CN110218767A - A method of low dosage gamma ray radiation, which is evaluated, using glutathione peroxidase 4 damages - Google Patents
A method of low dosage gamma ray radiation, which is evaluated, using glutathione peroxidase 4 damages Download PDFInfo
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Abstract
The present invention relates to a kind of methods for evaluating the damage of low dosage gamma ray radiation using glutathione peroxidase 4.The present invention characteristic very sensitive to low dosage gamma ray radiation using human peripheral blood AHH1 lymphocyte Glutathione Peroxidase 4(GPX4), the dose-effect relationship of the up-regulation rate of the relative expression quantity of the low dosage gamma ray radiation dosage and GPX4 that are received according to human peripheral blood AHH1 lymphocyte, by the up-regulation rate of the relative expression quantity of GPX4 in detection human peripheral blood AHH1 lymphocyte, biological damage of the low dosage gamma ray radiation to blood of human body is assessed.It is multiple that this method has the advantages that quick, low in cost, high sensitivity easy to operate, accuracy is good, environmental risk is small etc..
Description
Technical field
The invention belongs to low dosage gamma ray radiation Damage Evaluation technical fields, and in particular to a kind of to utilize human peripheral
Blood AHH1 lymphocyte Glutathione Peroxidase 4(GPX4) relative expression quantity up-regulation rate penetrated to evaluate low dosage gamma
The method of beta radiation damage.
Background technique
In extensive Nuclear Accidents medical rescue, to the radioactive dose and biology of the receiving of ionising radiation exposed population group
It is the key that treatment that damage, which carries out quickly and effectively estimation,.With the fast development of Protocols in Molecular Biology, the life of gene level
Object marker because its quickly, easy and sensitive feature due to be possible to be used as evaluating low dose radiation damage.
Peripheral blood lymphocytes is very sensitive for ionising radiation, since it can be obtained from blood, is also easy to cultivate, at
The best object of radiation injury detection.Some researches show that DDB2, CDKN1A and XPC gene exist in peripheral blood lymphocytes
After the high dose ionising radiation of 0.2 2 Gy, between dose of radiation and the expression quantity of mRNA there is linear dosage effect to close
System.However for low dose radiation under the conditions of, the radiation injury evaluation method based on molecular marked compound is not yet established.
Due to the extensive use of nuclear energy, radioactive isotope and radiodiagnosis and treatment, the blood of human body is in many feelings
The radiation that will receive low dosage gamma ray under condition causes the various complication of blood.The radiation injury of blood causes people
Common concern, need screening can evaluate low dose radiation damage molecular marked compound, to the low dose radiation of blood of human body
Damage is evaluated.
Summary of the invention
For above situation, human peripheral blood AHH1 lymphocyte GSH-PX activity peroxide is utilized the present invention provides a kind of
Compound enzyme 4(GPX4) relative expression quantity up-regulation rate come evaluate low dosage gamma ray radiation damage method.Benefit of the invention
It is very quick to low dosage gamma ray radiation with human peripheral blood AHH1 lymphocyte Glutathione Peroxidase 4(GPX4)
The characteristic of sense, the opposite table of the low dosage gamma ray radiation dosage and GPX4 that are received according to human peripheral blood AHH1 lymphocyte
Up to the dose-effect relationship of the up-regulation rate of amount, pass through the relative expression quantity of GPX4 in detection human peripheral blood AHH1 lymphocyte
Up-regulation rate, evaluate biological damage of the low dosage gamma ray radiation to blood of human body.Its principle is glutathione peroxidase
Enzyme 4(GPX4) reduced glutathione is aoxidized by catalytic action by hydrogen peroxide, make organic hydroperoxide and
Lipid peroxide is restored, and plays the role of protecting cells from oxidative damage.GPX4 is lipid peroxidation process
Inhibit albumen, it being capable of degradation of small molecular peroxide and relative complex lipid peroxide.Low dose radiation causes to turn
The ferric ion of Human Placental Ferritin Receptor transhipment becomes ferrous ion by reduction, can occur with intracellular hydrogen peroxide fragrant
Reaction of pausing generates active oxygen radical ROS, promotes the death of cell.And cell itself is not broken to protect by oxygen radical
It is bad, activate glutathione peroxidase 4(GPX4), it is reacted using reduced glutathione (GSH) and hydrogen peroxide, shape
At oxidized form of glutathione (GSSH) and water, reaction equation H2O2+2GSH→2H2O+GSSH subtracts to remove body active oxygen
Peroxidation that is light and preventing active oxygen.Therefore, in certain radiated by gamma-ray dosage range, radiation that body is subject to
Damage more serious, the relative expression quantity of GPX4 is higher, glutathione peroxidase 4(GPX4) height of protein expression and people
Body radiation effect degree of injury is closely related, this method have quick, low in cost, high sensitivity easy to operate, accuracy it is good,
The multiple advantages such as environmental risk is small.
It comprises the concrete steps that:
(1) culture of human peripheral blood AHH1 lymphocyte；
(2) radiated by gamma-ray of lymphocyte；
(3) detection of the relative expression quantity up-regulation rate of GPX4；
(4) radiation injury overall merit.
Its further step is:
The culture of the human peripheral blood AHH1 lymphocyte method particularly includes:
Human peripheral blood lymphocytes AHH-1(number ATCC CRL-8146) is placed in containing the 10% serum (Australia Gibco tire
Ox) Iscove ' s Modified Dubecco ' s Medium (IMDM) culture solution in, in 37 DEG C, gas concentration lwevel
To be cultivated in 5% incubator, a culture solution was changed every 3 days, and a cell passage was carried out every 3-5 days, takes logarithm raw
Long-term cell is tested.
The radiated by gamma-ray of the lymphocyte method particularly includes:
Cell density is adjusted to 6 × 10 using cell counter5Cell/ml, then take 6 × 106A cell, i.e. 10 ml cells
Solution moves it to new culture bottle, carries out radiated by gamma-ray to it with the dosage rate of 14 μ Gy/h, respectively collect irradiation 0,7,
21, the peripheral blood AHH1 lymphocyte after 42 and 56 days (irradiation dose is respectively 0,2.4,7.2,14.1 and 18.8 mGy).
The detection of the relative expression quantity up-regulation rate of the GPX4 method particularly includes:
The peripheral blood AHH1 lymphocyte for receiving low dosage radiated by gamma-ray is subjected to protein extraction, total protein concentration is surveyed
Fixed, protein denaturation, gel electrophoresis, transferring film, immune response, chemiluminescence reaction, gel imaging scanning analysis, finally carry out
The relative expression quantity up-regulation rate of TFRC1 is analyzed.
Specific step is as follows for the protein extraction:
Culture solution is outwelled, bottle is tipped upside down on blotting paper, blotting paper is allowed to blot culture solution, every bottle of cell adds 3 ml 4oC pre-cooling
PBS gently shakes 1 min of washing, then discards washing lotion after laying flat.It repeats above operation twice, washes altogether three times to wash away culture
Liquid.Culture bottle is placed on ice after PBS is abandoned only.1 ml lysate is added in 10 ul PMSF, shakes up and is placed on ice.To every
Bottle plus lysate of 400 ul containing PMSF, in cracking 30 min on ice；To crack cell sufficiently, culture bottle will often shake back and forth
It is dynamic.After having cracked, centrifuge tube is moved to, is centrifuged 5 min, supernatant is taken to save in centrifuge tube.
Specific step is as follows for the total protein concentration measurement:
The BSA standard solution of 0.2 mg/ml is added into dilution, is successively diluted to 0,5,10,20,50,100,150 and 200
The standard solution of μ g/ml.Then their absorbance values at 562 nm are measured, standard curve is drawn.Measure each sample
Solution is after the absorbance value under 562 nm, then calculates by standard curve the concentration of protein in sample.
Specific step is as follows for the protein denaturation:
5 × protein sample-loading buffer is added according to the ratio of 4:1 in protein solution, boiling water bath is denaturalized 15 min, is put into -20
DEG C refrigerator saves backup.
Specific step is as follows for the gel electrophoresis:
30% acrylamide, 1.5 M TRIS-HCl, 10%SDS, AP and TEMED are configured to 10% point according to a certain percentage
From glue and 5% concentration glue；After gelling to be separated is solid, electrophoresis liquid is added in electrophoresis tank, the protein example after denaturation is added
In electrophoresis hole, electrophoresis is carried out.Concentrate glue voltage is adjusted to 75 V, separation gel voltage is adjusted to 120 V, electrophoresis to bromophenol blue just run to
Separation gel bottom can terminate electrophoresis, carry out transferring film.
Specific step is as follows for the transferring film:
First pvdf membrane is activated using methanol, then places the filter paper of 67 × 9 cm in the bottom of film, is together placed in
In transferring film instrument, transferring film liquid, 300 mA constant current transferring film half an hour are added.In During migration, transferring film slot is placed in ice water and is cooled down.
Specific step is as follows for the immune response:
5% skim milk (0.5% TBST dilution) of the film to take a turn for the better is closed, 1 h is mixed on decolorization swinging table.Using TBST
5% skim milk of dissolution carries out 1:100 concentration dilution to TFRC primary antibody (mouse is anti-human), then carries out it with the film closed
It mixes, is incubated overnight at 4 DEG C.It is cleaned with the film that 2% TBST is incubated overnight to TFRC primary antibody (mouse is anti-human), is existed at room temperature
It is cleaned on decolorization swinging table three times, every time 5 min.3000 times finally are diluted to secondary antibody (sheep anti mouse) with TBST, is incubated for 30 at room temperature
After min, cleaned on decolorization swinging table three times, every time 5 min.
Specific step is as follows for the chemiluminescence reaction:
It is by two kinds of reagents of ECLA and ECLB in the medium volume mixture of centrifuge tube in darkroom, the albumen of pvdf membrane is face-up, add
Enter after the ECL solution mixed sufficiently reacts 1-2 min, is put into gel imaging system and is scanned analysis.
Specific step is as follows for the gel imaging scanning analysis:
It is taken pictures using gel imaging system to protein band, then using the OD value of Alpha software analysis object tape.It is logical
It crosses to scan GPX4 albumen gray value and obtains GPX4 expressing quantity, internal reference Protein G APDH albumen gray value is scanned and is obtained
GAPDH expressing quantity；GPX4 expressing quantity is obtained to the opposite table of GPX4 divided by internal reference Protein G APDH expressing quantity
Up to amount.
Specific step is as follows for the relative expression quantity up-regulation rate analysis of the GPX4:
GPX4 relative expression quantity up-regulation rate is calculated by formula (1):
(1)
The method of the radiation injury overall merit is:
Radiation injury of the low dosage gamma ray radiation to human peripheral blood is evaluated using GPX4 relative expression quantity up-regulation rate.When
When GPX4 relative expression quantity up-regulation rate in human peripheral blood AHH-1 lymphocyte is less than 10%, show low dosage gamma ray spoke
It penetrates not damaged to human peripheral blood；When human peripheral blood AHH-1 lymphocyte GPX4 relative expression quantity up-regulation rate is small greater than 10%
When 30%, the impairment scale of low dosage gamma ray radiation at this time isGrade；When in human peripheral blood AHH-1 lymphocyte
GPX4 relative expression quantity up-regulation rate is greater than 30% when less than 60%, and the impairment scale of low dosage gamma ray radiation at this time is
Grade；When the GPX4 relative expression quantity up-regulation rate in human peripheral blood AHH-1 lymphocyte is greater than 60%, low dosage gal at this time
The impairment scale of horse ray radiation isGrade.When the impairment scale of low dosage gamma ray radiation reachesWhen grade, occupation work people
Member should reinforce radiation protection；When the impairment scale of low dosage gamma ray radiation reachesWhen grade, occupation work personnel should stop in time
Breath a period of time；When the impairment scale of low dosage gamma ray radiation reachesWhen grade, occupation work personnel should be transferred from former work
Post, and receive appropriate treatment.
The evaluation method of human peripheral blood radiation injury caused by low dosage gamma ray radiation proposed by the present invention is benefit
With the characteristic very sensitive to low dosage gamma ray radiation of the GPX4 in human peripheral blood lymphocytes AHH-1, according to gamma
The docs-effect between GPX4 relative expression quantity up-regulation rate in ray radiation dosage and human peripheral blood lymphocytes AHH-1
Relationship calculates GPX4 relative expression quantity up-regulation rate, using relative expression quantity up-regulation rate to caused by low dosage gamma ray radiation
Radiation injury is evaluated.This method have it is easy to operate quickly, that high sensitivity, accuracy are good, environmental risk is small etc. is multiple excellent
Point, long, complicated for operation, the specific difficulties such as not strong of period needed for solving existing low dosage gamma ray radiation damage evaluation method
Topic.
Specific embodiment
Specific embodiments of the present invention will be further explained below.
I material composition
Human peripheral blood AHH-1 lymphocyte, 10% serum, carbon dioxide incubator, radiated by gamma-ray source.
II implementation method
Human peripheral blood lymphocytes AHH-1(number ATCC CRL-8146) is placed in containing the 10% serum (Australia Gibco tire
Ox) IMDM culture solution in, cultivated in the incubator that temperature is 37 DEG C, gas concentration lwevel is 5%, changed one every 3 days
Secondary culture solution, a cell passage was carried out every 3-5 days, and the cell of logarithmic growth phase is tested.Using cell counter
Cell density is adjusted to 6 × 105Cell/ml, then take 6 × 106A cell, i.e. 10 ml cell solutions, move it to new culture
Bottle, carries out radiated by gamma-ray to it with the dosage rate of 14 μ Gy/h, collects (the irradiation agent of irradiation 0,7,21,42 and 56 day respectively
Amount be respectively 0,2.4,7.2,14.1 and 18.8 mGy) after peripheral blood AHH1 lymphocyte.Low dosage gamma will be received to penetrate
The peripheral blood AHH1 lymphocyte of line irradiation carries out protein extraction, total protein concentration measurement, protein denaturation, gel electrophoresis, turns
Film, immune response, chemiluminescence reaction, gel imaging scanning analysis finally divide the relative expression quantity up-regulation rate of GPX4
Analysis.
(1)
Radiation injury of the low dosage gamma ray radiation to human peripheral blood is evaluated using the relative expression quantity up-regulation rate of GPX4.
When the relative expression quantity up-regulation rate of the GPX4 in human peripheral blood lymphocytes AHH-1 is when within 10%, show low dosage gamma
Ray radiation is not damaged to human peripheral blood；When the relative expression quantity of the GPX4 in human peripheral blood lymphocytes AHH-1 raises
Rate is greater than 10% when less than 30%, and the impairment scale of low dosage gamma ray radiation at this time isGrade；When human peripheral hemolymph
The relative expression quantity up-regulation rate of GPX4 in cell AHH-1 be greater than 30% and when less than 60%, low dosage gamma ray spoke at this time
The impairment scale penetrated isGrade；When the relative expression quantity up-regulation rate of the GPX4 in human peripheral blood lymphocytes AHH-1 is greater than 60%
When, the impairment scale of low dosage gamma ray radiation at this time isGrade.When the impairment scale of low dosage gamma ray radiation reaches
It arrivesWhen grade, occupational staff should reinforce radiation protection；When the impairment scale of low dosage gamma ray radiation reachesWhen grade, professional people
Member should rest a period of time in time；When the impairment scale of low dosage gamma ray radiation reachesWhen grade, occupational staff should be transferred from
Work position, and receive appropriate treatment.
III embodiment
Embodiment 1
Human peripheral blood lymphocytes AHH-1(number ATCC CRL-8146) is placed in containing the 10% serum (Australia Gibco tire
Ox) IMDM culture solution in, cultivated in the incubator that temperature is 37 DEG C, gas concentration lwevel is 5%, changed one every 3 days
Secondary culture solution, a cell passage was carried out every 3-5 days, and the cell of logarithmic growth phase is tested.Using cell counter
Cell density is adjusted to 6 × 105Cell/ml, then take 6 × 106A cell, i.e. 10 ml cell solutions, move it to new culture
Bottle carries out radiated by gamma-ray to it with the dosage rate of 14 μ Gy/h, collects chronic exposure 7 days, and 2.4 mGy's of irradiation dose is outer
All blood AHH1 lymphocytes.The peripheral blood AHH1 lymphocyte for having received low dosage radiated by gamma-ray of collection is carried out albumen to mention
It takes, total protein concentration measurement, protein denaturation, gel electrophoresis, transferring film, immune response, chemiluminescence reaction, gel imaging scanning
Analysis, the relative expression quantity up-regulation rate analysis that GPX4 is finally carried out according to formula (1).
Peripheral blood AHH1 lymphocyte irradiates 7 d under the dosage rate of 14 μ Gy/h, receives the low dosage gal of 2.4 mGy
After horse x ray irradiation x, the relative expression quantity up-regulation rate of GPX4 is 5.3%, and low dosage radiated by gamma-ray is to human peripheral blood at this time
It is not damaged.
Embodiment 2
Human peripheral blood lymphocytes AHH-1(number ATCC CRL-8146) is placed in containing the 10% serum (Australia Gibco tire
Ox) IMDM culture solution in, cultivated in the incubator that temperature is 37 DEG C, gas concentration lwevel is 5%, changed one every 3 days
Secondary culture solution, a cell passage was carried out every 3-5 days, and the cell of logarithmic growth phase is tested.Using cell counter
Cell density is adjusted to 6 × 105Cell/ml, then take 6 × 106A cell, i.e. 10 ml cell solutions, move it to new culture
Bottle carries out radiated by gamma-ray to it with the dosage rate of 14 μ Gy/h, collects chronic exposure 21 days, 7.2 mGy's of irradiation dose
Peripheral blood AHH1 lymphocyte.The peripheral blood AHH1 lymphocyte for having received low dosage radiated by gamma-ray of collection is subjected to albumen
Extraction, total protein concentration measurement, protein denaturation, gel electrophoresis, transferring film, immune response, chemiluminescence reaction, gel imaging are swept
The relative expression quantity up-regulation rate analysis retouched analysis, finally carry out GPX4 according to formula (1).
Peripheral blood AHH1 lymphocyte irradiates 21 d under the dosage rate of 14 μ Gy/h, receives the low dosage gal of 7.2 mGy
After horse x ray irradiation x, the relative expression quantity up-regulation rate of GPX4 is 13.3%, and the impairment scale of low dosage radiated by gamma-ray is at this timeGrade, occupation work personnel answer enhanced rad to protect.
Embodiment 3
Human peripheral blood lymphocytes AHH-1(number ATCC CRL-8146) is placed in containing the 10% serum (Australia Gibco tire
Ox) IMDM culture solution in, cultivated in the incubator that temperature is 37 DEG C, gas concentration lwevel is 5%, changed one every 3 days
Secondary culture solution, a cell passage was carried out every 3-5 days, and the cell of logarithmic growth phase is tested.Using cell counter
Cell density is adjusted to 6 × 105Cell/ml, then take 6 × 106A cell, i.e. 10 ml cell solutions, move it to new culture
Bottle carries out radiated by gamma-ray to it with the dosage rate of 14 μ Gy/h, collects chronic exposure 42 days, 14.1 mGy of irradiation dose
Peripheral blood AHH1 lymphocyte.The peripheral blood AHH1 lymphocyte for having received low dosage radiated by gamma-ray of collection is subjected to egg
White extraction, total protein concentration measurement, protein denaturation, gel electrophoresis, transferring film, immune response, chemiluminescence reaction, gel imaging
Scanning analysis, the relative expression quantity up-regulation rate analysis that GPX4 is finally carried out according to formula (1).
Peripheral blood AHH1 lymphocyte irradiates 42 d under the dosage rate of 14 μ Gy/h, receives the low dosage of 14.1 mGy
After radiated by gamma-ray, GPX4 relative expression quantity up-regulation rate is 42.8 %, at this time the impairment scale of low dosage radiated by gamma-ray
ForGrade, occupation work personnel should rest a period of time in time.
Embodiment 4
Human peripheral blood lymphocytes AHH-1(number ATCC CRL-8146) is placed in containing the 10% serum (Australia Gibco tire
Ox) IMDM culture solution in, cultivated in the incubator that temperature is 37 DEG C, gas concentration lwevel is 5%, changed one every 3 days
Secondary culture solution, a cell passage was carried out every 3-5 days, and the cell of logarithmic growth phase is tested.Using cell counter
Cell density is adjusted to 6 × 105Cell/ml, then take 6 × 106A cell, i.e. 10 ml cell solutions, move it to new culture
Bottle carries out radiated by gamma-ray to it with the dosage rate of 14 μ Gy/h, collects chronic exposure 56 days, 18.8 mGy of irradiation dose
Peripheral blood AHH1 lymphocyte.The peripheral blood AHH1 lymphocyte for having received low dosage radiated by gamma-ray of collection is subjected to egg
White extraction, total protein concentration measurement, protein denaturation, gel electrophoresis, transferring film, immune response, chemiluminescence reaction, gel imaging
Scanning analysis, the relative expression quantity up-regulation rate analysis that GPX4 is finally carried out according to formula (1).
Peripheral blood AHH1 lymphocyte irradiates 56 d under the dosage rate of 14 μ Gy/h, receives the low dosage of 18.8 mGy
After radiated by gamma-ray, GPX4 relative expression quantity up-regulation rate is 69.6 %, at this time the impairment scale of low dosage radiated by gamma-ray
ForGrade, occupation work personnel should be transferred from former work position, and receive appropriate treatment.
It is above only better embodiment of the invention, according to the above-mentioned design, those skilled in the art
Can also to this various modification can be adapted and transformation.For example, the class of the type of transformation irradiated rays, transformation human peripheral blood lymphocytes
Type adjusts radiation dose rate, using different irradiation times and irradiation dose, transformation radiation injury appraisement system etc..However, this
A little similar transformation and modification belong to essence of the invention.
Claims (2)
1. it is a kind of using glutathione peroxidase 4 evaluate low dosage gamma ray radiation damage method, using human body outside
Characteristic GPX4 very sensitive to low dosage gamma ray radiation in all blood AHH1 lymphocytes is drenched according to human peripheral blood AHH1
The dose-effect relationship of the up-regulation rate of the relative expression quantity of low dosage gamma ray radiation dosage and GPX4 that bar cell receives,
By the up-regulation rate of the relative expression quantity of GPX4 in detection human peripheral blood AHH1 lymphocyte, low dosage gamma ray spoke is assessed
The biological damage to blood of human body is penetrated,
It comprises the concrete steps that:
(1) culture of human peripheral blood AHH1 lymphocyte；
(2) gamma ray radiation；
(3) detection of the relative expression quantity up-regulation rate of GPX4；
(4) radiation injury overall merit；
Its further step is:
The culture of the human peripheral blood AHH1 lymphocyte method particularly includes:
Human peripheral blood AHH-1 lymphocyte, with containing in 10% serum free culture system liquid, being placed in 37 DEG C, 5% carbon dioxide incubator, often
A culture solution was changed every 3 days, and a cell passage was carried out every 3-5 days, logarithmic growth phase cell is tested,
The gamma ray radiation method particularly includes:
Cell density is adjusted to 6 × 10 using cell counter5Cell/ml, then take 6 × 106A cell i.e. 10 ml cells
Solution moves it to new culture bottle, is placed under the gamma ray that dosage rate is 14 μ Gy/h and carries out long lasting for irradiation, respectively
Collect chronic exposure 0,7,21,42 and 56 day, irradiation dose is respectively 0,2.4,7.2,14.1 and 18.8 mGy, peripheral blood
AHH1 lymphocyte,
The detection of the relative expression quantity up-regulation rate of the GPX4 method particularly includes:
The peripheral blood AHH1 lymphocyte for having received low dosage radiated by gamma-ray of collection is subjected to protein extraction, total protein concentration
Measurement, gel electrophoresis, transferring film, immune response, chemiluminescence reaction, gel imaging scanning analysis, finally carries out protein denaturation
The relative expression quantity up-regulation rate of GPX4 is analyzed,
Specific step is as follows for the protein extraction:
Culture solution is outwelled, and bottle, which is tipped upside down on blotting paper, makes blotting paper blot culture solution, every bottle of cell adds 3ml 4oC pre-cooling
PBS, which is laid flat, gently shakes 1 min washing, then discards washing lotion, repeats above operation twice, is washed altogether three times to wash away culture solution,
Culture bottle is placed on ice after PBS is abandoned only, 1ml lysate adds 10ulPMSF, shakes up and is placed on ice, and every bottle plus 400 μ l contains
The lysate of PMSF, in cracking 30 min on ice, to crack cell sufficiently, culture bottle wants frequent waggle, after having cracked,
Centrifuge tube is moved to, 5 min are centrifuged, supernatant is saved in centrifuge tube,
Specific step is as follows for the total protein concentration measurement:
Dilution is added successively to be diluted to 0,5,10,20,50,100,150 and 200 μ g/ 0.2 mg/ml BSA standard solution
Then the standard solution of ml measures their absorbance values at 562 nm, draw standard curve, it is molten to measure each sample
After absorbance value under 562 nm of liquid, the concentration of protein in sample is calculated finally by standard curve,
Specific step is as follows for the protein denaturation:
5 × protein sample-loading buffer is added according to the ratio of 4:1 in protein solution, boiling water bath is denaturalized 15 min, is put into -20
DEG C refrigerator saves backup,
Specific step is as follows for the gel electrophoresis:
30% acrylamide, 1.5M TRIS-HCl, 10%SDS, AP and TEMED are configured to 10% point according to a certain percentage
After electrophoresis liquid is added in electrophoresis tank, protein example after denaturation is added after waiting separation gelling admittedly good from glue and 5% concentration glue
Enter in electrophoresis hole, carries out electrophoresis, concentrate glue voltage is adjusted to 75 V, separation gel voltage is adjusted to 120 V, and electrophoresis to bromophenol blue has just been run
To separation gel bottom, electrophoresis can be terminated, carries out transferring film,
Specific step is as follows for the transferring film:
First pvdf membrane is activated using methanol, then places the filter paper of 67 × 9 cm in the bottom of film, is together placed in
In transferring film instrument, transferring film liquid is added, 300 mA constant current transferring film half an hour in During migrations, transferring film slot were placed in ice water and is cooled down,
Specific step is as follows for the immune response:
5% skim milk (0.5% TBST dilution) of the film to take a turn for the better is closed, 1 h is mixed on decolorization swinging table, using TBST
5% skim milk of dissolution carries out 1:100 concentration dilution to GPX4 primary antibody, then mixes it with the film closed, 4 DEG C
It is incubated overnight, is cleaned with the film that 2% TBST is incubated overnight to GPX4 primary antibody, at room temperature, three are cleaned on decolorization swinging table
Secondary, 5 min, finally dilutes 3000 times to secondary antibody with TBST, after being incubated for 30 min at room temperature, three is cleaned on decolorization swinging table every time
It is secondary, 5 min every time,
Specific step is as follows for the chemiluminescence reaction:
It is by two kinds of reagents of ECLA and ECLB in the medium volume mixture of centrifuge tube in darkroom, the albumen of pvdf membrane is face-up, add
Enter after the ECL solution mixed sufficiently reacts 1-2 min, be put into gel imaging system and be scanned analysis,
Specific step is as follows for the gel imaging scanning analysis:
It is taken pictures using gel imaging system to protein band, then using the light of Alpha software processing system analysis object tape
Density value obtains GPX4 expressing quantity by scanning to GPX4 albumen gray value, sweeps to internal reference Protein G APDH albumen gray value
It retouches and obtains GAPDH expressing quantity；GPX4 expressing quantity is obtained into GPX4 phase divided by internal reference Protein G APDH expressing quantity
To expression quantity,
Specific step is as follows for the relative expression quantity up-regulation rate analysis of the GPX4:
GPX4 relative expression quantity up-regulation rate is calculated by formula (1),
GPX4 relative expression quantity up-regulation rate is calculated by formula (1),
(1)
The method of the radiation injury overall merit is:
Biological damage of the low dosage gamma ray radiation to human peripheral blood is assessed using GPX4 relative expression quantity up-regulation rate, when
When GPX4 relative expression quantity up-regulation rate in human peripheral blood AHH-1 lymphocyte is less than 10%, show low dosage gamma ray spoke
It penetrates not damaged to human peripheral blood；When human peripheral blood AHH-1 lymphocyte GPX4 relative expression quantity up-regulation rate is small greater than 10%
When 30%, the impairment scale of low dosage gamma ray radiation at this time is I grade；When in human peripheral blood AHH-1 lymphocyte
GPX4 relative expression quantity up-regulation rate is greater than 30% when less than 60%, and the impairment scale of low dosage gamma ray radiation at this time is II
Grade；When the GPX4 relative expression quantity up-regulation rate in human peripheral blood AHH-1 lymphocyte is greater than 60%, low dosage gal at this time
The impairment scale of horse ray radiation is III grade.
2. a kind of utilization glutathione peroxidase 4 according to claim 1 is evaluated low dosage gamma ray radiation and is damaged
The method of wound, which is characterized in that I grade of degree of injury of the impairment scale is lower than II grade of impairment scale, II grade of impairment scale damage
Degree is lower than III grade of impairment scale.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910549847.8A CN110218767B (en) | 2019-06-24 | 2019-06-24 | Method for evaluating low-dose gamma ray radiation damage by using glutathione peroxidase 4 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910549847.8A CN110218767B (en) | 2019-06-24 | 2019-06-24 | Method for evaluating low-dose gamma ray radiation damage by using glutathione peroxidase 4 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110218767A true CN110218767A (en) | 2019-09-10 |
| CN110218767B CN110218767B (en) | 2023-02-28 |
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