CN103792364A - Reagent for detection of ROR1 protein of circulating tumor cells in peripheral blood and application thereof - Google Patents
Reagent for detection of ROR1 protein of circulating tumor cells in peripheral blood and application thereof Download PDFInfo
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Abstract
The invention discloses a reagent for detection of an ROR1 protein in circulating blood and an application thereof, and particularly discloses the reagent for the detection of the ROR1 protein of circulating tumor cells in peripheral blood. The reagent includes an ROR1 antibody, a buffer solution, a marker, a nuclear dye and a lymphocyte identification antibody. A characteristic that ROR1 is only expressed in cancer cells and is not expressed in normal adult tissue cells is utilized, and the reagent is applied to detect the expressed ROR1 protein of the circulating tumor cells or tumor stem cells in the circulating blood, wherein the circulating tumor cells or the tumor stem cells fall off from a primary tumor nidus into the blood; limitations of a traditional method are overcome, and the accuracy and the reliability of the detection of the circulating tumor cells are greatly improved. The reagent can be used for helping detect the tumor cells in the circulating blood of a tumor patient, and is a potential breakthrough point for tumor disease diagnosis, evaluation of therapeutic effects and cancer metastasis monitoring.
Description
Technical field
The present invention relates to technical field of protein detection, in particular to a kind of reagent and application thereof for detection of circulating tumor cell ROR1 albumen in peripheral blood.
Background technology
In the growth course of tumour, tumour cell can come off and enter into the circulation system and become " circulating tumor cell " from primary lesion, and detects TS test rating as a kind of.Metastases is that the tissue that circulating tumor cell can attach to far-end after primary tumo(u)r focus enters into blood circulation starts to grow into new tumour.Clinical datas show in a large number, and circulating tumor cell is to cause tumor patient main causes of death.The separation method for circulating tumor cell developing recently has very important meaning to the early diagnosis of tumour.Importantly circulating tumor cell quantitatively can be used for predicting tumors shift consequence.In addition, tumor stem cell (one of circulating tumor cell can be led oncogenic generation) can be used to the diagnosis of metastases and recurrence.Therefore, the detection of circulating tumor cell has been acknowledged as and can be effectively applied to external early-stage cancer diagnosis, the rapid evaluation of chemotherapeutics, and individualized treatment comprises the detection of clinical sieve medicine, drug resistance, the exploitation of the monitoring of tumor recurrence and tumour novel drugs etc.But how finding responsive special and stable circulating tumor cell label is a difficult problem that perplexs at present development cycle tumour cell detection technique accurately to identify circulating tumor cell.
The method report of catching circulating tumor cell is a lot, is substantially divided into three major types: antibody Direct Acquisition, enrichment, filters.By contrast, how accurately identification identifies that isolated cell is that circulating tumor cell report is very few, and main cause is to lack to stablize special tumour cell recognizate.Existing " cell searching system (CellSearch System) " method that comprises the approval of Bureau of Drugs Supervision of the U.S. all adopts cytokeratin (CK) and/or the EpCAM positive, and the karyocyte of CD45 feminine gender is as the criterion of identification circulating tumor cell.But increasing evidence is not that all cancer cells are all expressed EpCAM.In cancer development process or in the time shifting, the EpCAM of some cancer expresses and increases and the EpCAM reduction of some cancer.Cancer cell especially by epithelial cell source property to the interstitial cell transition period, CK and EpCAM express and reduce.EpCAM expresses low cancer cell and easily comes off from entity tumor, and the cytokeratin of special construction function can impel cancer cell to have more plasticity and animal migration.Although people attempt to strengthen with more tumor marker antibody accuracy and the susceptibility of identifying circulating tumor cell, the albumen of expressing due to these tumours or only express in part population or change with the different phase of tumor development, so testing cost increase can't resolve again root problem.
ROR belongs to film surface propylhomoserin kinases family, has ROR1 and ROR2 member, finds at first in brain glioblastoma cell.Both genes respectively on No. 1 and 9, chromosome, the albumen of coding 104KD.Think that two ROR acceptors are all " orphan " acceptor all the time, find that recently Wnt5A is the interior originality part of ROR2.But the interior originality part of ROR1 is much to seek so far.It is very high and disappear after maturation that ROR grows expression embryo nervous organ.Recently find ROR1 at acute and chronic leukemia cells and the growth of cancer cell is played a decisive role.But it is very few how to monitor ROR1 and the application in diagnosing tumor, treatment, transfer progress.
Summary of the invention
The present invention aims to provide a kind of reagent and application thereof of the ROR1 albumen for detection of tumor cells expression in blood circulation, easy to solve existing shortage, the technical matters of sensitive and reliable early diagnosis of cancer and cancer metastasis monitoring.
To achieve these goals, according to an aspect of the present invention, provide a kind of reagent for detection of circulating tumor cell ROR1 albumen in peripheral blood.This reagent comprises: ROR1 antibody, damping fluid, label, core dye well lymphocyte identification antibody.
Further, core dyestuff is DAPI; Lymphocyte identification antibody is CD45.
Further, ROR1 antibody is monoclonal antibody, polyclonal antibody or immune serum.
According to another aspect of the present invention, provide a kind of kit for detection of circulating tumor cell ROR1 albumen.This kit comprises as the reagent of claim 1 or 2.
According to a further aspect of the invention, provide the application of a kind of mentioned reagent in the kit of preparing cancer diagnosis.
According to a further aspect of the invention, provide a kind of mentioned reagent to detect in vitro the application of circulating tumor cell ROR1 albumen.
According to a further aspect of the invention, provide a kind of detection method that helps cancer diagnosis.The method comprises the following steps: 1) from peripheral blood or biological fluid, separate the non-leukocytic mixed cellularity group of non-red blood cell; 2) detect cell and whether express ROR1 gene and albumen; And 3) help cancer diagnosis according to ROR1 gene and/or protein expression information.
According to a further aspect of the invention, provide a kind of for helping the method for formulation, assessment of cancer result for the treatment of and survival rate of therapeutic scheme.The method comprises the following steps: separating tumor cell in the whole blood of the cancer patient after the treatment of 1) always hanging oneself or biological fluid; 2) ROR1 gene and/or protein expression level in monitoring tumour cell; And 3) according to the formulation of ROR1 gene and/or protein expression level help therapeutic scheme, judge treatment of cancer effect and survival rate, wherein treatment is selected from operation, radiation therapy, drug therapy, targeted therapy or their conjoint therapy.
According to a further aspect of the invention, provide a kind of for helping to assess the method for cancer metastasis, drug resistance and recurrence possibility.The method comprises the following steps: separating cycle tumour cell in the whole blood of the cancer patient after the treatment of 1) always hanging oneself or biological fluid; 2) ROR1 gene and/or protein expression level in monitoring circulating tumor cell; And 3) help assessment cancer metastasis, drug resistance and recurrence possibility according to ROR1 gene and/or protein expression level, wherein treatment is selected from operation, radiation therapy, drug therapy, targeted therapy or their conjoint therapy.
According to a further aspect of the invention, provide a kind of and help cancer diagnosis detection method, help the method for assessment of cancer result for the treatment of and survival rate or help assessment cancer metastasis, drug resistance and recurrence possibility, wherein, from biological fluid, separating tumor cell adopts the one being selected from antibody Direct Acquisition method, circulating tumor cell enrichment and separation method, filtration method; Preferably, biological fluid comprises blood circulation, marrow, hydrothorax, cerebrospinal fluid, lymph liquid, ascites, urine, saliva, sputum, punching filling liquid, seminal fluid, glandular secretion thing, inflammatory exudate; Preferably, ROR1 method of protein detection comprises: immunofluorescence technique as fluorescent microscope, Flow Cytometry, microfluid, immunoprecipitation, Western-style pastry ink dot method,, enzyme linked immunosorbent assay (ELISA), radio immunoassay and mass spectrometry method; Preferably, detect the expression of ROR1 gene by PCR and/or reverse transcriptase polymerase chain reaction; Preferably, blood circulation is taken from normal person or cancer patient, and cancer patient comprises the patient of the solid tumor of suffering from epithelial origin; Preferably, cancer patient comprises the patient who suffers from lung cancer, breast cancer, oophoroma, cervical carcinoma, prostate cancer, carcinoma of urinary bladder, carcinoma of testis, the cancer of the esophagus, colorectal cancer, the cancer of the esophagus, cancer of the stomach, liver cancer, the cancer of the brain, glioma, cancer of pancreas and/or cutaneum carcinoma; Preferably, by the protein expression level of ROR1 antibody test ROR1, adopt fluorescence labeling to carry out mark, fluorescence labeling adopts immunohistochemical staining, Immunofluorescence assay and the follow-up qualitative and quantitative analysis of being undertaken by fluorescent microscope and/or fluorescence analysis thereof.
A kind of method of the drug screening for antineoplastic or inhibition cancer cell transfer is provided according to a further aspect of the invention.The method comprises: 1) separating cycle tumour cell from the biological fluid of the animal used as test from trouble cancer; 2) ROR1 gene or protein expression level in monitoring circulating tumor cell; And 3) the animal used as test candidate compound of suffering from cancer according to preset frequency and consumption and administration time, 4) separating cycle tumour cell in the biological fluid of animal used as test of trouble cancer that gives candidate compound always again; 5) again monitor from giving ROR1 gene or protein expression level in the circulating tumor cell of animal used as test of candidate compound, wherein in circulating tumor cell ROR1 gene or protein expression level lower than in step 2) in the circulating tumor cell of monitoring the candidate compound of ROR1 gene or protein expression level be target compound.
Reagent for detection of ROR1 albumen in blood circulation of the present invention comprises ROR1 antibody, damping fluid, label, core dye well separation of lymphocytes antibody, utilize ROR1 only to express at cancer cell, and not in this characteristic of normal adult tissue cellular expression, applying reagent of the present invention detects in blood circulation from primary tumo(u)r focus and is shed to the ROR1 albumen that circulating tumor cell blood or tumor stem cell are expressed, overcome the limitation of classic method, greatly improved accuracy and reliability that circulating tumor cell detects.Clinical front result shows, that reagent of the present invention can provide is quantitative, repeatably result helps doctors to treat patient.Reagent of the present invention can be used in and helps to detect the tumour cell in tumour patient circulating, and this is for the medical diagnosis on disease of tumour, and the assessment of result for the treatment of and metastasis of cancer monitoring are potential breakthrough points.
Attached caption
Figure of description is used to provide a further understanding of the present invention, forms a part of the present invention, and schematic description and description of the present invention is used for explaining the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1 shows the circulating tumor cell that in embodiment 1, immunofluorescence staining is expressed for detection of ROR1 in cancer human blood;
Fig. 2 shows detection-flow cytometer method of ROR1 circulating tumor cell in cancer human blood in embodiment 2;
Fig. 3 shows the detection of circulating tumor cell in the different grade malignancies lung cancer patient of (clinical stages) in embodiment 4;
Fig. 4 shows in embodiment 5 ROR1 albumen in the expression of results of hepatocarcinoma patient cancerous tissue; And
Fig. 5 shows in embodiment 6 testing result of the circulating tumor cell of the ROR1 positive in tumor-bearing mice blood.
Embodiment
It should be noted that, in the situation that not conflicting, the feature in embodiment and embodiment in the present invention can combine mutually.Describe below with reference to the accompanying drawings and in conjunction with the embodiments the present invention in detail.
Reagent for detection of circulating tumor cell ROR1 albumen in blood circulation of the present invention comprises ROR1 antibody, damping fluid, label, core dye well separation of lymphocytes antibody, can be used for Case treatment effect, the assessment of survival rate and the monitorings of cancer metastasis such as lung cancer, breast cancer, oophoroma, prostate cancer, cancer of the stomach, colorectal cancer.
Reagent for detection of circulating tumor cell ROR1 albumen in blood circulation of the present invention can use separately, also can identify whole blood or isolated circulating tumor cell with specific recognition with other tumor marker couplings.For example be combined with ROR1 then with circulating tumor cell quantitative ROR1 expression in the technology identification blood such as immunofluorescence with ROR1 antibody.In the present invention, be to utilize first ROR1 antibody, damping fluid and label to detect the ROR1 albumen that in blood circulation, circulating tumor cell is expressed.
Monoclonal antibody, polyclonal antibody, humanized antibody that ROR1 antibody of the present invention can be anti-human or animal, or immune serum etc.Reagent for detection of circulating tumor cell ROR1 albumen in blood circulation of the present invention is including, but not limited to following composition: the IgG (as fluorescence, enzyme etc.) of ROR1 antibody, damping fluid, mark.Fluoroscopic examination way comprises: immunohistochemical staining, Immunofluorescence assay and follow-up by fluorescent microscope and/or fluorescence analysis, as flow cytometer (Flow Cytometry), fluorospectrophotometer and microfluid (microfluidics) etc., the qualitative and quantitative analysis of carrying out.
The circulating tumor cell that reagent for detection of ROR1 albumen in blood circulation of the present invention can separate with various ways for whole blood, comprise antibody Direct Acquisition method (as " the cell searching system " of FDA approval), various circulating tumor cell enrichment and separation methods and various filter methods etc.
Can the reagent for detection of circulating tumor cell ROR1 albumen in blood circulation of the present invention can screen for Healthy People, whether suffer from cancer according to capturing cell (circulating tumor cell or other cells as stem cell etc.) the assisted diagnosis experimenter who expresses ROR1.
Reagent for detection of ROR1 albumen in blood circulation of the present invention can be for helping to carry out curative effect and survival rate assessment.Cancer patient is after treatment (comprising operation, radiation therapy, drug therapy, targeted therapy or above conjoint therapy), according to the circulating tumor cell that can capture ROR1 expression, cell quantity and ROR1 expression help to judge the cancer patient susceptibility to applied therapeutic scheme, result for the treatment of and survival rate.
Reagent for detection of ROR1 albumen in blood circulation of the present invention can be for helping monitoring to judge the possibility of cancer metastasis, resistance and recurrence.According to the circulating tumor cell that can capture ROR1 expression, cell quantity and/or ROR1 expression help to judge after cancer patient's treatment whether have the degree that shifts and shift, and whether produce resistance in therapeutic process, and the early warning of whether recurring during survival course after treatment.
ROR1 method of protein detection includes but not limited to following method: immunofluorescence technique is as fluorescent microscope or Flow Cytometry (Flow Cytometry), microfluid (microfluidics), immunoprecipitation, Western-style pastry ink dot method (immunoblotting, WesternBlotting), enzyme linked immunosorbent assay (ELISA) (ELISA), radio immunoassay (RIA) and mass spectrometry method etc.
The present invention also provides a kind of kit for detection of ROR1 gene expression dose.Gene tester includes but not limited to following methods: PCR (PCR) and reverse transcriptase polymerase chain reaction (RT-PCR) etc.Be applicable to the biological fluid extract obtaining from experimenter's (normal or cancer patient), include but not limited to blood, marrow, hydrothorax, cerebrospinal fluid, lymph liquid, ascites, urine, saliva, sputum, punching filling liquid, seminal fluid, glandular secretion thing, inflammatory exudate etc.This kit can use separately, also can combine use with existing and leaved for development other clinical tumor detection method of other tumor markers or hospital.
Reagent, kit in the present invention can be for detection of the circulating tumor cell of animal used as test for anti-cancer agent exploitations, and screening comprises the assessment of antineoplastic and inhibition cancer cell diversion medicaments.Animal used as test includes but not limited to the mouse (tumour comes from the humanized's of inoculation clone or the primary tumor tissues of people or cell) of immunity shortcoming of lotus knurl or the mouse of nonimmune shortcoming (spontaneous tumor, drug-induced, transgenosis or caused by gene alteration, or inoculation homology is cytogenetic).
Detection-the immunofluorescence staining of embodiment 1.ROR1 on circulating tumor cell
Circulating tumor cell ROR1 protein assay reagent comprises: immobile liquid, damping fluid, the anti-ROR1 antibody of fluorescence coupling, anti-CD45 antibody mixed liquor, DAPI fluorescein stain and the mounting liquid of fluorescence coupling.ROR1 antibody is that the recombinant protein obtaining from Chinese hamster ovary celI transfected with human ROR1 extracellular fragment is prepared from (ROR1 antibody recombinant protein sequence is as follows).By being fixed on the lung cancer circulating tumor cell of the enrichment on slide and PBS incubated at room after 5 minutes, inhale and abandon PBS.Add the anti-ROR1 antibody of 1:200Alexa Flour 488 couplings and the anti-CD45 antibody mixed liquor (0.5%BSA-PBS) of 1:400Alexa Flour 594 couplings, room temperature lucifuge is hatched 30 minutes.Antibody is abandoned in suction.Use 0.5%BSA-PBS washing slide 3 times, each 3 minutes.Each slide drips 20 microlitres containing DAPI mounting liquid, mounting.Fluorescence microscopy Microscopic observation.As shown in Figure 1, tumour cell is ROR1+(green)/DAPI+/CD45-, leucocyte is ROR1-/DAPI+/CD45+ (redness).ROR1 antibody recombinant protein sequence is as follows:
1 mhrprrrgtr ppllallaal llaargaaaq etelsvsael vptsswniss elnkdsyltl
61 depmnnitts lgqtaelhck vsgnppptir wfkndapvvq eprrlsfrst iygsrlrirn
121 ldttdtgyfq cvatngkevv sstgvlfvkf gppptaspgy sdeyeedgfc qpyrgiacar
181 fignrtvyme slhmqgeien qitaaftmig tsshlsdkcs qfaipslchy afpycdetss
241 vpkprdlcrd eceilenvlc qteyifarsn pmilmrlklp ncedlpqpes peaancirig
301 ipmadpinkn hkcynstgvd yrgtvsvtks grqcqpwnsq yphthtftal rfpelngghs
361 ycrnpgnqke apwcftlden fksdlcdipa cds
The circulating tumor cell that embodiment 2. flow cytometer methods are expressed for detection of ROR1 in cancer human blood
The circulating tumor cell of cancer patient and fluorescently-labeled ROR1 antibody or control antibodies (Isotype IgG) 4 ° of C in Incubating Solution are hatched 20 minutes.Incubating Solution is containing the phosphate buffer (pH7.4) of 3%FBS and 1M HEPES.Washing twice with phosphate buffer measures afterwards with flow cytometer.Result is with FlowJo software analysis and shown in Fig. 2.Figure is the circulating cells test result of representative number.The left side is the cell (blueness) of fluorescently-labeled homotype control antibodies dyeing; Right side is the cell (redness) of fluorescently-labeled ROR1 antibody staining.
The detection of embodiment 3. lung cancer patient circulating tumor cells: the comparison of ROR1 kit and classic method
The 25 routine patients that clinical case are diagnosed as to primary lung cancer have carried out comparative study.From suspending, the tumour cell of every routine lung cancer patient enrichment divides equally two parts.A part is expressed with the keratin (CK) of existing technology for detection circulating tumor cell; The ROR1 albumen of technology for detection circulating tumor cell of the present invention for another part.After acetone is fixing, the former uses the anti-CK mixtures of antibodies (anti-CK18 etc.) of FITC-coupling and the anti-CD45 antibody staining of DyLight 549-coupling, and nucleus dyes with DAPI; The ROR1 protein reagent that the latter describes with embodiment 1 detects.Circulating tumor cell is CK or the ROR1 positive, CD45 feminine gender, and the nuclear staining positive.As shown in table 1, the quantity of the circulating tumor cell (CK18+/DAPI+/CD45-) detecting by currently available technology is obviously less than the quantity of the tumour cell (ROR1+/DAPI+/CD45-) arriving by technology for detection of the present invention.The circulating tumor cell quantity detecting with the present invention also becomes positive correlation with Malignancy of Lung Cancer.
Table 1
The detection of embodiment 4. circulating tumor cells in the lung cancer patient of normal person and different grade malignancy (clinical stages)
The cell of enrichment from the whole blood of different grade malignancy lung cancer patients (clinical scale I-IV) is carried out to circulating tumor cell identification and analysis.Identify that the standard of circulating tumor cell, for expressing ROR1, has core, and without leucocyte feature, i.e. ROR1 stained positive, negative (ROR1+/DAPI+/CD45-) expressed in DAPI stained positive and CD45 dyeing.Testing result shows in Fig. 3.The circulating tumor cell of varying number in the cancer patient of 136 routine different grade malignancies, detected.Even existing circulating tumor cell occurs at peripheral blood in a lot of patients of precancerous lesion and the patient of I phase carcinoma in situ, it is significant to early diagnosis cancer.Its result can help making a definite diagnosis of cancer etc. with other clinical detection results as imaging monitor, biopsy etc. are combined with.In addition, above result shows that the quantity of circulating tumor cell in blood and cancer staging are that grade malignancy is proportionate.Clinical especially III phase and its cancer cell of IV phase patient have been transferred to its hetero-organization or organ, and the content of circulating tumor cell in blood obviously increased compared with I phase and II phase patient.Therefore, testing result of the present invention can help to determine the clinical stages of cancer as imaging monitor, biopsy etc. are combined with other clinical detection results.
Also 31 routine non-tumours being contrasted to experimenters' (two examples are non-cancer pneumonia patient, and 29 examples are NHS) by technology of the present invention is studied.Result demonstration, none contrast experimenter detects tumour cell (ROR1+/DAPI+/CD45-).
After patient's liver cancer tissue paraffin section de-waxing and aquation, with PBS(PH 7.4) rinse three times after antigen hot repair multiple.By the activity of 3% superoxol blocking-up endogenous peroxydase, PBS rinses three times.Get rid of after PBS, drip normal goats serum confining liquid, room temperature 20 minutes.After getting rid of unnecessary liquid, drip ROR1 antibody or homotype IgG contrast, 4 ℃ are spent the night.Rinse and get rid of that rear dropping biotinylation two is anti-hatches 25 ℃, 20 minutes with PBS.After washing 4 times, PBS carries out DAB colour developing.After distillation washing, redye 2 minutes, hydrochloride alcohol differentiation with haematoxylin.Then dehydration, transparent, mounting, microscopy.Fig. 4 A shows hepatocarcinoma patient liver cancer tissue cellular expression ROR1 albumen, and 4B is homotype IgG negative control.
In embodiment 6. tumor-bearing mice blood, express the detection of the circulating tumor cell of ROR1
ROR1 detection method can be applicable to new medicament screen for detection of the circulating tumor cell of animal used as test, comprises the assessment of antineoplastic and inhibition cancer cell diversion medicaments.
Fluorescein-labeled human breast cancer cell (MDA-MB-231-Luc) is inoculated into after immunity shortcoming mammary gland of mouse fat pad, mouse generation tumor in situ, treat that then tumor growth different times collection whole blood detects the expression (seeing embodiment 1 or 2, immunofluorescence technique or flow cytometer method) of ROR1 to the circulating tumor cell separating.Fig. 5 result shows that then we detect weekly circulating tumor cell and the cardinal principle imaging monitor metastases (4,6 and 8 weeks) in blood at 3 mouse (1,2,3) subcutaneous vaccination tumour cell.After another 3 (mouse 4,5,6) subcutaneous vaccination tumour cells, detect weekly the circulating tumor cell in blood, but started to accept antineoplastic at the 6th week (Erlotinib, Tarceva, 100mg/kg, once a day) treatment finishes to experiment.Result shows that circulating tumor cell appears in tumor-bearing mice after surrounding in blood, and this in stage substantially image detect and there is no sign and show to have transfer sign (table 2).By the 5th week, existing massive tumor cell appeared in blood, and image just detects lymphatic metastasis substantially.These all prove that the present invention can monitor metastases more sensitively.Table 2 shows circulating tumor cell that ROR1 in tumor experiment animal model expresses and can be used as the detection means of metastasis of cancer sensitivity.In the mouse of accepting antineoplaston, the circulating tumor cell amount in blood obviously reduces as can be seen from Figure 5.Treat in the animal blood having after two weeks and even can not find out tumour cell, continue to increase and now do not accept circulating tumor cell number in the animal blood of oncotherapy (solvent contrast).These evidences show that in blood, circulating tumor cell can be used as tumor pharmacother recruitment evaluation.
Table 2
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (11)
1. for detection of a reagent for circulating tumor cell ROR1 albumen in peripheral blood, it is characterized in that, comprising: ROR1 antibody, damping fluid, label, core dye well lymphocyte identification antibody.
2. reagent according to claim 1, is characterized in that, described core dyestuff is DAPI; Lymphocyte identification antibody is CD45.
3. reagent according to claim 1, is characterized in that, described ROR1 antibody is monoclonal antibody, polyclonal antibody or immune serum.
4. for detection of a kit for circulating tumor cell ROR1 albumen, it is characterized in that, comprise reagent as claimed any one in claims 1 to 3.
5. the application of reagent in the kit of preparing cancer diagnosis as claimed any one in claims 1 to 3.
One kind as claimed any one in claims 1 to 3 reagent detect in vitro the application of circulating tumor cell ROR1 albumen.
7. a detection method that helps cancer diagnosis, is characterized in that, comprises the following steps:
1) from peripheral blood or biological fluid, separate the non-leukocytic mixed cellularity group of non-red blood cell;
2) detect described cell and whether express ROR1 gene and/or albumen; And
3) help cancer diagnosis according to described ROR1 gene and/or protein expression information.
8. for helping the method for formulation, assessment of cancer result for the treatment of and survival rate for therapeutic scheme, it is characterized in that, comprise the following steps:
1) separating tumor cell in cancer patient's whole blood after treatment or biological fluid of always hanging oneself;
2) monitor ROR1 gene and/or protein expression level in described tumour cell; And
3) according to the formulation of described ROR1 gene and/or protein expression level help therapeutic scheme, judge described treatment of cancer effect and survival rate,
Wherein said treatment is selected from operation, radiation therapy, drug therapy, targeted therapy or their conjoint therapy.
9. for helping to assess a method for cancer metastasis, drug resistance and recurrence possibility, it is characterized in that, comprise the following steps:
1) the separating cycle tumour cell in cancer patient's whole blood after treatment or biological fluid of always hanging oneself;
2) monitor ROR1 gene and/or protein expression level in described circulating tumor cell; And
3) help assessment cancer metastasis, drug resistance and recurrence possibility according to described ROR1 gene and/or protein expression level,
Wherein said treatment is selected from operation, radiation therapy, drug therapy, targeted therapy or their conjoint therapy.
10. according to the method for the help cancer diagnosis detection method described in claim 7 to 9 any one, the method that helps assessment of cancer result for the treatment of and survival rate or help assessment cancer metastasis, drug resistance and recurrence possibility, it is characterized in that, described from biological fluid separating tumor cell adopt and be selected from one in antibody Direct Acquisition method, circulating tumor cell enrichment and separation method, filtration method; Preferably, described biological fluid comprises blood circulation, marrow, hydrothorax, cerebrospinal fluid, lymph liquid, ascites, urine, saliva, sputum, punching filling liquid, seminal fluid, glandular secretion thing, inflammatory exudate; Preferably, ROR1 method of protein detection comprises: immunofluorescence technique is as fluorescent microscope, Flow Cytometry, microfluid, immunoprecipitation, Western-style pastry ink dot method, enzyme linked immunosorbent assay (ELISA), radio immunoassay and mass spectrometry method; Preferably, detect the expression of described ROR1 gene by PCR and/or reverse transcriptase polymerase chain reaction; Preferably, described blood circulation is taken from normal person or cancer patient, and described cancer patient comprises the patient of the solid tumor of suffering from epithelial origin; Preferably, described cancer patient comprises the patient who suffers from lung cancer, breast cancer, oophoroma, cervical carcinoma, prostate cancer, carcinoma of urinary bladder, carcinoma of testis, the cancer of the esophagus, colorectal cancer, the cancer of the esophagus, cancer of the stomach, liver cancer, the cancer of the brain, glioma, cancer of pancreas and/or cutaneum carcinoma; Preferably, by the protein expression level of ROR1 antibody test ROR1, adopt fluorescence labeling to carry out mark, described fluorescence labeling adopts immunohistochemical staining, Immunofluorescence assay and the follow-up qualitative and quantitative analysis of being undertaken by fluorescent microscope and/or fluorescence analysis thereof.
11. 1 kinds of methods for the drug screening of antineoplastic or inhibition cancer cell transfer, comprising:
1) separating cycle tumour cell from the biological fluid from trouble cancer animal used as test;
2) monitor ROR1 gene or protein expression level in described circulating tumor cell; And
3) give the animal used as test candidate compound of described trouble cancer according to preset frequency and consumption and administration time,
4) separating cycle tumour cell in the biological fluid of described trouble cancer animal used as test that gives described candidate compound always again;
5) again monitor from ROR1 gene or protein expression level in the described circulating tumor cell of the described described animal used as test that gives candidate compound, in wherein said circulating tumor cell, ROR1 gene or protein expression level are lower than in step 2) in the described circulating tumor cell of monitoring the candidate compound of ROR1 gene or protein expression level be target compound.
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