CN108287239A - Application of the change of serum C A2 albumen in preparing diagnosing cancer of liver or prognosis evaluation reagent kit - Google Patents
Application of the change of serum C A2 albumen in preparing diagnosing cancer of liver or prognosis evaluation reagent kit Download PDFInfo
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- CN108287239A CN108287239A CN201711297274.1A CN201711297274A CN108287239A CN 108287239 A CN108287239 A CN 108287239A CN 201711297274 A CN201711297274 A CN 201711297274A CN 108287239 A CN108287239 A CN 108287239A
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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
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
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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
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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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
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
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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
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/988—Lyases (4.), e.g. aldolases, heparinase, enolases, fumarase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Abstract
The application and kit that the present invention relates to change of serum C A2 albumen in preparing diagnosing cancer of liver or prognosis evaluation reagent kit.The beneficial effects are mainly as follows：1, CA2 can be used as a kind of new diagnosing cancer of liver biomarker.2, blood testing is at low cost, sample collection and processing are simple, it is in office why must in the case of repeatable sampling can be used as ideal disease diagnosis and prognosis judgement material, CA2 can be as the marker of prognosis in hcc, and the prognosis by regulating and controlling the biological function influence liver cancer of liver cancer cells.
Description
(1) technical field
The application and kit that the present invention relates to change of serum C A2 albumen in preparing diagnosing cancer of liver or prognosis evaluation reagent kit.
(2) background technology
Liver cancer is one of digestive system common cancer, and grade malignancy height, poor prognosis, incidence become in rising year by year
Gesture.China is High Phc Incidence Area, counts and shows according to newest Cancer in China, the new cases and death toll of China's liver cancer occupy generation
Boundary is the first, seriously threatens people's health and life.Complex treatment based on operation excision at present, is still liver cancer treatment
Main Patterns and most effective means, however easily transfer and relapse after Liver Cancer Operation, the recurrence and metastatic rate in postoperative 1 year are up to 20%-
30%, the recurrence and metastatic rate in 5 years is more up to 50%-70%.Therefore it is current that liver cancer, which easily turns the biological characteristics that recurrence moves,
The bottleneck problem for further increasing liver cancer treatment effect, reducing mortality of liver cancer, improving liver cancer long term survival rate.If can be in HCC
It before relapse and metastasis, predicts early, diagnose and active and effective measure is taken to be prevented in time, control further increasing liver cancer
Therapeutic effect will be of great significance.
Carbonic anhydrase II (Carbonic Anhydrase II, CA2) is strongest a member of activity in acid anhydrides enzyme family, ginseng
With the functions such as the secretion of body gas transport, acid-base accommodation and tissue, play a significant role in terms of the stabilization of environment in maintenance.
Since CA2 can efficient catalytic CO2Hydration reaction, generate H+ and HCO3, HCO3It is exchanged with intracellular Cl- to remain intracellular
Alkaline environment, intracellular H+ are transported extracellular by H+-Na+ ion pump modes, will extracellularly become acidic environment.Therefore,
CA2 is likely to be is catalyzed CO by invertibity2And HCO3Mutually convert, participate in the acidification of extracellular microenvironment.This shows CA2
The generation of tumour can be influenced by adjusting the pH of tumour cell microenvironment；And the low pH of extracellular microenvironment is then generally considered to be swollen
Tumor is imbued with a kind of mark of invasion and poor prognosis, therefore tumour occurs for the change of CA2 expressions and its biological behaviour
It has a major impact.
(3) invention content
Purpose of the present invention is to diagnosing cancer of liver and prognostic marker be used as to study CA2, change of serum C A2 albumen is provided
Application in preparing diagnosing cancer of liver or prognosis evaluation reagent kit and kit.
The technical solution adopted by the present invention is：
Change of serum C A2 (Carbonic Anhydrase II) albumen is in preparing diagnosing cancer of liver or prognosis evaluation reagent kit
Using.
The invention further relates to a kind of diagnosing cancer of liver or prognosis evaluation reagent kit, the kit is with the spy of change of serum C A2 albumen
It is detection marker to levy peptide fragment, includes mainly：According to the peptide fragment of mark again of the feature peptide fragment of change of serum C A2 albumen synthesis (smart ammonia is marked again
Acid or lysine13C,15N), detection reagent and the ELISA kit of CA2 albumen.
Specifically, the feature peptide section sequence of the change of serum C A2 albumen is：GGPLDGTYR or SADFTNFDPR.
The detection reagent is the common agents in the Mass Spectrometer Method of this field, including PBS (pH7.4), pancreatin (sequencing grade),
Ammonium hydrogen carbonate (500mM), water (mass spectrum grade), acetonitrile (chromatographic grade), formic acid.
The ELISA kit of the CA2 albumen can be prepared voluntarily, and commodity purchased in market can also be used.
The present invention using iTRAQ (isobaric tags for relative and absolute quantitation,
ITRAQ MS/MS technologies detection hepatocarcinoma patient cancerous tissue, cancer beside organism and remote cancerous tissue secretory protein) are combined and is tied using iTRAQ
It closes MS/MS technologies and detects follow-up serum before hepatocarcinoma patient postoperative recurrence, it is common to find biomarker CA2.It is in liver cancer tissue
It is apparently higher than remote cancerous tissue with the secretory volume in cancer beside organism, and the secretion of liver cancer tissue is significantly higher than cancer beside organism, and CA2
Concentration is apparently higher than the serum-concentration for not recurring patient to albumen in serum before hepatocarcinoma patient recurs, through ELISA and parallel reaction
Monitoring (Parallel reaction monitoring, PRM) verification shows that CA2 albumen concentration in In Sera of Patients With Hepatocarcinoma is apparent
Higher than Healthy People, serum-concentration, which is apparently higher than, in recurring patient does not recur patient.
The beneficial effects are mainly as follows：The present invention uses the quantitative proteomics skill marked based on iTRAQ
Art, intergrant Extracellular proteins group and internal serum photeomics data carry out comprehensive and systematic grind to hepatocarcinoma patient
Study carefully, provides new application and kit of the change of serum C A2 albumen as In Sera of Patients With Hepatocarcinoma diagnosis and prognostic marker, the marker
It is as a result more reliable by ELISA and mass spectrum PRM verifications, valuable information can be provided for clinical application.
(4) it illustrates
Fig. 1 is liver cancer tissue secretory protein group work flow diagram；
Fig. 2 is the PSM collection of illustrative plates of the peptide fragment GGPLDGTYR and SADFTNFDPR of CA2 albumen；
Fig. 3 is CA2 mass spectrum PRM verification results；
Fig. 4 is the CA2 concentration in PRM detection hepatocarcinoma patients and Healthy Human Serum；*P<0.05, * * P<0.01, * * * P<
0.001；
Fig. 5 is CA2 in the assessment of the ROC curve of diagnosing cancer of liver medium sensitivity and specificity；
Fig. 6 is ELISA detections recurrence/non-recurrent hepatic cancer patients serum CA2 concentration；*P<0.05, * * P<0.01, * * * P<
0.001；
Fig. 7 is the ROC curve of CA2 diagnosing liver cancer postoperative recurrences；
Fig. 8 is the relationship that Kaplan-Meier methods analyze CA2 serum-concentrations and hepatocarcinoma patient postoperative recurrence.
(5) specific implementation mode
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
Reagent source in embodiment：
Key instrument
Embodiment 1：
The secretory protein CA2 concentration of secretory protein group technology detection hepatocarcinoma patient cancerous tissue is apparently higher than cancer beside organism and remote
Cancerous tissue secretes concentration.
1. detecting sample：
Choose liver and gall surgical department of Medical University Of Fujian Meng Chao liver and gall hospitals in November, 2013~2014 year accept for medical treatment December it is preoperative
Without the secretory protein of the first tissue culture supernatant extraction for controlling hepatocarcinoma patient of operative treatment or chemotherapy.
2. detection method：
(1) lysate is replaced, FASP enzymolysis is carried out；
(2) above-mentioned tissue of patient culture supernatant extraction secretory protein enzymolysis peptide fragment quantifies 100 μ g, according to iTRAQ kits
Specification marks the mixture of all remote cancerous tissue secretory proteins with iTRAQ reagents 113 respectively, is corrected for internal reference, 114-119
For the label (liver cancer tissue, cancer beside organism and remote cancerous tissue, 2 biology repeat) of individual specimen, 5 iTRAQ are carried out altogether
Kit marks, and amounts to 10 biology and repeats, and 10 technologies repeat, and then mixes, is lyophilized；
(3) desalination is carried out to the peptide fragment after label with Sep-Pak Vac C18 extraction columns；
(4) the peptide fragment 95%H after desalination2O+5%ACN, PH=10 are resuspended, and then use and are connected to C18 reversed-phase columns
HPLC (high performance liquid chromatography) system detached under high ph-values, collects 80 pipe fractions；
(5) 32 μ L0.1%FA+99.9%H of each fraction2O is resuspended, and online EFI is used in combination in nanoliter liquid phase separation
Mist tandem mass spectrometer is analyzed；
(6) mass spectrometric data uses 1.4 softwares of Thermo Scientific Proteome Discoverer version
Search, matches with UniprotKB human proteome datas library；
(7) analysis of biological information SecretomeP 2.0Server software detections signal peptide, prediction secretory protein；With
Scaffold_4.3.2 softwares obtain quantitative protein list and each secretory protein each group sample expression quantity, draw protein diversities
Express spectra, screening significant difference express secretory protein.
3. testing result：
CA2 equal up-regulated expressions in C/F groups (liver cancer tissue is than remote cancerous tissue), P/F groups (cancer beside organism is than remote cancerous tissue),
Peptide fragment ratio is respectively 2.50,3.09 in two groups of co-expression differential secretion albumen, shows the change of CA2 expressions to liver
The generation of cancer and its biological behaviour have a major impact.
Embodiment 2：
Haemocyanin CA2 concentration, which is apparently higher than, before the detection hepatocarcinoma patient recurrence of serum photeomics technology does not recur patient
Concentration.
1. detecting sample：
4 hepatocarcinoma patient (A, B, C, D) root value criterions 1,3,6 of Medical University Of Fujian Meng Chao liver and gall hospital, September are collected respectively
Follow-up serum.Limosis vein blood 5mL is extracted to biochemical tube to satisfactory patient, ice chest fetches rear 3000rpm, 4 DEG C of centrifugations
10 minutes, -80 DEG C of refrigerators are stored in after taking upper serum to dispense.
2. detection method：
(1) Gao Feng in multiple affine 14 liquid-phase chromatographic columns of the removal system MARS-Human removal serum of Agilent is used
Spend albumen；
(2) every group of serum quantifies 100 μ g, and trypsin digestion is carried out according to iTRAQ kit specifications.With 2 iTRAQ
Kit, one iTRAQ kit of patient's A, B low-abundance protein peptide fragment, patient's C, D low-abundance protein peptide fragment is with one
ITRAQ kits.A4 follow-up serum of patient is respectively labeled as 113,114,115,116, and 4 follow-up serum of patient B are marked respectively
It is denoted as 117,118,119,121；4 follow-up serum of patient C are respectively labeled as 113,114,115,116,4 follow-ups of patient D
Serum is respectively labeled as 117,118,119,121.
(3) the iTRAQ label samples 95%H of combination drying2O+5%ACN, pH=10 redissolve, and pass through C18 reverse-phase chromatographies
Column will mix 80 fractions of sample point；
(4) each fraction carries out the identification of low ph value nanoliter Liquid Chromatography-Tandem Mass Spectrometry respectively.
(5) mass spectrometric data uses 1.4 softwares of Thermo Scientific Proteome Discoverer version
Search, matches with UniprotKB human proteome datas library；
3. testing result：
Mass spectral results show that haemocyanin CA2 concentration, which is apparently higher than, before hepatocarcinoma patient recurrence does not recur patient's concentration.
Embodiment 3：PRM technologies detection In Sera of Patients With Hepatocarcinoma CA2 concentration is apparently higher than Healthy People concentration.
1. detecting sample：
Take the preoperative serum of HBV associated hepatocellular carcinoma patients and the PRM analyses of pairing Healthy Human Serum row.Wherein hepatocarcinoma patient totally 49
Example, including 24 recurrence patients and 25 do not recur patient.Preoperative not capable any antineoplaston, Follow-up Data are complete.Together
When collect 23 pairing Healthy Human Serums as a contrast.
2. detection method：
(1) according to the data of liver cancer tissue secretory protein group early period, the characteristic peptide fragment that CA2 is uniquely expressed is chosen
Peptide fragment (marking arginine 13C15N again) is marked in GGPLDGTYR and SADFTNFDPR, synthesis again.The characteristic peptide fragment of CA2 albumen
The PSM collection of illustrative plates of GGPLDGTYR (left side) and SADFTNFDPR (right side) are referring to Fig. 2.
(2) the multiple affine exclusion of high performance liquid chromatograph and Agilent companies removal 14 kinds of high-abundance proteins of people is utilized
System (Human 14Multiple Afinity Removal System, MARS), removes above-mentioned hepatocarcinoma patient and healthy population
Serum sample in 14 kinds of high-abundance proteins such as albumin, IgG and antitrypsin, obtain low-abundance protein, and carry out pancreas
Enzymic digestion.
(3) the mark peptide fragment again of synthesis is incorporated into as internal standard in the serum sample of removal high-abundance proteins, utilizes targeting
Quantitative proteomics technology-parallel reaction monitoring (Parallel Reaction Monitoring, PRM) carries out Mass Spectrometric Identification
With quantitative, the serum-concentration of CA2 in detection hepatocarcinoma patient.CA2 mass spectrum PRM verification results are referring to Fig. 3.
(4) data analysis：The initial data that PRM is obtained Thermo Scientific Proteome
2.1 softwares of Discoverer carry out searching library, matching Uniprot people's source protein matter group database (http://
Www.uniprot.org/uniprot), result can be as new database, then the initial data that PRM is obtained imports
Skyline softwares carry out interpretation of result, confidence level 99%, analysis target peptide fragment and the content ratio for marking peptide fragment again, and then carry out
Compare the absolute quantitation value of target peptide fragment between each group.
3. testing result：
As a result, it has been found that (referring to Fig. 4), CA2 concentration mean values in the serum of hepatocarcinoma patient are 13.72pg/mL, and CA2 is in health
In the serum of crowd concentration mean value be 6.357pg/mL, concentration of the CA2 in the serum of hepatocarcinoma patient be significantly higher than respectively its
Concentration (P in normal human serum<0.01).
CA2 in the ROC curve assessment result of diagnosing cancer of liver medium sensitivity and specificity referring to Fig. 5, as shown in figure 5, ROC points
The area under the curve for analysing CA2 diagnosis is 0.715, has statistical significance (P ＜ 0.05).Diagnostic method show it is highly sensitive and
Good specificity.
Embodiment 4：Change of serum C A2 concentration is apparently higher than that not recur patient's same time dense before ELISA detection hepatocarcinoma patient recurrences
Degree
1. detecting sample：
Serum sample is taken between 2 months to 2014 in Septembers, 2016 in Meng Chao liver and gall hospital of Medical University Of Fujian row root
The property controlled resection operation and the postoperative serum of patient HCC of rule follow-up.82 liver cancer clinical samples are had collected altogether, wherein not recurring liver
23 people of carninomatosis people, 59 people of recurrent hepatic cancer patient.2. detection method：
(1) the ELISA kit reagent of CA2 albumen is slowly balanced to room temperature.
(2) standard items are prepared according to kit：Standard dilutions 1mL is added in 1 pipe standards product, and gently mixing, room temperature are quiet
It sets 10 minutes, is made into 20ng/mL standard items concentrates.By specification method doubling dilution standard items successively.Standard dilutions
Composition：Sample Diluent(LifeSpan Biosciences,Inc.Human CA2/Carbonic AnhydraseⅡ
ELISA Kit(Sandwich ELISA)Catalog NO.LS-F4349)。
(3) prepared by secondary antibody working solution：150 μ l secondary antibody diluents are added in 1 pipe secondary antibody freeze-dried powder and are made into 100 × concentrate,
Use 100 times of dilution secondary antibody concentrates of secondary antibody diluent at secondary antibody working solution before use.Secondary antibody diluent forms：Assay
Diluent A(LifeSpan Biosciences,Inc.Human CA2/Carbonic AnhydraseⅡELISA Kit
(Sandwich ELISA)Catalog NO.LS-F4349)。
(4) prepared by Streptavidin HRP working solutions：100 times of dilution Streptavidin of HRP dilutions are used before use
HRP concentrates, mixing.HRP dilutions form：Assay Diluent B(LifeSpan Biosciences,Inc.Human
CA2/Carbonic AnhydraseⅡELISA Kit(Sandwich ELISA)Catalog NO.LS-F4349)。
(5) cleaning solution is prepared：25mL concentrated cleaning solutions are diluted to 500mL with 475mL distilled water.
(6) serum is diluted 60 times with sample diluting liquid, 100 μ l standard items and serum is taken to sequentially add 96 hole elisa Plates
On, closing paper is covered, 37 DEG C are incubated 2 hours.Concentrated cleaning solution forms：Wash Buffer(30×)(LifeSpan
Biosciences,Inc.Human CA2/Carbonic Anhydrase ⅡELISA Kit(Sandwich ELISA)
Catalog NO.LS-F4349)。
(7) it inhales and abandons all liq, wash 3 times.
(8) add 100 μ l secondary antibody working solutions per hole, cover closing paper, 37 DEG C are incubated 1 hour.
(9) it inhales and abandons all liq, wash 3 times.
(10) 100 μ l HRP working solutions are added per hole, cover closing paper, 37 DEG C are incubated 30 minutes.
(11) it inhales and abandons all liq, wash 3 times.
(12) 100 μ l substrates are added per hole, liquid slowly becomes blue, is incubated 10 minutes at room temperature.
(13) 100 μ l terminate liquids are added per hole, liquid becomes yellow from blue.
(14) microplate reader surveys OD values with 450nm wavelength.
(15) it calculates：According to standard concentration and corresponding OD values, standard curve is calculated using CurveExpert softwares
Linear regression equation calculates the CA2 concentration of sample using sample OD values on regression equation again.
3. testing result：
As a result it shows that recurrence group patient is recurred CA2 concentration in preceding serum and is apparently higher than and does not recur patient, CA2 is not recurring disease
Concentration 29.96 ± 10.9ng/mL of average out in the postoperative serum of people, and in the postoperative serum of recurrence patient, then up to 48.96
± 30.4ng/mL has significant difference (P through both statistics<0.05), referring to Fig. 6.
The ROC curve of CA2 diagnosing liver cancer postoperative recurrences (does not recur recurrence and the CA2 contents in patients serum referring to Fig. 7
ROC analyses are carried out, as a result show that CA2 has statistical significance (P in terms of predicting recurrence of PHC<0.05), under curve
Area (AUC) is 0.723), CA2 serum-concentrations and the relationship of hepatocarcinoma patient postoperative recurrence (pass through Kaplan-Meier referring to Fig. 8
Whether survivorship curve analyzes CA2 postoperative serum-concentration and the recurrence-free survival rate that patient HCC is postoperative related.As a result (Fig. 8) is aobvious
Show CA2 low concentration groups (CA2<41.8ng/mL), 10 months, 20 months, 30 months recurrence-free survival rates be respectively 50.0%,
44%, 44%, and CA2 high concentrations group (CA2 >=41.8ng/mL) 10 months, 20 months, 30 months recurrence-free survival rate difference
It is 17%, 15%, 15%, two groups are compared with statistical significance (P<0.001).
Claims (4)
- Change of serum C A2 1. (Carbonic Anhydrase II) albumen answering in preparing diagnosing cancer of liver or prognosis evaluation reagent kit With.
- 2. application as described in claim 1, it is characterised in that the kit is detection with the feature peptide fragment of change of serum C A2 albumen Marker.
- 3. application as claimed in claim 2, it is characterised in that the feature peptide section sequence of the change of serum C A2 albumen is： SADFTNFDPR or GGPLDGTYR.
- 4. a kind of diagnosing cancer of liver or prognosis evaluation reagent kit, the kit include mainly：According to the feature peptide of change of serum C A2 albumen The peptide fragment of mark again and detection reagent of Duan Hecheng；The feature peptide section sequence of the change of serum C A2 albumen is：SADFTNFDPR or GGPLDGTYR。
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| CN114113439A (en) * | 2021-06-17 | 2022-03-01 | 福建医科大学孟超肝胆医院(福州市传染病医院) | Application of serum LRG1 protein in preparation of intrahepatic bile duct cancer lymph node diagnosis kit |
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