CN110261518A - A kind of screening of human body effect of low dose radiation molecular injury marker taurine and verification method - Google Patents
A kind of screening of human body effect of low dose radiation molecular injury marker taurine and verification method Download PDFInfo
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- CN110261518A CN110261518A CN201910549849.7A CN201910549849A CN110261518A CN 110261518 A CN110261518 A CN 110261518A CN 201910549849 A CN201910549849 A CN 201910549849A CN 110261518 A CN110261518 A CN 110261518A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8696—Details of Software
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
Abstract
The present invention relates to a kind of screening of human body effect of low dose radiation molecular injury marker taurine and verification methods.The exposure group using front-line workers as low dose radiation as a control group with clerk carries out the screening of low dose radiation molecular injury marker based on non-targeted metabolism group method to low dose radiation exposed population group.Screen and demonstrate taurine can be used as human body low dose radiation damage molecular marker, compared with other radiation injury molecular markers, taurine more can system reflection professional population low dose radiation irradiation under biological damage effect；Taurine detect used in sample, such as blood, urine is all easier to obtain, more be of practical significance in the application, has the advantages that short required time, high specificity, high reliablity, environmental risk are small etc. multiple.
Description
Technical field
The invention belongs to low dose radiation Damage Evaluation technical fields, and in particular to a kind of effect of low dose radiation damage point
The screening of sub- marker taurine and verification method.
Background technique
Ionising radiation generates adverse effect by generating free radicals directly or indirectly damage cell and tissue, and to body,
Cause inflammation, cancer even dead.With the extensive use of nuclear power, isotope and radiotherapy, people are by low dosage electricity
More and more from the chance of radiation, from after Fukushima nuclear accident in 2011, influence of the low dose radiation to human health has caused
Extensive concern.
The health effect of high dose ionising radiation is more clear, but imitates about biology caused by low dose radiation
It answers and damage markers is not verified yet.After individual is exposed to effect of low dose radiation, it may will not go out within a few houres or several days
Now apparent symptom, it is also possible to the progressive damage and carcinogenic effect of hemopoietic system and immune system are just shown after the several years.
We cannot still assess the harm risk of low dose radiation exposure at present, this is since we are to low to a certain extent
The understanding of biological effect caused by dose is insufficient, and correlative study also more lacks.Therefore, reliable biology effect is found
Marker is answered, the medical care triage management of assessment and exposed population group to early stage low dose radiation risk all has highly important meaning
Justice.
In conventional method, cytogenetics is the standard method of radiation injury biological effective biomarkers research.But work as low dose
When molecular changes caused by amount ionising radiation are more slight, cytogenetic methods then lack certain sensibility.Metabolism group
Can in biological sample slight change small-molecule substance carry out it is qualitative and be quantitatively evaluated, quickly measure metabolite content,
It is successfully applied to the fields such as toxicity evaluation, medical diagnosis on disease and marker searching.
Non-targeted metabolism group method is mainly used for the extensive evaluation and screening of metabolin, there are poor repeatability and is easy to produce
The shortcomings that raw peak alignment error；The quantitative analysis that metabolism group method is used for specific metabolite is targeted, there are metabolins to study model
The case where enclosing relative narrowness.Therefore, it is studied, can quickly and accurately be sieved in conjunction with targeting and non-targeted metabolism group method
Choosing and verifying low dose radiation damage markers, and the metabolic pathway disorder of low dose radiation induction is assessed.
Currently, the metabolic pathway disorder effect of low dose radiation induction and the research of radiation injury biomarker are usually adopted
It is carried out with mouse, rat and non-primate.But the metabolism of different genera can have a certain difference, thus to the phase of animal
The mankind can not be completely suitable for by closing result of study.
Summary of the invention
For above situation, the present invention provides a kind of human body effect of low dose radiation molecular injury marker taurines
Screening and verification method.The exposure group using uranium mine front-line workers as low dose radiation first, using clerk as control
Group carries out potential low dose radiation molecular injury mark to low dose radiation exposed population group using non-targeted metabolism group method
The extensive screening of will object is then based on non-targeted metabolism group result of study and potential low dose radiation molecular injury marker
Related physiological meaning, further using targeting metabolism group method targeting quantitative verification is carried out to the metabolin filtered out.
It is united on this basis using non-targeted and targeting metabolism group method to the expression of taurine in two groups of crowd's blood
Meter analysis.In non-targeted metabolism group statistic analysis result, the taurine of screening meetsp< 0.05 and variation multiple be less than
0.82 or be greater than 1.2, and targeting metabolism group result in, there were significant differences in two groups of crowds for taurine mean value (p<
0.05).It is thus identified that taurine can be used as human body effect of low dose radiation molecular injury marker.This method can be used to sieve
The molecular marker of choosing and verifying low dose radiation damage has institute to assess the low dose radiation degree of impairment of professional population
The multiple advantages such as short, high specificity, high reliablity, the environmental risk of taking time be small.
Specific steps include:
(1) object is included in；
(2) sample collection；
(3) non-targeted metabolism group primary dcreening operation；
(4) metabolism group quantitative verification is targeted；
(5) it statisticallys analyze and determines low dose radiation molecular injury marker.
Its further step is:
What the object was included in method particularly includes:
It saves certain uranium mine at certain to record by consulting mine, using 32 front-line workerss for having radioactive exposure as exposure group, by nothing
As a control group, two groups of crowds are similar on age and sex composition by 32 clerks of radioactive exposure.It is included in standard:
1. whole staff of the uranium mine；2. the uranium mine workers ' health information table questionnaire data fills in complete person (seeing attached list 1)；
3. the person that is collected into blood sample.Exclusion criteria: 1. there is glycosuria patient；2. there is serious blood systemic disease person；3. there is Long-term taking medicine
Shi Zhe.
The sample collection method particularly includes:
The centrifuge tube for being equipped with sodium heparin anticoagulant carries out empty stomach sampling, acquires 2 ml of whole blood, and collected blood sample exists
It is centrifuged 5 min(3000 rpm at room temperature), take the supernatant of 0.2 ml to freeze loaded on 4 pipes, -80 DEG C of refrigerators in centrifuge tube, are filled altogether
It deposits.
The non-targeted metabolism group primary dcreening operation method particularly includes:
Sample process before loading: being placed on after -80 DEG C of sample thaws at 4 DEG C, pipette 100 μ l samples into EP pipe,
300 μ l methanol and 10 μ l internal standards are added, are vortexed after mixing 30 s, ultrasonic 10 min(ice-water baths), then stood at -20 DEG C
1 h.Liquid will be stood at 4 DEG C again and is centrifuged 15 min under the revolving speed of 13000 rpm, takes out 200 μ l supernatants in included interpolation pipe
Sample injection bottle in.
2. upper machine testing: 1) chromatographic condition: using Waters ACQUITY UPLC(chromatographic column for UPLC HSS T:1.7
μm mm of 2.1 mm × 100) it is used as analysis instrument, the group of positive ion mode mobile phase becomes 0.1% aqueous formic acid (A)-acetonitrile
(B), negative ion mode flowing phase composition is 5 mM ammonium acetate aqueous solutions (A)-acetonitrile (B).Gradient is 0 min, 1% B；1
Min, 1% B；8 min, 99% B；10 min, 99% B；10.1 min, 1% B；12 min, 1% B.Flow velocity is 0.5 ml/min,
Sample volume is 1 μ l.2) Mass Spectrometry Conditions: being acquired data using Thermo Q Exactive Orbitrap, it is positive and negative from
Sub- spray voltage is respectively 3.8 and 3.1 kV, and capillary temperature is 320 DEG C, and sheath gas is 40 arb, and auxiliary gas is 15 arb, is swept
Retouching range is 70-1000, resolution ratio 70000, collision voltage 20/40/60eV.
3. data processing and substance verifying: the mass spectrum initial data of acquisition changes into mzML lattice using ProteoWizard software
Formula recycles XCMS to do retention time correction, peak filtering, peak identification, peak extraction, peak integral, peak alignment etc..Use Compond
(Version 1.0, Dalian are limited up to large information technology by Discoverer (Version 2.0, Thermo) and OSI-SMMS
Company) software cooperation mzCloud database and Self-built Database carry out substance verifying.
4. the screening of potential effect biomarker and metabolic pathway analysis
Using t method of inspection, the differential expression situation of exposure group and control group metabolin is analyzed using two independent samples.With variation times
Number (fold change) is that (variation multiple is greater than in rising compared with the control group come the level for indicating exposure group difference metabolin
Or downward trend (variation multiple less than 0.82) 1.2).The screening criteria of this difference metabolin are as follows:p< 0.05 and become
Change multiple less than 0.82 or greater than 1.2.
By the difference metabolin sieved in biomolecule information database (Kyoto Encyclopedia of Genes and
Genomes (KEGG) database, Human Metabolome Database(HMDB) database and PubChem database)
Middle progress substance annotation obtains corresponding ID number and relevant information.For the physiology for further exploring the difference metabolin annotated
It meaning and finds and by low dose radiation is influenced higher signal path, we are further by these substances on KEGG mapper
It is labeled and carries out metabolic pathway analysis using Metabo Analyst.
5. quality controls: respectively taking 20 μ l to be mixed into QC sample in each sample to be tested, then take 200 μ l from QC sample
Upper machine testing is with the stability for measuring system.
The targeting metabolism group method quantitative verification method particularly includes:
Sample process before loading: 100 μ l samples are pipetted into EP pipe after being placed on -80 DEG C of sample defrosting, 300 μ are added
L methanol is vortexed after mixing 30 s, 1 h is stood at -20 DEG C, and liquid then will be stood at 4 DEG C under the revolving speed of 13000 rpm
15 min are centrifuged, 200 μ l supernatants is taken out in the sample injection bottle of included interpolation pipe, carries out subsequent high performance liquid chromatography mass spectral analysis,
The supernatant of 10 times and 200 times of dilution is taken to do high performance liquid chromatography mass spectral analysis respectively again.
2. standard solution is prepared: when preparing titer, accurately weighing the standard items of corresponding amount respectively in 10 ml volumetric flasks
In, it is configured to the standard items stock solution of 10 mmol/L, then takes the standard items stock solution of corresponding amount in 10 ml volumetric flasks again,
It is configured to mixed standard solution, the standard solution is finally successively diluted and obtains a series of calibration solution.
3. upper machine testing: 1) chromatographic condition: use 1290 Infinity II series of Agilent (chromatographic column for
Waters ACQUITY UPLC HSS T3 T:1,8 μm, 100 × 2.1 mm) Ultra Performance Liquid Chromatography instrument is as analyzer
Device.The group of mobile phase becomes 0.1% aqueous formic acid (A)-methanol (B), and chromatography gradient is shown in Table 1.Column oven temperature is 35 DEG C, sample
Product disk is set as 4 DEG C, and sample volume is 1 μ l.2) it Mass Spectrometry Conditions: (is equipped with using 6460 triple quadrupole mass spectrometer of Agilent
AJS-ESI ion source), it is analyzed by mass spectrometry with multiple-reaction monitoring (MRM) mode.Source parameters is as follows: capillary voltage be+
4000/-3500 V, spray voltage are+500/-500 V, and auxiliary gas (nitrogen) temperature is 300 DEG C, assist gas (nitrogen) flow velocity
For 5 L/min, sheath gas (nitrogen) temperature is 250 DEG C, and sheath gas (nitrogen) flow velocity is 11 L/min, nebulizer pressure 45
psi.。
1 liquid chromatogram gradient of table
| Time | A (H2O) | B (MeOH) | Flow |
| 0.00 min | 98.00% | 2.00% | 0.30 mL/min |
| 1.00 min | 98.00% | 2.00% | 0.30 mL/min |
| 5.00 min | 70.00% | 30.00% | 0.30 mL/min |
| 7.00 min | 20.00% | 80.00% | 0.30 mL/min |
| 8.00 min | 20.00% | 80.00% | 0.30 mL/min |
| 8.20 min | 98.00% | 2.00% | 0.30 mL/min |
| 13.00 min | 98.00% | 2.00% | 0.30 mL/min |
Before carrying out high performance liquid chromatography mass spectral analysis, target compound criteria solution is introduced into mass spectrum.Entire detection process
In, the acquisition of all mass spectrometric datas and the quantitative analysis of target compound pass through Agilent MassHunter Work
Station Software(B.08.00, Agilent Technologies) it completes.In detection process, if detection is to be measured
Object concentration is higher by calibration curve range, redeterminates again after diluting suitable multiple again, and quantitative result is institute after final dilution
The data measured.
It is described to statistically analyze the specific steps for determining low dose radiation molecular injury marker are as follows:
In non-targeted metabolism group result, the metabolin of screening needs to meetp< 0.05 and variation multiple it is less than 0.82 or big
In 1.2, and in targeting metabolism group result, there were significant differences for the mean value of exposure group and two groups of control group the crowd metabolin (p
< 0.05), that is, confirm that the metabolin is low dose radiation molecular injury marker.Generation is screened and targeted through non-targeted metabolism group
Xie Zuxue verifying, taurine can be used as human body effect of low dose radiation molecular injury marker.
The present invention is included in using object, sample collection, non-targeted thank group learn screening and targeting metabolism group verifying combines
Method, screen and demonstrate taurine can be used as human body low dose radiation damage molecular marker, with other radiation injuries
Molecular marker is compared, taurine more can system reflection professional population low dose radiation irradiation under biological damage effect；Ox sulphur
Acid detects used sample, such as blood, urine, is all easier to obtain, and is more of practical significance in the application, has and is taken
Between the multiple advantage such as short, high specificity, high reliablity, environmental risk be small.
Specific embodiment
Embodiment 1
It saves certain uranium mine at certain to record by consulting mine, using 32 front-line workerss for having radioactive exposure as exposure group, with not
Receive to radiate 32 clerks irradiated as a control group, two groups of crowds are similar on age and sex composition.It is included in mark
It is quasi-: 1. whole staff of the uranium mine；2. uranium mine workers ' health information table questionnaire data is filled in complete person and (is seen attached list
1)；3. the person that is collected into blood sample.Exclusion criteria: 1. there is glycosuria patient；2. there is serious blood systemic disease person；3. there is long-term clothes
Medicine history person.The centrifuge tube for being equipped with sodium heparin anticoagulant carries out empty stomach sampling, acquires 2 ml of whole blood, the blood sample after acquisition
It is centrifuged 5 min(3000 rpm at room temperature), take the supernatant of 0.2 ml to freeze loaded on 4 pipes, -80 DEG C of refrigerators in centrifuge tube, are filled altogether
It deposits.Primary dcreening operation is carried out to low dose radiation molecular injury marker using non-targeted metabonomic technology, using t method of inspection, is utilized
The differential expression situation of two independent samples analysis exposure group and control group metabolin, the difference of exposure group is indicated with variation multiple
Metabolite level is in rising (variation multiple is greater than 1.2) or to decline becoming for (variation multiple being less than 0.82) compared with the control group
Gesture, the screening criteria of difference metabolin are as follows:p< 0.05 and variation multiple less than 0.82 or be greater than 1.2.Analyze the metabolism of primary dcreening operation
Expression after the related physiological meaning of object, using targeting metabolism group to the metabolin filtered out in two groups of crowd's blood
Quantitative comparison is carried out, there were significant differences in exposure group and two groups of crowds of control group for metabolin mean value (p< 0.05), i.e., confirmation should
Metabolin is low dose radiation molecular injury marker.Finally, the present embodiment confirmation taurine can be used as the ionization of human body low dosage
Radiation injury molecular marker, taurine is non-targeted and targeting metabolism group statistic analysis result sees attached list 2.
2 taurine of table is non-targeted and targets metabolism group statistic analysis result
Note:* P< 0.05, difference is statistically significant compared with the control group for exposure group.
Claims (1)
1. screening and the verification method of a kind of human body effect of low dose radiation molecular injury marker taurine, first with a line work
People is as low dose radiation exposure group, as a control group with clerk, based on non-targeted metabolism group method to low dosage
The screening of radioactive exposure crowd progress low dose radiation molecular injury marker, which is characterized in that be then based on non-targeted metabolism
Group learns the related physiological meaning of result of study and potential low dose radiation molecular injury marker, further utilizes targeting generation
Xie Zuxue method carries out targeting quantitative verification to the metabolin filtered out, utilizes non-targeted and targeting metabolism group on this basis
Method is for statistical analysis to the expression of taurine in two groups of crowd's blood, and confirmation taurine is ionized as human body low dosage
Radiation injury molecular marker, specific steps include:
(1) object is included in；
(2) sample collection；
(3) non-targeted metabolism group primary dcreening operation；
(4) metabolism group quantitative verification is targeted；
(5) it statisticallys analyze and determines low dose radiation molecular injury marker,
Its further step is:
What the object was included in method particularly includes:
Using front-line workers as exposure group, as a control group with clerk, two groups of crowds phase on age and sex composition
Seemingly, it is included in standard: 1. whole staff of the uranium ore factory；2. uranium mine workers ' health information table questionnaire data is filled in completely
Person；3. the person that is collected into blood sample；Exclusion criteria: 1. there are diabetes；2. there is serious blood systemic disease；3. there is Long-term taking medicine
History,
The sample collection method particularly includes:
Before being equipped with the centrifuge tube progress empty stomach of sodium heparin anticoagulant, 2 ml of whole blood is acquired, the blood sample after acquisition is in room
Lower 3000 rpm of temperature are centrifuged 5 min, take the supernatant of 0.2 ml loaded in centrifuge tube, fill 4 pipes altogether, and -80 DEG C of refrigerators freeze,
The non-targeted metabolism group primary dcreening operation method particularly includes:
Sample process before loading: it is placed on after -80 DEG C of sample thaws at 4 DEG C, pipettes 100 μ l samples into EP pipe, add
Enter 300 μ l methanol and 10 μ l internal standards, is vortexed after mixing 30 s, 10 min of ultrasound, then -20 DEG C of 1 h of standing, then at 4 DEG C
Under the conditions of, 13000 rpm of liquid will be stood and be centrifuged 15 min, take out 200 μ l supernatants in the sample injection bottle of included interpolation pipe,
2. upper machine testing:
Chromatographic condition: using Waters ACQUITY UPLC as analysis instrument, the group of positive ion mode mobile phase becomes 0.1%
Aqueous formic acid (A)-acetonitrile (B), it is 5mM ammonium acetate aqueous solution (A)-acetonitrile (B), elution that negative ion mode, which flows phase composition,
Gradient are as follows: 0 min, 1% B；1 min, 1% B；8min, 99% B；10 min, 99% B；10.1 min, 1% B；12 min, 1%
B, flow velocity 0.5ml/min, sample volume are 1 μ l,
Mass Spectrometry Conditions: being acquired data using Thermo Q Exactive Orbitrap, negative ions spray voltage point
Not Wei 3.8 and 3.1 kV, capillary temperature be 320 DEG C, sheath gas be 40 arb, auxiliary gas be 15 arb, scanning range 70-
1000, resolution ratio 70000, collision voltage 20/40/60eV,
3. data processing and substance verifying: the mass spectrum initial data of acquisition changes into mzML format using ProteoWizard software,
It recycles XCMS to do retention time correction, peak filtering, peak identification, peak extraction, peak integral, peak alignment, uses Compond
Discoverer and OSI-SMMS software cooperates mzCloud database and Self-built Database to carry out substance verifying,
4. the screening of potential effect biomarker and metabolic pathway analysis
The differential expression situation that exposure group and control group metabolin is calculated using two independent samples t tests, with variation multiple come table
Showing difference metabolin to be compared with the control group in exposure group level is in rising or downward trend, studies the sieve of difference metabolin
Select standard are as follows:p< 0.05 and variation multiple less than 0.82 or be greater than 1.2,
By the difference metabolin sieved in biomolecule information database database, Human Metabolome Database database
And carry out substance annotation in PubChem database and obtain corresponding ID number and relevant information, further these substances are carried out
Metabolic pathway analysis is marked and carries out,
5. quality controls: respectively taking 20 μ l to be mixed into QC sample in each sample to be tested, then from taking machine on 200 μ l in QC sample
It detects to be used to measure the stability of system,
The targeting metabolism group method quantitative verification method particularly includes:
1. sample process before loading: pipetting 100 μ l samples into EP pipe after being placed on -80 DEG C of sample defrosting, 300 μ l are added
Methanol is vortexed after mixing 30 s, -20 DEG C of 1 h of standing, then will stand 13000 rpm of liquid under the conditions of 4 DEG C and be centrifuged 15min,
200 μ l supernatants are taken out in the sample injection bottle of included interpolation pipe, carry out subsequent high performance liquid chromatography mass spectral analysis, then take respectively dilute
It releases 10 times and 200 times of supernatant and does high performance liquid chromatography mass spectral analysis respectively,
2. standard solution is prepared: the standard items for accurately weighing corresponding amount when preparing titer respectively are prepared in 10 ml volumetric flasks
At the standard items stock solution of 10 mmol/L, the standard items stock solution of corresponding amount is then taken to be configured to mix in 10 ml volumetric flasks again
Standardization solution finally successively dilutes the standard solution and obtains a series of calibration solution,
3. upper machine testing:
Chromatographic condition: using liquid chromatograph as analysis instrument, column oven temperature is 35 °C, and sample disc is set as 4 °C, sample volume
For 1 μ l,
Mass Spectrometry Conditions: mass spectrograph is used, is analyzed by mass spectrometry with multiple-reaction monitoring pattern, source parameters is as follows: capillary electricity
Pressure is+4000/-3500 V, and spray voltage is+500/-500 V, and auxiliary temperature degree is 300 DEG C, and secondary air speed is 5 L/
Min, sheath temperature degree are 250 DEG C, and sheath gas is 11 L/min, and atomizer voltage is 45 psi,
The statistical analysis determines the specific steps of low dose radiation molecular injury marker are as follows:
In non-targeted metabolism group result, the metabolin of screening needs to meetp< 0.05 and variation multiple it is less than 0.82 or big
In 1.2, and in targeting metabolism group result, which has significance difference in exposure group and two groups of crowds of control group
It is different, that is, confirm that the metabolin is low dose radiation molecular injury marker, screens and target metabolism group through non-targeted metabolism group
Verifying, taurine is as human body effect of low dose radiation molecular injury marker.
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| CN114609284A (en) * | 2022-03-16 | 2022-06-10 | 广东工业大学 | Screening and verifying method of occupational combined pollution exposure biomarker based on human urine |
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| US20080187952A1 (en) * | 2007-02-05 | 2008-08-07 | Wisconsin Alumni Research Foundation | Biomarkers of Ionizing Radiation Response |
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| US20080187952A1 (en) * | 2007-02-05 | 2008-08-07 | Wisconsin Alumni Research Foundation | Biomarkers of Ionizing Radiation Response |
| CN103529469A (en) * | 2013-10-14 | 2014-01-22 | 苏州大学 | Method for screening radiation-damaged biomarkers at early stage of rat and application |
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| CN114214324A (en) * | 2021-11-23 | 2022-03-22 | 中国辐射防护研究院 | miR-219a-2-3p and application thereof as molecular marker for early diagnosis of radiation damage |
| CN114609284A (en) * | 2022-03-16 | 2022-06-10 | 广东工业大学 | Screening and verifying method of occupational combined pollution exposure biomarker based on human urine |
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