CN103575905A - Platelet-derived growth factor receptor beta detection kit and application thereof - Google Patents
Platelet-derived growth factor receptor beta detection kit and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biomedicine and in particular relates to a platelet-derived growth factor receptor beta detection kit and application thereof. The invention aims to provide a novel selection for prognostic analysis of non-muscle invasive bladder cancer. According to the technical scheme, the platelet-derived growth factor receptor beta detection kit comprises a detection agent and a catching agent which can specifically identify a tumor protein marker, namely the platelet-derived growth factor receptor beta in urine. The platelet-derived growth factor receptor beta detection kit has significance for clinically judging prognosis of bladder cancer patients and provides a novel selection for the prognostic analysis of non-muscle invasive bladder cancer.
Description
Technical field
The invention belongs to field of biomedicine technology, be specifically related to platelet derived growth factor receptor β detection kit and uses thereof.
Background technology
The microenvironment situation of urinary system has vital role for generation and the evolution of Patients with Urinary System Tumors.Urine albumen can reflect the microenvironment situation of urinary system, the simple and convenient not damaged and urine is originated, and patient is acceptant, without sense of discomfort, there is no contraindication.Therefore urine is a kind of desirable biological sample for diagnosis of urinary diseases.
By detecting every biochemical indicator of urine, reflect clinically at present the change of urinary system function, assist clinic diagnosis.But these indexs are to point out the generation of Patients with Urinary System Tumors or development.To this, a large amount of research concentrates on and in urine, finds some diseases associated protein mark, or can point out delicately in early days the generation of Patients with Urinary System Tumors and provide reference to its prognosis.In recent years, some protein markers that had bibliographical information.Yet, because the source of urine albumen is very complicated, and affected by the factors such as environment diet, physiology fluctuation is very huge, thereby has caused these disease related protein marks because the problems such as specificity and stability are not all by wide clinical application.
Summary of the invention
The object of the invention is provides a kind of new selection for the prognostic analysis of non-flesh layer aggressive carcinoma of urinary bladder.
Technical scheme of the present invention is platelet derived growth factor receptor β detection kit, the detection agent and the agent for capturing that comprise Tumor marker PDGFRB in energy specific recognition urine.
Concrete, described agent for capturing behaviour PDGFRB protein antibodies.
Further, described antibody is monoclonal antibody, polyclonal antibody or recombinant antibodies.
Concrete, described detection agent is detectable label.
Further, described detectable label composition comprises enzyme, prothetic group, fluorescent material, luminescent substance, bioluminescence material or radioactivity material.
Further, described enzyme is horseradish peroxidase, alkaline phosphatase, beta-galactase or acetylcholine vinegar enzyme.
Further, described prothetic group is streptomycete avidin/biotin albumen or avidin/biotin albumen.
Further, described fluorescent material is umbelliferone, fluorescein, fluorescein isothiocyanic acid vinegar, rhodamine, dansyl Cl or phycoerythrin.
Further, described luminescent substance is luminol.
Further, described bioluminescence material is luciferase, fluorescein or aequorin.
Further, described radioactivity material is
125i,
35s,
14c,
3h or
52mg.
The present invention also provides the purposes of described kit in non-flesh layer aggressive carcinoma of urinary bladder prognostic analysis.
Above-mentioned agent for capturing can with the packing of PDGFRB albumen, also can coupling.
Detection sample of the present invention is the urine of non-flesh layer aggressive bladder cancer patients; Above-mentioned detection kit also contains required dilution reagent or the buffer reagent of detection being necessary, and is used as the PDGFRB protein standard substance of the certain concentration of diagnostic criteria purposes.
Above-mentioned antibody can obtain by commercial sources, the conventional method that can also prepare by antibody completes, as the Hangzhoupro body of preparing by recombination method, as phage displaying antibody, the antibody of separation from transgenic animals (as mouse), the antibody of expressing with the recombinant expression carrier that proceeds to host cell, separated antibody from restructuring combinatorial antibody library.In addition, also can be by expressing and the above-mentioned people of purifying source PDGFRB albumen, with its immune animal, with adaptive immune serum, then obtain with the antibody in conventional method purified blood serum.
Prepare detection kit of the present invention, can obtain agent for capturing and the necessary dilution buffer liquid in kit by market business approach, what wherein the seizure reagent in embodiments of the invention adopted is the specific antibody of commercial source, and the method that routinely prepared by antibody prepares.Each component of mentioned reagent box only needs conventional use, without special operation, can form that reagent operation instruction is carried out or according to the operation of normal experiment method, those skilled in the art can correctly use detection kit in conjunction with the feature of sample according to each.
The recognition detection carrier of above-mentioned PDGFRB albumen is not limited to the mode of kit, and other detects carrier also can be used to detect above-mentioned protein marker as protein chip, Test paper etc.
Above-mentioned PDGFRB albumen has the purposes for the preparation of the specific antibody of non-flesh layer aggressive carcinoma of urinary bladder prognostic analysis, the testing products such as kit, protein chip or Test paper that utilize its specific antibody to judge for the preparation of non-flesh layer aggressive carcinoma of urinary bladder prognosis.
The present invention by extensive healthy population urine is carried out to proteomics systematic analysis, GO analyzes, tumor correlated albumen interphase interaction (PPI) network analysis, and verify by enzyme linked immunosorbent assay, finally searched out the marker protein of PDGFRB as non-flesh layer aggressive bladder tumor recurrence.PDGFRB detection kit of the present invention will have vital role to the prognosis of clinical judgment bladder cancer patients, and the prognostic analysis of flesh layer aggressive carcinoma of urinary bladder provides a kind of new selection, and will produce great marketable value.The present invention utilizes biochemical test to analyze the PDGFRB protein marker in non-flesh layer aggressive transitional cell bladder carcinoma, reaches the object of the bladder tumor recurrence possibility that prestores, and detection method is unique, and specificity is high, therefore has great market outlook.
Accompanying drawing explanation
Fig. 1 is for analyzing healthy population urine process flow diagram by protein group system.Wherein: HPLC, high performance liquid chromatography; MW, molecular weight; RP, reverse-phase chromatography; MS, mass spectrum; LC, liquid chromatography; MARs, high-abundance proteins is removed system; CHIP, micro-fluidic chip; SCX, strong cation exchange.
Fig. 2 is 592 high-abundance proteins with 4 above unique peptide sections or 10 above spectroscopic datas that identify of choosing, and by Cytoscape software, is undertaken after BinGOplug-in analysis, and wherein 373 protein labelings are extracellular protein or plasmosin.
Fig. 3 is that above-mentioned 373 albumen are analyzed to draw out by STRING and included 312 nodes, with 1779 interactional protein-protein interphase interaction networks.
Fig. 4 is for to analyze retrieval to 6 Tumor-assaciated keywords in KEGG database to above-mentioned protein-protein interphase interaction network by Cytoscape and ClueGO+Cluepediaplug-in software.
Fig. 5 is the protein-protein interphase interaction network that comprises 15 Tumor-assaciateds urine albumen by STRING software building.
Fig. 6 is before enzyme linked immunosorbent assay detects bladder cancer patients electrocision and postoperative urine PDGFRB level.
Fig. 7 is bladder patients with recurrent and patients with recurrent urine PDGFRB protein level not in postoperative 3 years.
Fig. 8 is that PDGFRB is as experimenter's working curve (ROC) of the detection index of bladder tumor recurrence.
Fig. 9 is the experimenter working curve (ROC) of PDGFRB after in conjunction with clinical indices such as patient age, diameter of tumor, cancer kitchen range quantity.
Embodiment
Inventor, through extensive healthy population urine sample is carried out to proteomics systematic analysis, tumor correlated albumen interphase interaction (PPI) network analysis, filters out APC, APC2, CDH1, COL4A1, COL4A2, COL4A3, COL4A6, CSF1R, CTNNA3, EGF, FGFR2, FN1, KLK3, abundant tumor correlated albumen in these 15 urines of LAMC1 and PDGFRB.Further research is by detecting the ELISA of PDGFRB expression in 185 non-flesh layer aggressive transitional cell bladder carcinomas, and patient is carried out to 3 years follow up a case by regular visits to by a definite date, confirmed that in urine, the relapse of PDGFRB albumen relative concentration and non-flesh layer aggressive carcinoma of urinary bladder is close.
Platelet derived growth factor receptor (platelet-derivedgrowthfactorreceptor, PDGFR) belong to receptor tyrosine kinase transmembrane glycoprotein dimer molecule, in the various kinds of cell such as normal endothelial cell, smooth muscle cell and fibroblast and neurocyte, all there is distribution, and be high expressed at various human tumour cell.PDGFR mainly comprises receptor subtype PDGFR-α (PDGFRA) and the PDGFR-β (PDGFRB) of two kinds of structural similarities.Dimeric ligand molecular makes the acceptor molecule of combination with it also form homology or heterodimer.
PDGFR and respective ligand generation specific binding make its dimerization, and cause the autophosphorylation of PDGFR and the interior tyrosine kinase of born of the same parents to activate, tyrosine residue exposes SH2 domain/PTB domain binding site and the combination of substrate protein molecule, thus signal transduction pathway in active cell.Two hypotypes of PDGFR with can inducing cell after ligand binding occur ruffling phenomenon and stress fiber disappearance, regulate the effects such as alcium and phosphor metabolization, the chemotactic that promotes cell and intercellular signal exchange.The excessive activation of PDGFR can promote propagation, conversion, the migration of tumour cell, and strengthens tumor vessel nucleus formation, and the generation of tumour, development and invasion and attack are had to vital role.
Inventor has not only verified the feasibility of the PDGFRB in urine as prediction bladder tumor recurrence possibility and prognosis judgement by experiment, and has proposed its diagnostic criteria and developed corresponding detection kit.
For further setting forth the present invention, reach technological means and the beneficial effect that predetermined goal of the invention is taked, below and by reference to the accompanying drawings and preferred embodiment describe.By bioinformatic analysis, search out the process of tumor correlated albumen mark in urine albumen, and the specificity PDGFRB expression occurring in bladder tumor recurrence Urine in Patients changes, identify the experimental result of its change with the method for application immuno-enzymatic connection absorption, thus better explain PDGFR that the present invention proposes as a kind of new Biomarkers the technical scheme for carcinoma of urinary bladder prognosis judgement.
The screening of tumor correlated albumen mark in embodiment 1 urine albumen
Choose each 100 examples of healthy population masculinity femininity, get its urine specimen, by 4 different proteins group separation methods (Fig. 1), in healthy population urine, identify altogether 1641 high confidence level albumen (table 1).Choose the unique peptide section that identifies more than 4 or spectrum number the high-abundance proteins more than 10, totally 592.
By Cytoscape software, 592 high-abundance proteins are carried out after BinGOplug-in analysis, wherein 373 protein labelings are extracellular protein or plasmosin (Fig. 2).Further by STRING, analyze to draw out and include 312 nodes, with 1779 interactional protein-protein interphase interaction networks (Fig. 3).By Cytoscape and ClueGO+Cluepediaplug-in software, 6 Tumor-assaciated keywords in KEGG database are analyzed to retrieval to above-mentioned protein-protein interphase interaction network, and the final protein-protein interphase interaction network that comprises 15 Tumor-assaciateds urine albumen by STRING software building (table 2, Fig. 5).
Table 1 identifies altogether 1641 high confidence level albumen technical methods in healthy population urine
Note: in upper table+representing and carried out this operation, this step operation is not carried out in-representative.
The protein-protein interphase interaction network of a table 215 Tumor-assaciated urine albumen
Embodiment 2 urine PDGFR levels and bladder tumor recurrence relation
Choose 185 and be diagnosed as non-flesh layer aggressive carcinoma of urinary bladder by pathologic finding, and accept per urethra carcinoma of urinary bladder electrocision, the patient of the interior mitomycin C of rule row bladder and Doxorubicin perfusion therapy in postoperative 1 year.Patient's basic condition is in Table 3.All patients that include this research in all accept 3 years follow up a case by regular visits to by a definite date.In 3 years, patient is again diagnosed as the carcinoma of urinary bladder of identical or lower clinical stages through pathological diagnosis and is defined as bladder tumor recurrence.
Table 3 patient basic condition
| ? | Recurrence | Not recurrence |
| Patient's |
68 | 117 |
| Age (≤60/ > 60) | 28/40 | 52/65 |
| Sex (male/female) | 57/11 | 103/14 |
| Pathology grade Ta/T1 | 25/43 | 49/68 |
| Clinical stages (I/II) | 24/44 | 43/74 |
| Single cancer kitchen range/a plurality of cancer kitchen ranges | 30/38 | 75/42 |
| Diameter of tumor (≤3cm/ > 3cm) | 43/25 | 96/21 |
| Recurrence time (moon) | 11.2±4.3 | N/A |
185 pathology including research in are diagnosed as to non-flesh layer aggressive bladder cancer patients, before electrocision and within postoperative 3 days, collect continuously its urine specimen.The mornings 8, ︰ 00 collected stage casing urina sanguinis 10mL, under 4 ℃ of conditions, 3000 revs/min centrifugal 20 minutes, leave and take supernatant, with 10kDa molecular sieve concentrate with desalting processing after, carry out protein quantification.Afterwards, urine concentrating sample is in-80 ℃ of preservations.Adopt enzyme linked immunosorbent assay to detect PDGFRB protein concentration in its urine.In order to verify the PDGFRB marker protein relative concentration of screening in the present invention, by the urine albumen of handling well, with after Bradford method protein quantification, protein concentration furnishing is consistent.By ELISA kit (U.S. Uscn Life Science company), quantitatively urinate the PDGFRB relative concentration in albumen.ELISA detection kit the result shows PDGFRB albumen in the preoperative and is postoperative without significant difference (Fig. 6).
3 years follow up a case by regular visits to demonstration, wherein 106 (57.92%) routine patients are diagnosed as bladder tumor recurrence.And retrospective research shows, the urine albumen PDGFR relative concentration of recurrence group is significantly higher than not recurrence group, and (intermediate value is respectively 358.9ng/mg, 210.9ng/mg, interquartile-range IQR is respectively 283.36-434.91ng/mg, 152.88-229.56ng/mg), this explanation urine PDGFR level and bladder tumor recurrence (Fig. 7) in close relations.
By urine PDGFRB level, sex, age, diameter of tumor, knurl kitchen range quantity, classification, by stages etc. correlative factor and tumor recurrence carry out regretional analysis, prompting PDGFRB, diameter of tumor, knurl kitchen range quantity have appreciable impact (table 4) to tumor recurrence probability, wherein, PDGFRB concentration and recurrence correlativity be (OR=3.575, p<0.001) significantly.
The Logistic regretional analysis of table 4 urine PDGFRB level and other clinical indices and bladder tumor recurrence correlativity
| Correlative factor | Odds ratio (OR) | Standard error (SE) | P |
| Urine PDGFRB concentration | 3.575 | .213 | <0.001 |
| Sex (women/male sex) | 2.307 | .547 | .127 |
| Age | 1.730 | .402 | .172 |
| Primary tumo(u)r diameter | 3.297 | .456 | .009 |
| Pathological grading | 1.042 | .476 | .930 |
| Clinical stages | 1.811 | .484 | .220 |
| Primary carcinoma kitchen range quantity | 2.680 | .431 | .022 |
Urine PDGFRB relative concentration is as experimenter's working curve demonstration of bladder tumor recurrence probability judge index, and area under curve (AUC) is 0.64(Fig. 7).Adopt 253.76ng/mg as diagnosis index, its detection sensitivity is 80.6%, and specificity is 83.1%.As by urine PDGFRB relative concentration in conjunction with age, primary tumo(u)r diameter, three clinical indices of primary carcinoma kitchen range quantity, experimenter's working curve AUC is 0.81(Fig. 8), sensitivity is 62.7%, specificity is 85.5%.
According to variation tendency and the detection kit the result of transitional cell bladder carcinoma PDGFRB albumen in this experiment, think that the recurrence of urine PDGFRB protein level and carcinoma of urinary bladder is closely related, and can predict bladder tumor recurrence possibility as Proteinchip profiling.Specific antibody for PDGFRB albumen, as catching reagent, can quantitatively be detected to PDGFRB albumen relative concentration in urine in conjunction with suitable detection reagent.Antibody can be used with the coupling of detection reagent, to improve the specificity of detection efficiency and diagnosis.
The preparation of embodiment 3 urine PDGFRB detection kit
Prepare urine PDGFRB detection kit of the present invention, can obtain Anti-Human PDGFRB protein monoclonal antibody as the agent for capturing in kit by market business approach, the conventional method that can also prepare by antibody completes, as the Hangzhoupro body of preparing by recombination method, as phage displaying antibody, the antibody of separation from transgenic animals (as mouse), the antibody of expressing with the recombinant expression carrier that proceeds to host cell, separated antibody from restructuring combinatorial antibody library.In addition, also can be by expressing and the marker protein in the above-mentioned people of purifying source, with its immune animal, with adaptive immune serum, then obtain with the antibody in conventional method purified blood serum.
Detection agent and necessary dilution buffer liquid, what wherein the agent for capturing in embodiments of the invention adopted is the humanized PDGFRB specific antibody of commercial source, the method that routinely prepared by antibody prepares.Each component of mentioned reagent box only needs conventional use, without special operation, can form that reagent operation instruction is carried out or according to the operation of normal experiment method, those skilled in the art can correctly use detection kit in conjunction with the feature of sample according to each.
The recognition detection carrier of above-mentioned marker protein is not limited to the mode of kit, and other detects carrier also can be used to detect above-mentioned protein marker as protein chip, Test paper etc.
Above-mentioned marker protein has the purposes for the preparation of the specific antibody of disease in the urological system early screening, utilizes its specific antibody can be for the preparation of the testing products such as kit, protein chip or Test paper of disease in the urological system early screening.
The above, it is only better embodiment of the present invention, not the present invention is done to any pro forma restriction, therefore not in order to limit the present invention, any those skilled in the art, do not departing within the scope of technical solution of the present invention, any simple modification, equivalent variations and the modification above embodiment done according to technical spirit of the present invention, all still belong in the scope of technical solution of the present invention.
Claims (12)
1. platelet derived growth factor receptor β detection kit, is characterized in that: the detection agent and the agent for capturing that comprise Tumor marker platelet derived growth factor receptor β in energy specific recognition urine.
2. platelet derived growth factor receptor β detection kit as claimed in claim 1, is characterized in that: described agent for capturing behaviour platelet derived growth factor receptor β protein antibodies.
3. platelet derived growth factor receptor β detection kit as claimed in claim 2, is characterized in that: described antibody is monoclonal antibody, polyclonal antibody or recombinant antibodies.
4. the platelet derived growth factor receptor β detection kit as described in claim 1～3 any one, is characterized in that: described detection agent is detectable label.
5. platelet derived growth factor receptor β detection kit as claimed in claim 4, is characterized in that: described detectable label composition comprises enzyme, prothetic group, fluorescent material, luminescent substance, bioluminescence material or radioactivity material.
6. platelet derived growth factor receptor β detection kit as claimed in claim 5, is characterized in that: described enzyme is horseradish peroxidase, alkaline phosphatase, beta galactose enzyme or acetylcholine vinegar enzyme.
7. platelet derived growth factor receptor β detection kit as claimed in claim 5, is characterized in that: described prothetic group is streptomycete avidin/biotin albumen or avidin/biotin albumen.
8. platelet derived growth factor receptor β detection kit as claimed in claim 5, is characterized in that: described fluorescent material is umbelliferone, fluorescein, fluorescein isothiocyanic acid vinegar, rhodamine, dansyl Cl or phycoerythrin.
9. platelet derived growth factor receptor β detection kit as claimed in claim 5, is characterized in that: described luminescent substance is luminol.
10. platelet derived growth factor receptor β detection kit as claimed in claim 5, is characterized in that: described bioluminescence material is luciferase, fluorescein or aequorin.
11. platelet derived growth factor receptor β detection kit as claimed in claim 5, is characterized in that: described radioactivity material is
125i,
35s,
14c,
3h or
52mg.
The purposes of kit described in 12. claim 1～11 any one in non-flesh layer aggressive carcinoma of urinary bladder prognostic analysis.
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Citations (4)
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|---|---|---|---|---|
| CN1128496A (en) * | 1994-01-07 | 1996-08-07 | 休正公司 | Treatment of platelet derived growth factor related disorders such as cancers using inhibitors of platelet derived growth receptor |
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| CN102439454A (en) * | 2009-02-11 | 2012-05-02 | 卡里斯Mpi公司 | Molecular profiling of tumors |
| WO2012135844A2 (en) * | 2011-04-01 | 2012-10-04 | Cornell University | Circulating exosomes as diagnostic/prognostic indicators and therapeutic targets of melanoma and other cancers |
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2013
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1128496A (en) * | 1994-01-07 | 1996-08-07 | 休正公司 | Treatment of platelet derived growth factor related disorders such as cancers using inhibitors of platelet derived growth receptor |
| CN102439454A (en) * | 2009-02-11 | 2012-05-02 | 卡里斯Mpi公司 | Molecular profiling of tumors |
| WO2012052757A1 (en) * | 2010-10-20 | 2012-04-26 | Astrazeneca Ab | Tumour phenotype patient selection method |
| WO2012135844A2 (en) * | 2011-04-01 | 2012-10-04 | Cornell University | Circulating exosomes as diagnostic/prognostic indicators and therapeutic targets of melanoma and other cancers |
| WO2012135844A3 (en) * | 2011-04-01 | 2012-11-22 | Cornell University | Circulating exosomes as diagnostic/prognostic indicators and therapeutic targets of melanoma and other cancers |
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Application publication date: 20140212 |