II level.
Fig. 9 is the cancer of the esophagus sample that shows the III level that uses WHO pathological classification, utilizes anti-SLC38A4 protein antibody to carry out the microphotograph of the result of immuning tissue's dyeing.In figure, show typical 4 examples of III level.
Figure 10 is the figure that shows the score of the tissue staining of each grade of the cancer of the esophagus sample that utilizes anti-SLC38A4 protein antibody.
Figure 11 shows to use anti-SLC38A4 protein antibody Alimentary System sample 6 examples (sdenocarcinoma of stomach, colorectal adenocarcinoma, the carcinoma of the rectum, cancer of pancreas, hepatocellular carcinoma, kidney) except cancer of the esophagus to be carried out to the microphotograph of the result of immuning tissue's dyeing.In figure, show the typical case of the tissue staining of each cancer sample.
Embodiment
The inspection method > of < cancer of the esophagus
The 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 " refers to the cell or tissue that becomes sample (object) detect SLC38A4 protein expression in inspection method of the present invention time.To the cell or tissue application the present invention from the isolated state of biosome." isolate from subject " and refer to by extracting cell or tissue from biosome, thus the state that the biosome in this cell or tissue and its source is isolated completely.As the subject of extraction cell or tissue, be not limited to cancer patient, also can be to be good for ordinary person's (comprise and may suffer from cancer person) as object.By in biopsy (Biopsy), gather, the internal organs of subject and/or a part for tissue supply with the inspection method of being invented.
Pathological tissue conventionally be present in state in biosome, with around Cell binding state under (as tissue) preparation, use in the method for the invention, but also can use in the method for the invention after pathological tissue is isolated from cell around.
In the case of with by the testing result of SLC38A4 protein expression for being diagnosed as object of cancer of the esophagus, as pathological tissue, preferably use by other diagnosises be judged as YES cancer tissue, be judged as YES the tissue that the probability of cancer is high or be likely the tissue of cancer.The tissue using is preferably by other diagnosises and is judged as YES the tissue of cancer or is judged as YES the high tissue of probability of cancer.Here, as other diagnosises, for example can enumerate, use the diagnosis of x-ray imaging inspection, endoscopy, ultrasonic inspection, CT examination, MRI inspection, PET inspection, tumor markers etc.Conventionally, use by more than one in above diagnosis to suspect it is the tissue of cancer.
In the present invention, detect the protein that " the SLC38A4 protein " of expressing comprises the amino acid sequence that sequence number 1 records typically.But the amino acid sequence of protein may be due to sudden change of its encoding gene etc. and is suddenlyd change at occurring in nature (, inhuman building site).Therefore, in the present invention, the natural mutation of such SLC38A4 protein also can become detected object.
In the present invention, " the detection SLC38A4 protein expression " detection having or not that comprises 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.In the situation that grasping relative quantity, for example, can relatively judge with the SLC38A4 protein expression amount of the standard model of preparing." standard model " is to have determined in advance the sample of whether expressing SLC38A4 protein.For example, can be using the pathological tissue that is defined as the position that has cancer of the esophagus as standard model of the present invention.In addition the tissue (normal structure) of, not suffering from cancer also can be used as standard model of the present invention.
The detection of the SLC38A4 protein expression in the present invention is preferably undertaken by 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, the associativity (binding capacity) taking this antibody to SLC38A4 protein is index, detects SLC38A4 protein.Here " contact " refer under anti-SLC38A4 protein antibody can be identified the physiological condition of SLC38A4 protein and place this antibody and SLC38A4 protein.For example, in the case of using this antibody to carry out the dyeing of the SLC38A4 protein on cell surface, refer to and will infiltrate the solution that contains antibody from the isolated cell or tissue of subject, or the solution that contains antibody is fully dripped or is sprayed in this cell or tissue, be placed on this antibody and can identify under the physiological condition that is present in the SLC38A4 in 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 be also favourable.
As the anti-SLC38A4 protein antibody using in detection method of the present invention, as long as there is the specific binding to SLC38A4 protein, its kind and/or source are not particularly limited.Be preferably the antibody of the amino acid sequence " GIGNSEKAAMSSQFANEDT " (sequence number 2) of the 29th～the 47th of identification SLC38A4 protein (sequence number 1).
If as anti-SLC38A4 protein antibody, use and be combined with the antibody of mark substance, can directly measure by detecting this mark and the antibody amount of SLC38A4 protein bound, be easy.But then, also there is the antibody trouble of preparing to be combined with mark substance in this method, and the general lower such problem of detection sensitivity.Therefore in the present invention, preferably utilize the two anti-methods that are combined with mark substance, and/or utilize two anti-combinations with mark substance and must the Indirect Detecting Method such as method of polymkeric substance.Here " two is anti-" is the antibody that shows specific bond for anti-SLC38A4 protein antibody.For example, in the situation that preparing anti-SLC38A4 protein antibody as rabbit antibody, resist and can use anti-rabbit igg antibody as two.For the antibody that derives from the various biological species such as rabbit, goat, mouse, commercially available have can use two anti-, can select according to the source biological species difference of anti-SLC38A4 protein antibody suitable two anti-ly, uses in the present invention.Can also replace two to resist by the albumin A, the Protein G etc. that are combined with mark substance.
The material that serves as a mark, can enumerate peroxidase, beta-D-galactosidase, microperoxisome, horseradish peroxidase (HRP), fluorescein isothiocynate (FITC), rhodamine isothiocyanate (RITC), alkaline phosphatase, biotin and radiomaterial etc.In the present invention, if use the biotin material that serves as a mark, make itself and avidin peroxidase reaction, can be with the antibody of high-sensitivity detection and SLC38A4 protein bound.
The immunohistochemical staining of bio-tissue is generally undertaken by 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 a middle fringe compiled, and learns border enterprise picture).
(1) fixing paraffin embedding
The tissue samples gathering from biosome by surgery is fixing with formalin or paraformaldehyde, absolute ethyl alcohol etc.Then use paraffin embedding.Generally after dehydration of alcohols, use dimethylbenzene processing, finally use paraffin embedding.The slice that sample after paraffin embedding is cut into required thickness (for example, 3～5 μ m are thick) stretches it on microslide.In addition, sometimes also can use alcohol fixed preparation, the dry sample sealing, freeze the replacement paraffin embedding samples such as sample.
(2) de-paraffin
Generally process successively by dimethylbenzene, alcohol and purified water.
(3) pre-treatment (antigen retrieval)
Carry out as required enzyme processing, heat treated and/or pressurized treatments etc. for antigen retrieval.
(4) endogenous peroxydase is removed
Use peroxidase at the mark substance as when dyeing, in advance with hydrogen peroxide water treatment to remove endogenous peroxidase activity.
(5) inhibition of nonspecific reaction
For example will cut into slices, by bovine serum albumin solution (, 1% solution) processing a few minutes～dozens of minutes to suppress nonspecific reaction.In addition,, if use the antibody-solutions that contains bovine serum albumin(BSA) to carry out ensuing primary antibodie reaction, can omit this operation.
(6) antibody response
The antibody that is diluted to debita spissitudo is added drop-wise in the section on microslide, then reacts dozens of minutes～several hours.After reaction finishes, by 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, the in the situation that of the anti-SLC38A4 protein antibody of mark not, can also use and be combined with Protein G, the albumin A etc. that two of peroxidase resists, is combined with peroxidase.For example, using two anti-in the situation that, in the two anti-sections that are added drop-wise on microslide that have peroxidase, then reacting dozens of minutes～several hours.After reaction finishes, by suitable buffer solution for cleaning such as phosphate buffers.
(8) chromogenic reaction
In Tris damping fluid, dissolve DAB (3,3 '-diaminobenzidine).Then add aquae hydrogenii dioxidi.Make the colour developing solution of modulation like this soak into section a few minutes (for example 5 minutes), make its colour developing.After colour developing, section is fully cleaned with tap water, to remove DAB.
(9) nuclear staining
Mayer haematine is reacted and carry out nuclear staining several seconds～tens seconds.Clean and develop the color (conventionally a few minutes) with flowing water.
(10) dehydration, sharpening, sealing
With after dehydration of alcohols, carry out sharpening processing with dimethylbenzene, finally with the sealing such as synthetic resin or glycerine, mountant.
Cell or tissue is the cancerization position in probability and/or the tissue of cancer, has strong correlativity with the ratio of the staining power of immunostaining and/or the cell that is colored by immunostaining.Therefore, in the present invention, by detecting the specific of cancerization position evaluation and/or the tissue of the SLC38A4 protein expression probability that can to carry out from the isolated cell or tissue of subject be cancer.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 because SLC38A4 protein expression is detected with high positive rate in the cancer of the esophagus in the early stage differentiated stage, thereby the method according to this invention, can judge the stage (differentiated stage) of pathological tissue.Compared with the late cancer developing with grade, the cancer cell in differentiated stage is more with normal squamous cell similar point in cellular morphology, being difficult to realize cancer distinguishes with normal, but utilize the tissue staining of the present invention of anti-SLC38A4 protein antibody, extremely useful in the detection by visual and normal nondescript early carcinoma.In the present invention, " early stage cancer of the esophagus " specifically do not reach the cancer of the esophagus in the stage of III level, is preferably the cancer of the esophagus of I level or II level.Implement the information that obtains of said method taking cancer patient as object, can be in utilization in the evaluation of the evaluation of this patient's the state of an illness or grasp, result for the treatment of etc.For example, if implement concurrently method of the present invention with treatment, can be taking the information that as a result of obtains as basic evaluation result for the treatment of.Specifically, can study the chromatic variation in pathological tissue by implement method of the present invention after granting medicament, judge result for the treatment of by the variation of the increase and decrease of dyeing part.Can utilize like this monitor of method of the present invention as result for the treatment of.On the other hand, with the people except patient, do not regard as the information that the artificial object of cancer obtains, can be in utilization in the judgement evaluation having or not of suffering from of cancer of the esophagus etc.If based on method of the present invention, can the index based on the such objectivity excellence of dyeability carry out the diagnosis of cancer.
The diagnosis of the cancer in subject normally (comprises the people who is subject to doctor's instruction by doctor.Lower same.) carry out, obtain by method of the present invention, about the data of the SLC38A4 protein expression amount in pathological tissue, doctor's diagnosis is played a role.Therefore, method of the present invention also can be described as the method for collection, prompting data that doctor's diagnosis is played a role.
< cancer of the esophagus detects with composition and manufacture method > thereof
The invention provides cancer of the esophagus detection composition, wherein, comprise anti-SLC38A4 protein antibody.As long as anti-SLC38A4 protein antibody has the specific bond to SLC38A4 protein, its kind and/or source are not particularly limited.Detecting with the antibody using in composition in cancer of the esophagus of the present invention, can be monoclonal antibody, can be also polyclonal antibody.In addition can be the polymeric form (for example, dimer, tripolymer, the tetramer, polymer) of functional fragment and/or the functional fragment of antibody in cancer of the esophagus detection of the present invention with the antibody using in composition.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 " refers to the Fab of the monovalence of the immunoglobulin (Ig) of a part that comprises a light chain and heavy chain.Can and obtain by recombination method by the papain digestion of antibody.More than one or 2 halfcystine of the hinge area that " Fab ' " comprises antibody, different from Fab owing to having added a small amount of residue at the carboxyl terminal of heavy chain CH1 domain." F (ab ') 2 " is the Fab of the divalence of the immunoglobulin (Ig) of the part that comprises two light chains and two heavy chains." Fv " is the minimum antibody fragment with complete antigen recognizing and binding site.Fv is the dimer being formed by connecting securely by non-covalent bond by variable region of heavy chain and variable region of light chain.The variable region of heavy chain that " scFv " comprises antibody and variable region of light chain, these regions are present in single polypeptied chain." sc (Fv) 2 " is that He Liangge variable region of light chain, two variable region of heavy chain is combined into strand by joint (linker) and obtains." dsFv " is the Fv of disulfide bond stabilization." bivalent antibody " is the little antibody fragment with two antigen binding sites, and this fragment is included in the variable region of heavy chain of being combined with variable region of light chain in same polypeptied chain, and the complementary region of each region and other chains forms pairing.
As anti-SLC38A4 protein antibody, be preferably the antibody of the amino acid sequence " GIGNSEKAAMSSQFANEDT " (sequence number 2) of the 29th～the 47th of identification SLC38A4 protein (sequence number 1).
Cancer of the esophagus of the present invention detects and comprises by the manufacture method of composition: 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 other compositions that allow as composition.
It is known with the preparation method for antibody using in composition in this area that cancer of the esophagus of the present invention detects, 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.
The preparation of polyclonal antibody can be carried out according to following steps.First prepare antigen (for example, the total length of the SLC38A4 protein that sequence number 1 is recorded or its part), then use this antigen to carry out immunity to animals such as rabbits.SLC38A4 protein as antigen can be prepared by separation and purification from biosome sample.In addition, also can be used as SLC38A4 protein prepared by recombinant protein as antigen.Recombinant protein is prepared as follows: the gene of coding SLC38A4 protein or its part, with in the state insertion vector that can express, are imported to suitable host by this carrier, then the protein of expressing in host is carried out to separation and purification.Peptide region that can also be by SLC38A4 protein, be 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 be by the separation and purification easily of general method.
Repeat as required immunity, the moment blood sampling of fully rising tiring of antibody, obtains serum by centrifugal treating etc.The serum obtaining can be by utilizing the affinity chromatography of Protein G, albumin A etc. to make IgG fraction.Can be by utilizing SLC38A4 protein or its a part of affinity purification, further separation and purification and the antibody as immunogenic SLC38A4 protein or its a part of combination from antiserum, IgG fraction.
Detect in cancer of the esophagus of the present invention the peptide that is particularly preferably the amino acid sequence " GIGNSEKAAMSSQFANEDT " (sequence number 2) of the 29th～the 47th that immunity comprises SLC38A4 protein with the polyclonal antibody using in composition, as the antibody separation and purification of being combined with this peptide and must polyclonal antibody.
On the other hand, monoclonal antibody can be according to following step preparation.First implement immune operation according to step similar to the above.Repeat as required immunity, the moment of fully rising at antibody titer is won antibody produced cell from immune animal.Then the antibody produced cell of gained and myeloma cell are merged and acquisition hybridoma.Then select to produce clone target protein to the antibody of high specific.Can, after by hybridoma monoclonal, by the selected clone's of purifying nutrient solution, obtain target antibody.On the other hand, also can make hybridoma propagation to more than required number, be for example then transplanted to, in the abdominal cavity of animal (mouse) and make it in ascites internal breeding, then ascites be carried out to purifying, thereby obtain target antibody.In the purifying of above-mentioned nutrient solution or the purifying of ascites, preferably use the affinity chromatography of having utilized Protein G, albumin A.Also can use antigen immobilization in addition affinity chromatography.Can also use in addition the methods such as ion-exchange chromatography, gel permeation chromatography, ammonium sulfate classification and centrifuging.These methods can be used separately or combination in any is used.
Taking the antibody that obtains like this or its gene as basis, can prepare functional fragment and/or its polymer (for example, dimer, tripolymer, the tetramer and polymer) of the antibody such as Fab, Fab ', F (ab ') 2, Fv, scFv, sv (Fv) 2, dsFv and bivalent antibody.
As mentioned above, in the case of the antibody amount of direct-detection and SLC38A4 protein bound, the anti-SLC38A4 protein antibody of gained can directly pass through the marks such as enzyme, fluorescence and use.On the other hand, implementing to utilize two anti-grades while detecting with the Indirect Detecting Method of the antibody amounts of SLC38A4 protein bound, the also anti-SLC38A4 protein antibody of mark (primary antibodie) not, and to identifying the two anti-marks that carry out of this antibody.
As cancer of the esophagus detection composition 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-mannital, white sugar etc.As disintegrant, can use starch, carboxymethyl cellulose, calcium 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, Carboxymethyl Cellulose, Carboxymethyl Cellulose, lauryl sodium sulfate etc.As stabilizing agent, can use propylene glycol, diethylin sulphite, ascorbic acid etc.As preservative agent, can use phenol, Benzalkonii Chloridum, phenmethylol, methaform, methyl p-hydroxybenzoate etc.As antiseptic, can use Benzalkonii Chloridum, P-hydroxybenzoic acid, methaform etc.
< cancer of the esophagus detects uses kit >
The present invention also provides the cancer of the esophagus detection kit that comprises above-mentioned cancer of the esophagus detection composition.In kit of the present invention, except cancer of the esophagus detects with composition (antibody standard substance), substrate, positive control and/or the negative control that detection that can composite marking is required or the damping fluid that uses in the dilution of reagent and/or washing etc.Taking unlabelled antibody as antibody standard substance in the situation that, kit of the present invention for example can combine, by the material of being combined with this antibody (, two anti-, Protein G, albumin A etc.) marking and material.And kit of the present invention can comprise the operation instructions of this kit.Kit of the present invention is for example useful in the diagnosis of cancer of the esophagus.
[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 the screening technique taking SLC38A4 protein as target, the method that comprises following operation: SLC38A4 protein or its a part of operation are provided, the operation that candidate compound is contacted with SLC38A4 protein or its part, and the operation of the compound of selection and SLC38A4 protein or its a part of combination.By the method, can obtain and the compound of 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.In the situation that using antibody as candidate compound, can measure by EIA, ELISA etc. the combination activity of candidate compound.Screening with the artificial synthetic compound of protein bound by the high flux method by combinatorial chemistry technique is also 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 obtaining so also can use in the target screening of cancer of the esophagus.
Another way is the screening technique taking SLC38A4 protein expression as index, it is the method that comprises following operation: the operation of cancer of the esophagus animal pattern (except people) being granted to candidate compound or contrast, separate the operation of the tissue of the esophageal of this animal pattern, and detect separate tissue in SLC38A4 protein expression, with compare, select the operation of the compound that reduces SLC38A4 protein expression.This method can, with the above-mentioned screening technique taking SLC38A4 protein as target in conjunction with enforcement, also can independently be implemented.
In the method, first prepare cancer of the esophagus animal model, for example, can, at subcutaneous injection ethanol or the acetaldehyde of waiting of ALDH2 knock-out mice, bring out dermoid cancer, obtain the animal model (for example,, with reference to TOHKEMY 2005-110601 communique) of cancer of the esophagus.In addition also can in immunodeficient mouse, make cancer of the esophagus animal model by people's esophageal cancer cell is implanted into.
Then, this animal pattern is granted to 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.In contrast, can use positive control and/or negative control.Here as negative control, conventionally use physiological saline etc..Also can be to have distinguished the material that there is no result for the treatment of.As positive control, use and distinguished there is the material of result for the treatment of 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 selection.
Then, the tissue of the esophageal of disjunctive model animal.Separated is organized in the situation of supplying with immunostaining, as mentioned above, can be fixed the processing such as paraffin embedding.
Finally, detect the SLC38A4 protein expression in the tissue separating, select the compound that reduces compared with the control SLC38A4 protein expression.In the situation that detecting SLC38A4 protein expression by immunostaining, grant the colored graph picture of group and candidate compound and grant the colored graph picture of group by positive control relatively, select dyeing that candidate compound grants group for more negative (for example, a little less than staining power, or the ratio of the cancer cell of strong dyeing is few) compound.Colored graph picture in contrast, also can prepare positive control and grant the colored graph picture of group (have the colored graph picture of granting group of the material of result for the treatment of: standard colored graph picture) and replace negative control to grant the colored graph picture of group, select to bring above on an equal basis the compound of negative colored graph picture.
Embodiment
Illustrate in greater detail by the following examples the present invention, but the invention is not restricted to these embodiment.
(1) making of antibody
According to the amino acid sequence information of SLC38A4 protein, carry out secondary structure prediction by computing machine, expose on contact rate, flexibility, the surface that combines itself and solvent, whole factors of antigenicity, water wettability and polarity, and result judges the position antigenicity the highest (Fig. 1) of the amino acid sequence " GIGNSEKAAMSSQFANEDT " (sequence number 2) of the 29th～the 47th.The peptide of the artificial synthetic amino acid sequence that comprises the 29th～the 47th makes it be combined with KLH, then mixes with Freund's complete adjuvant, 6 times two above rabbits is carried out to immunity week about.In the antiserum that uses immobilization to have the sepharose 4B post of antigenic peptides to obtain, be purified into the IgG of antigentic specificity from immunity.For IgG purification, use immobilization to have 96 orifice plates of antigenic peptides to carry out ELISA, in the time of antigen concentration 0.4 μ g/ml, its tire (absorbance OD450) is 1.0 above in the situation that, for later experiment.Tiring of the IgG fraction of the antigentic specificity obtaining in the present embodiment is 1.2 (Fig. 2).
(2) confirming performance of antibody
The antibody of gained preferably shows reactive in the cell dyeing of the 293T cultured cell of the total length to forced expression SLC38A4 protein and flow cytometer (FCM).Expression vector has used pcDNA3.1 (invitrogen company).Load as the IRES sequence of RES in the downstream of SLC38A4 gene and load the gene of egfp Azami-Green (Amalgam company) in the tight downstream of this IRES sequence.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 SLC38A4 gene.
First, for the 293T cell (1 × 10 after importing at gene
5individual cell) cell membrane on perforate, the paraformaldehyde/phosphate buffer (PBS) with 4% is fixed 15 minutes at 4 DEG C, then under room temperature, processes 15 minutes with 0.1% TritonX-100/PBS.Then, at room temperature make the antibody-solutions (diluting) of 1 μ g/ml and cell effect 1 hour in the PBS that contains 1% BSA and 0.1%TritonX-100 of 100 μ l, then as two anti-anti-rabbit igg antibody (BECKMANCOULTER company system, the 200 times of dilutions that make PE mark.Dilution is identical with primary antibodie dilution) reaction.In the time of antibody and antigen presentation cell effect, because the luminous cell of Azami-Green (green) is simultaneously because of PE (ruddiness) luminous (, in FCM data, loose point moves to upper right).Staining cell is launched with scatter diagram, result loose point in the case of being used as the rabbit igg of negative control use does not move to upper right, on the other hand, in the case of being used as the myc tag antibody of positive control (MBL system: PL14), with use in the situation of anti-SLC38A4 protein antibody, loose point moves (Fig. 3) to upper right.In addition, by importing in contrast the 293T cell of simulation gene and the 293T cell that imported SLC38A4/SNAT4 gene with above-mentioned similarly process 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 fluorescence (redness) detected (Fig. 4) in the 293T cell that has imported SLC38A4/SNAT4 gene.From the result judgement of Fig. 2～4, the antibody of preparation is identified SLC38A4 protein specifically.
(3) utilize the histotomy (paraffin embedding) of antibody to dye
In order to make sample unification derive from Asian, all histotomies (having carried out paraffin embedding) are bought from the Outdo company (China) in Shanghai.First, use the normal portion of esophagus (left side) and each 2 examples of esophagus dermoid cancer portion (right side) to carry out chromatic confirmation.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) evaluate the progression rates of esophagus dermoid cancer.In this evaluation, using " differentiated (well differentiaed) " in the staging of WHO as I level, " middle differentiation (moderately differentiaed) ", as II level, " low differentiation (poorly differentiaed) " is as III level (following identical).Patient Com01-D7 and Com01-D8 are the esophagus dermoid cancer of WHO pathological classification I level.For histotomy is taken off to Treating Cuttings with Paraffin Wax, by the processing of 5 minutes in dimethylbenzene carry out 3 times, by the processing of 5 minutes in 100% ethanol carry out 2 times, by the processing of 5 minutes in 95% ethanol carry out 1 time, by the processing of 5 minutes in 90% ethanol carry out 1 time, by the processing of 5 minutes in 80% ethanol carry out 1 time, by the processing of 5 minutes in 70% ethanol carry out 1 time, then the processing of 5 minutes in PBS is carried out to (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) of the Tween20 that contains 0.05%, uses autoclave to process 5 minutes at 125 DEG C.In order to eliminate endogenic peroxidase activity, in the PBS that contains 3% aquae hydrogenii dioxidi, at room temperature process 10 minutes, then at room temperature process 30 minutes with the PBS (lock solution) of the BSA that contains 5% normal goats serum and 0.5%.The excessive solution that uses cloth to wipe with, adds the appropriate anti-SLC38A4 protein antibody (fully soaking the degree of histotomy) that is diluted to 1 μ g/ml by lock solution, at room temperature reacts 2 hours.At room temperature wash 3 times with the PBS that contains 0.05% Tween20, each 5 minutes, then as the stoste (fully soaking the degree of histotomy) of the appropriate Histostar (Ms+Rb) (MBL company) of two anti-interpolations, at room temperature react 60 minutes.At room temperature wash 3 times with the PBS that contains 0.05% Tween20, each 5 minutes, then react 10 minutes with DAB substrate solution (MBL company).Stop reaction by washing histotomy with water.After brazilwood extract dyeing, carry out processed with ethanol and dimethylbenzene, make sample with preparation of specimen's liquid (loose unrestrained nitre).Carry out microscopy and record with (OLYMPUS IX71) under bright-field microscope, results verification anti-SLC38A4 protein antibody do not react with normal esophageal, and react (Fig. 5) with esophagus dermoid cancer.
Then, use by method similar to the above the 96 routine esophageal cancer patients samples of recording in table 1, table 2 (is I level 27 examples of well-differentiated in the pathological classification of WHO, I-II level 4 examples between differentiated and middle differentiation, II level 36 examples of middle 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.; the situation that tested cancerous tissue entirety dyes very is doughtily strong positive (++); the situation positive (+) of tested cancerous tissue bulk dyeing; the situation that the dyeing of tested cancerous tissue part of the whole or entirety dye weakly is the weak positive (+-), the complete achromophil situation of tested cancerous tissue negative (-) in addition.Consequently, in the tested tissue samples of the 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, strong positive to the weak positive is all added and and positive example add up to 52 examples, be equivalent to all 54%.In addition, according to WHO pathological classification rank, result is classified, 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.Distinguished by this result, in the cancer of the esophagus in the more early stage differentiated stage, utilize the positive rate of anti-SLC38A4 protein antibody high (Fig. 6).In addition, the positive example in each grade is classified according to staining power, in I level, strong positive is 25.8%, and the positive is 32.3%, and the weak positive is 25.8%, in II level, strong positive is 5.1%, and the positive is 30.8%, the weak positive is 20.5%, in III level, strong positive is 0% in addition, and the positive is 7.7%, the weak positive is 7.7%, has found the higher tendency (Figure 10) of staining power in lower grade tissue staining.Think thus, the cancer of the esophagus tissue staining that utilizes anti-SLC38A4 protein antibody is extremely useful to being visually difficult to the detection of the early carcinoma of normal differentiation.
In addition, in order to evaluate the dyeability of utilizing anti-SLC38A4 protein antibody in other Alimentary Systems, sdenocarcinoma of stomach, colorectal adenocarcinoma, rectal adenocarcinoma, cancer of pancreas, hepatocellular carcinoma and kidney are carried out to tissue staining (each 4 examples of each cancer kind) according to the method for above-mentioned Fig. 5.These samples are all purchased 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 in industry
As discussed above, according to the present invention, can and detect in early days taking SLC38A4 protein expression as index high precision cancer of the esophagus.Thus, can carry out at the commitment of the development of cancer of the esophagus the treatment of cancer of the esophagus, improve patient's treatment success ratio, and can seek the improvement of patient's prognosis.In addition, according to the present invention, can be taking SLC38A4 protein as target, and taking SLC38A4 protein expression as index, effectively carry out the screening of the curative of cancer of the esophagus.Like this, the present invention can make larger contribution to the exploitation of the diagnosis of cancer of the esophagus, treatment and curative etc.
Claims (9)
1. anti-SLC38A4 protein antibody detects the purposes with composition for the manufacture of cancer of the esophagus.
2. purposes according to claim 1, wherein, cancer of the esophagus is early stage cancer of the esophagus.
3. cancer of the esophagus detection composition, wherein, comprises antibody, and this antibody is the antibody in the identification region that comprises the amino acid sequence that the sequence number 2 in SLC38A4 protein records.
4. composition according to claim 3, wherein, cancer of the esophagus is early stage cancer of the esophagus.
5. the manufacture method of composition described in claim 3, wherein, comprises following operation:
(a) operation of the peptide that immunity comprises the amino acid sequence that the sequence number 2 in SLC38A4 protein records, and
(b) operation of the antibody of being combined in the region of the amino acid sequence that separation and/or the purifying sequence number 2 in comprising SLC38A4 protein is recorded.
6. manufacture method according to claim 5, wherein, composition is the detection composition of early stage cancer of the esophagus.
7. cancer of the esophagus detection kit, wherein, comprises the composition described in claim 3 or 4.
8. anti-SLC38A4 protein antibody detects the purposes with kit for the manufacture of cancer of the esophagus.
9. purposes according to claim 8, wherein, cancer of the esophagus is early stage cancer of the esophagus.
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| CN106811548B (en) * | 2017-04-11 | 2020-06-16 | 成都望路医药技术有限公司 | SLC38A4 as a target for diagnosis and treatment of colon adenocarcinoma |
| 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 |
| CN109142755B (en) * | 2018-10-09 | 2021-06-22 | 福建省立医院 | Four-autoantibody combined detection kit for diagnosing early esophageal squamous cell carcinoma and application |
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| Title |
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| M. Desforges等.SNAT4 isoform of system A amino acid transporter is expressed in human placenta.《Am J Physiol Cell Physiol》.2005,第290卷第C305-C312页. |
| SNAT4 isoform of system A amino acid transporter is expressed in human placenta;M. Desforges等;《Am J Physiol Cell Physiol》;20050907;第290卷;第C305-C312页 * |
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