CN107121551A - Biomarker combinations, detection kit and the application of nasopharyngeal carcinoma - Google Patents
Biomarker combinations, detection kit and the application of nasopharyngeal carcinoma Download PDFInfo
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- CN107121551A CN107121551A CN201710438981.1A CN201710438981A CN107121551A CN 107121551 A CN107121551 A CN 107121551A CN 201710438981 A CN201710438981 A CN 201710438981A CN 107121551 A CN107121551 A CN 107121551A
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- G—PHYSICS
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- 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
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- G—PHYSICS
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
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Abstract
The invention discloses the biomarker of nasopharyngeal carcinoma, detection kit and application.The present invention obtains blood serum designated object concentration level, and then carry out bioinformatic analysis by analyzing patients with nasopharyngeal carcinoma, choose mark and carry out the further checking of ELISA experiments, as a result show, the blood serum designated object TIMP 2 that the present invention is provided, IGF I, IL 8, SELL, CCL24, MMP3, MSP alpha, HCC 4 and nasopharyngeal carcinoma are closely related, available for clinical diagnosis and prevention detection, with good actual application value.And checked directly against the mark in serum so that it is easy to operate, quick, suitable for popularization and application.
Description
Technical field
The invention belongs to knubble biological technical field, it is related to the biomarker for nasopharyngeal carcinoma, detection kit and answers
With.
Background technology
Nasopharyngeal carcinoma (Nasopharyngeal carcinoma, NPC) is that one kind betides mucous membrane of nasopharynx, with higher pernicious
The malignant tumour of degree and extremely strong transfer ability.Nasopharyngeal carcinoma is multiple-factor inheritance disease, and it is fallen ill and (heredity is easily for inherent cause
Perception), the viral infection of EB, environmental factor, many factors such as eating habit it is relevant, early diagnosis, early treatment are redemption patients
Life and the most effective means improved the quality of living.Regrettably, nasopharyngeal carcinoma onset is hidden, with strong metastasis tendency.According to
Statistics, about 75% patient has just reached late period when medical, occurs regional nodes and/or DISTANT METASTASES IN, recurrence after treatment
Or shift then poor prognosis, the main cause as treatment of nasopharyngeal carcinoma failure.Therefore, the tumor marker of screening for nasopharyngeal cancer, strives
Take early detection, selection therapeutic regimen, prediction prognosis, monitoring recurrence or transfer that there is important clinic to nasopharyngeal carcinoma diagnosis and treatment
Meaning.
The content of the invention
It is an object of the invention to provide the biomarker combinations of nasopharyngeal carcinoma, detection kit and application.
The technical solution used in the present invention is:
At least one of specific recognition cell factor SELL, CCL24, MMP3, MSP-alpha, HCC-4 reagent are in system
Application in standby nasopharyngeal carcinoma auxiliary diagnostic box.
Can be to cell factor SELL, CCL24, MMP3, at least one of MSP-alpha, HCC-4 carry out quantitative reagent and existed
Prepare the application in nasopharyngeal carcinoma auxiliary diagnostic box.
Specific recognition cell factor TIMP-2, IGF-I, IL-8, SELL, CCL24, MMP3, MSP-alpha and HCC-4
Reagent prepare nasopharyngeal carcinoma auxiliary diagnostic box in application.
Cell factor TIMP-2, IGF-I, IL-8, SELL, CCL24, MMP3, MSP-alpha and HCC-4 can be determined
Application of the reagent of amount in nasopharyngeal carcinoma auxiliary diagnostic box is prepared.
A kind of auxiliary diagnostic box of nasopharyngeal carcinoma, the kit contains specific recognition cell factor SELL, CCL24,
At least one of MMP3, MSP-alpha, HCC-4 reagent.
A kind of auxiliary diagnostic box of nasopharyngeal carcinoma, the kit contain can to cell factor SELL, CCL24, MMP3,
At least one of MSP-alpha, HCC-4 carry out quantitative reagent.
A kind of auxiliary diagnostic box of nasopharyngeal carcinoma, the kit contains specific recognition cell factor TIMP-2, IGF-
I, IL-8, SELL, CCL24, MMP3, MSP-alpha and HCC-4 reagent.
A kind of auxiliary diagnostic box of nasopharyngeal carcinoma, the kit contain can to cell factor TIMP-2, IGF-I, IL-8,
SELL, CCL24, MMP3, MSP-alpha and HCC-4 carry out quantitative reagent.
Further, the Cleaning Principle of mentioned reagent box is ELISA principles.
The beneficial effects of the invention are as follows:
Present invention discover that blood serum designated object cell factor TIMP-2, IGF-I, IL-8, SELL, CCL24, MMP3, MSP-
Alpha and HCC-4 is closely related with nasopharyngeal carcinoma, available for clinical diagnosis and prevention detection, with good practical application valency
Value.And checked directly against the mark in serum so that it is easy to operate, quick, suitable for popularization and application.
Brief description of the drawings
Fig. 1 is the fluorescent scanning detection figure after cell factor chip is detected;
Fig. 2 is the box traction substation of protein chip data;Horizontal line in box traction substation is the median for the data entirely organized.
Fig. 3 is the non-hierarchical clustering figure of differential expression cell factor;Green average value represents low-abundance protein, black
Median levels are represented, red represents high-abundance proteins.
Fig. 4 is the expression quantity of ELISA method detection cell factor.
Embodiment
It is directed to a group-specific Tumor biomarkers to carry out examination to cancer there is provided one kind measurement in the present invention
Detection and quantitative approach.Compared with normal person, the cell that there is differential expression in the presence of some may be contained in patients with nasopharyngeal carcinoma
The factor.The evaluation of the albumen (biomarker) unique to the group will be supplemented routine diagnostic method and be conducive to cancer
Early diagnosis.
The present invention collect 12, do not receive chemotherapy with radiotherapy, be diagnosed as nose by SABC and pathological diagnosis
The blood sample of pharynx cancer patient, while the blood sample for collecting 12 Healthy Peoples is used as control.By using based on protein chip
Method, one group of nasopharyngeal carcinoma cancer biomarker is identified from the serum of patient in the present invention, relative to normal control sample
Sheet, including TIMP-2, SELL, CCL24, MMP3, IGF-I and IL-8 are raised in patients with nasopharyngeal carcinoma, and MSP-alpha
Lowered with HCC-4.
The specificity and accuracy of this group of nasopharyngeal carcinoma biomarker are demonstrated with ELISA method, and is used for examination together
Nasopharyngeal carcinoma.Data after test are analyzed with SPSS statistical softwares, and group difference is analyzed with Student ' s t test, p<
0.05 is considered as statistically significant.
With reference to specific embodiment, the present invention is further illustrated, but is not limited thereto.
In order to be more clearly understood that the technology contents of the present invention, it is described with reference to the accompanying drawings especially exemplified by following examples.
It should be understood that these embodiments are only illustrative of the invention and is not intended to limit the scope of the invention.It is unreceipted in the following example
The experimental method of actual conditions, generally according to normal condition, or according to the condition proposed by manufacturer.It is used in embodiment
Various conventional chemical reagent, be commercially available prod.
Embodiment 1
1) sample prepares
12 are collected from Department of Pathology of attached Foshan First People's Hospital of Nanfang Medical Univ, is not received chemotherapy and radiotherapy
, the blood sample of Nasopharyngeal Carcinoma Patients is diagnosed as by SABC and pathological diagnosis, while collecting the blood of 12 Healthy Peoples
Sample is as control, and sample message is shown in Table will.By blood sample centrifuging and taking upper serum.
The clinical sample information of table 1
Patient | ||
Quantity | 12 people | |
Age (mean ± SD), year | 49.4±8.9 | |
Sex | Male=50%, women=50% | |
Staging | II=75%, III=25% | |
Treatment | It is no | |
Normal healthy controls | ||
Quantity | 12 people | |
Age (mean ± SD), year | 44.1±5.1 | |
Sex | Male=50%, women=50% | |
P-value (age, patient vs normal healthy controls) | 0.085 |
2) cDNA microarray blood serum designated object
Experimental method:
The present embodiment uses RayBio companies human cell factor chip Human cytokine antibody array
(RayBio Human Cytokine Antibody Array G series 2000,Cat No:AAH-CYT-G2000,
Raybiotech Co, Norcross GA, USA) detect 174 kinds of cell factors in serum.The serum-dilution two that upper step is collected
After times, add and two hours are incubated in cell factor chip.After washing, the antibody complex room temperature of biotin couplings is added
It is incubated 2 hours.After washing, add Cy3-conjugated streptavidin and be incubated 2 hours to protein chip.Most
Afterwards, chip is scanned with GenePix 4000B scanners (Axon Instruments, GenePix version 5.0), signal is used
GenePix 4000B softwares are obtained, then with Raybiotech companies analysis tool analysis (Raybiotech analysis
Tool, Excel specific software).
Experimental result:
1) chip scanning
Chip scanning, scanning result such as Fig. 1 are carried out to above-mentioned 24 parts of serum samples (12 healthy persons, 12 Nasopharyngeal Carcinoma Patients)
Shown, protein abundance is directly proportional to fluorescence intensity, and each sample repeats detection 2 times in chip.It can see from fluorescence picture
There are obvious fluorescent differences in the fluorescence signal in square frame, point out in control group (Control) and Nasopharyngeal Carcinoma Patients group (NPC)
Illustrate that expression quantity of these cell factors in Nasopharyngeal Carcinoma Patients body there may be significant difference, wherein, TIMP-2, SELL,
CCL24, MMP3, IGF-I and IL-8 are raised in patients with nasopharyngeal carcinoma, and MSP-alpha and HCC-4 is lowered.
Further corresponding fluorescence signal is quantified, as shown in table 2, table 2 is the blood of 12 healthy persons to quantitative result
Fluorescence signal value of 8 kinds of cell factors on chip in clear, be 12 Nasopharyngeal Carcinoma Patients serum in 8 kinds of cell factors in core
Fluorescence signal value on piece.
3) chip scanning data analysis and the determination of mark
1. the t-test analyses of chip scanning data
Said chip fluorescent value is analyzed using the t-test methods in chip analysis software, wherein there are 8 kinds of cells
There is significant difference (P in normal population and Nasopharyngeal Carcinoma Patients in the fluorescence signal intensity (representing cytokine content) of the factor<
0.05) result such as table 2, is made a concrete analysis of.Analysis result shows up-regulated expression in the serum of 5 kinds of cell factor Nasopharyngeal Carcinoma Patients,
There are 3 kinds of cell factors to lower expression.
The protein chip data results of table 2 (Nasopharyngeal Carcinoma Patients VS normal healthy controls)
2. box traction substation is analyzed
After the statistics progress T check analyses of above-mentioned protein chip, partly there is significant difference (P between two groups<
0.05) blood serum designated object is stated with box traction substation (boxplot);Horizontal line in box traction substation is the median for the data entirely organized.
Box traction substation is as shown in Figure 2.
Analyzed using SPSS statistical softwares, compare between statistical method mean and examined using t, group difference is used
Student ' s t test are analyzed, p<0.05 is considered as statistically significant and P<0.01 (difference highly significant), which is set to, to be had
Statistical significance.As can be seen that analyzing 174 factors in the cleer and peaceful normal human serum of Blood of Tumor Patients from table 2 and Fig. 2 analysis
Expression, as a result display include TIMP2, SELL, CCL24, MMP3, IGF-I and IL-8 in patients with nasopharyngeal carcinoma on
Adjust, and MSP-alpha and HCC-4 is lowered.
3. non-hierarchical clustering
Non-hierarchical clustering (unsupervised-hierarchical is used with Cluster 3.0software softwares
Cluster analysis) analysis with notable differential expression 8 blood serum designated objects (TIMP-2, SELL, CCL24, MMP3,
IGF-I, IL-8, MSP-alpha and HCC-4), analysis result is as shown in figure 3, it can be seen that this 8 blood serum designated objects are complete
It all can reach and significantly distinguish NPC groups and control group.Illustrate the index by the use of above-mentioned 8 kinds of cell factors as diagnosis of nasopharyngeal carcinoma,
Work well.
Non-hierarchical clustering (unsupervised- is used with the software softwares of Cluster 3.0
Hierarchical cluster analysis) analysis 8 significant differences blood serum designated object, as a result show NPC groups and control
Group can significantly distinguish
(3) ELISA verifies blood serum designated object
Other 20 normal samples and 20 Nasopharyngeal Carcinoma Patients (NPC) serum samples are collected, with the examination of the commercialization of purchase
Agent box (Raybiotech, Norcross GA, USA), is operated according to producer's operation instruction, further detects above-mentioned in the presence of expression
The cell factor of difference, including TIMP-2, SELL, CCL24, MMP3, IGF-I, IL-8, MSP-alpha and HCC-4.
Serum sample after dilution is separately added into corresponding elisa plate, is incubated at room temperature 2.5 hours.Wash elisa plate
After three times, the antibody (biotin-conjugated antibody) for adding biotin coupling is incubated 2 hours.Wash ELISA
After plank three times, the Streptavidin (HRP-conjugated streptavidin) of horseradish peroxidase-labeled is added
It is incubated 30 minutes, three addition tmb substrate colour developings afterwards of washing.Finally, sulfuric acid (sulfuric acid) stopped reaction is added,
Optical density is analyzed with ELIASA microplate reader (Biorek, USA, ELx800NB).
Analyzed using SPSS statistical softwares, compare between statistical method mean and examined using t, group difference is used
Student ' s t test are analyzed, p<0.05 is considered as statistically significant and P<0.01 (difference highly significant) is set to
It is statistically significant.Analyze the expression of 174 cell factors in patients with nasopharyngeal carcinoma and normal human serum.
Testing result is as shown in figure 4, there it can be seen that TIMP-2, SELL, CCL24, MMP3, IGF-I and IL-8 exist
Raised in patients with nasopharyngeal carcinoma, and MSP-alpha and HCC-4 is lowered, and is examined and divided with T with cell factor chip testing result one
ELISA data after analysis checking, ELISA results are consistent with protein chip result.NPC groups and control group (P<0.05, Mann-
Whitney U test analysis)。
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (9)
1. it is prepared by least one of specific recognition cell factor SELL, CCL24, MMP3, MSP-alpha, HCC-4 reagent
Application in nasopharyngeal carcinoma auxiliary diagnostic box.
2. can be to cell factor SELL, CCL24, MMP3, at least one of MSP-alpha, HCC-4 carry out quantitative reagent in system
Application in standby nasopharyngeal carcinoma auxiliary diagnostic box.
3. specific recognition cell factor TIMP-2's, IGF-I, IL-8, SELL, CCL24, MMP3, MSP-alpha and HCC-4
Application of the reagent in nasopharyngeal carcinoma auxiliary diagnostic box is prepared.
4. cell factor TIMP-2, IGF-I, IL-8, SELL, CCL24, MMP3, MSP-alpha and HCC-4 can be quantified
Reagent prepare nasopharyngeal carcinoma auxiliary diagnostic box in application.
5. a kind of auxiliary diagnostic box of nasopharyngeal carcinoma, it is characterised in that:The kit contains specific recognition cell factor
At least one of SELL, CCL24, MMP3, MSP-alpha, HCC-4 reagent.
6. a kind of auxiliary diagnostic box of nasopharyngeal carcinoma, it is characterised in that:The kit contain can to cell factor SELL,
At least one of CCL24, MMP3, MSP-alpha, HCC-4 carry out quantitative reagent.
7. a kind of auxiliary diagnostic box of nasopharyngeal carcinoma, it is characterised in that:The kit contains specific recognition cell factor
TIMP-2, IGF-I, IL-8, SELL, CCL24, MMP3, MSP-alpha and HCC-4 reagent.
8. a kind of auxiliary diagnostic box of nasopharyngeal carcinoma, it is characterised in that:The kit contain can to cell factor TIMP-2,
IGF-I, IL-8, SELL, CCL24, MMP3, MSP-alpha and HCC-4 carry out quantitative reagent.
9. a kind of auxiliary diagnostic box of nasopharyngeal carcinoma according to claim 8, it is characterised in that:The detection of the kit
Principle is ELISA principles.
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Cited By (2)
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CN112034187A (en) * | 2020-06-04 | 2020-12-04 | 北京臻知医学科技有限责任公司 | Marker for predicting 2019 coronavirus disease cytokines and thrombus storm, application and kit |
CN116694768A (en) * | 2023-06-27 | 2023-09-05 | 清远市人民医院 | Application of human leukocyte antigen HLA-B5801 gene in nasopharyngeal carcinoma susceptibility screening |
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Cited By (3)
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CN112034187A (en) * | 2020-06-04 | 2020-12-04 | 北京臻知医学科技有限责任公司 | Marker for predicting 2019 coronavirus disease cytokines and thrombus storm, application and kit |
CN112034187B (en) * | 2020-06-04 | 2022-05-20 | 北京臻知医学科技有限责任公司 | Marker for predicting 2019 coronavirus disease cell factors and thrombus storm, application and kit |
CN116694768A (en) * | 2023-06-27 | 2023-09-05 | 清远市人民医院 | Application of human leukocyte antigen HLA-B5801 gene in nasopharyngeal carcinoma susceptibility screening |
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Effective date of registration: 20221020 Address after: No. 404, Zone E, Guangzhou International Business Incubator, No. 3, Juquan Road, Science City, Guangzhou Hi tech Industrial Development Zone, 510700 Guangdong Patentee after: Guangzhou Hongxiang Biomedical Technology Co.,Ltd. Address before: 528051 Room 1204, 12/F, Block 2, No. 13, Huabao South Road, Chancheng District, Foshan, Guangdong Patentee before: FOSHAN HEZHEN BIOTECHNOLOGY CO.,LTD. |