CN110221337B - Method for evaluating irradiation biological damage in uranium ore dust by using alpha-1 protease resistance - Google Patents
Method for evaluating irradiation biological damage in uranium ore dust by using alpha-1 protease resistance Download PDFInfo
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Abstract
The invention relates to a method for evaluating irradiation biological damage in uranium ore dust by utilizing alpha-1 protease resistance. According to the dose-effect relationship between the amount of uranium ore dust suspension subjected to tracheal instillation by WISTAR rats and the up-regulation rate of the relative expression amount of alpha-1 protease resistance in lung tissues, the biological damage caused by the irradiation in the uranium ore dust is evaluated through the up-regulation rate of the relative expression amount of alpha-1 protease resistance in the lung tissues of the WISTAR rats by utilizing the characteristic that alpha-1 protease resistance in the lung tissues of the WISTAR rats is very sensitive to biological damage caused by the irradiation in the uranium ore dust. The method has the advantages of high sensitivity, strong specificity, good reliability and the like on biological damage caused by internal irradiation of uranium ore dust.
Description
Technical Field
The invention belongs to the technical field of evaluation of irradiation biological damage in uranium ore dust, and relates to a method for evaluating the irradiation biological damage in the uranium ore dust by using a molecular marker, in particular to a method for evaluating the irradiation biological damage in the uranium ore dust by using the up-regulation rate of the relative expression quantity of alpha-1 protease resistance in lung tissues of WISTAR rats.
Background
A certain amount of water is produced in uranium exploration, mining, hydrometallurgy and tailing disposal processesUranium ore dust pollution, wherein the uranium ore dust contains radionuclides such as uranium, thorium, radium and the like, and once the uranium ore dust is absorbed into a human body, the radionuclides can be retained in a lung trachea, a bronchus and a lymph node for a long time to form potential long-term irradiation harm and generate biological damage. The ionizing radiation of uranium mine dust is mainly from radon and its daughter: (218Po、214Pb、214Bi and214po) decay, and it is difficult to perform detection and biological damage assessment because the radioactive dose generated by the inhaled dust is low and it is difficult to exhibit biological effects in a short time. Therefore, the method has very important significance in screening out the biomarkers which have high detection speed, high sensitivity, strong specificity and good reliability on biological damage caused by internal irradiation of uranium ore dust.
After an organism inhales uranium ore dust, it may respond to ionizing radiation by altering the expression level of proteins and their post-translational modification states. The iTRAQ technology is an emerging high-throughput proteomics method, and the relative content or the absolute content of proteins in 4 or 8 different samples can be simultaneously compared by specifically labeling amino groups of polypeptides and then performing tandem mass spectrometry. Therefore, the iTRAQ technique can be used to screen proteins differentially expressed in tissue samples to identify biomarkers related to biological damage caused by internal irradiation of uranium ore dust, and the screened biomarkers can be used to evaluate the biological damage caused by internal irradiation of uranium ore dust.
Disclosure of Invention
In view of the above circumstances, the present invention provides a method for evaluating biological damage caused by irradiation in uranium ore dust using the relative expression level up-regulation rate of Alpha-1-antiproteinase (A1AT) in lung tissue of WISTAR rats. According to the characteristic that alpha-1 protease resistance in lung tissues of WISTAR rats is very sensitive to biological damage caused by internal irradiation of uranium ore dust, biological damage caused by internal irradiation of the uranium ore dust is evaluated through the up-regulation rate of the relative expression quantity of alpha-1 protease resistance in lung tissues of the WISTAR rats according to the dose-effect relation between the quantity of uranium ore dust suspension (the uranium ore dust and physiological saline are prepared according to a certain proportion) which is subjected to tracheal instillation and the up-regulation rate of the relative expression quantity of the alpha-1 protease resistance in the lung tissues. The principle is that the inventor screens and finds out that the relative expression quantity of alpha-1 protease resistance in lung tissues of WISTAR rats after receiving uranium ore suspension instilled through an iTRAQ technology is very sensitive to uranium ore dust, and a certain dose-effect relation exists. Alpha-1 anti-proteases are proteins whose plasma concentrations vary in response to inflammation, and are involved in oxidative stress, inflammatory responses and apoptosis. Within a certain internal irradiation dose range, the larger the internal irradiation damage to an organism is, the higher the relative expression quantity of alpha-1 protease resistance is, the close correlation between the expression level of alpha-1 protease resistance protein and the biological damage degree caused by the internal irradiation of uranium ore dust is directly realized, and the method has the multiple advantages of high biological damage sensitivity, strong specificity, good reliability and the like on the internal irradiation of the uranium ore dust.
The method comprises the following specific steps:
(1) establishing a rat model of uranium ore suspension tracheal instillation WISTAR;
(2) collecting lung tissues;
(3) detecting the up-regulation rate of the relative expression quantity of the alpha-1 protease;
(4) and (4) comprehensively evaluating internal irradiation biological damage.
The further measures are as follows:
the specific method for establishing the uranium ore suspension tracheal instillation WISTAR rat model comprises the following steps:
adult male WISTAR rats weighing between 180-220 g are selected, and divided into 4 groups randomly, 6 mice in each group, a control group is subjected to tracheal instillation of a suspension of normal saline and silica (the particle size of the silica is 200 meshes), and experimental groups are subjected to tracheal instillation of uranium ore dust suspensions (the particle size of the uranium ore dust is 200 meshes) with the concentration of 1.25, 2.5 and 5.0 mg/ml respectively, 0.2 ml of uranium ore dust is instilled for each time, 3 times are instilled for one week, and the continuous 5 weeks are carried out.
The specific method for collecting the lung tissue comprises the following steps:
the rats were sacrificed by cervical dislocation, lung tissues were taken after dissection, each tissue was divided into 3 equal portions, blood was removed by normal saline, water was removed, the frozen tubes were filled, and immediately stored in a refrigerator at-80 ℃.
The specific method for detecting the up-regulation rate of the relative expression quantity of the alpha-1 protease comprises the following steps:
and (3) irradiating WISTAR rat lung tissues in the collected uranium-removed ore dust to perform protein extraction, total protein concentration determination, protein denaturation, gel electrophoresis, film transfer, immunoreaction, chemiluminescence reaction, gel imaging scanning analysis and finally performing alpha-1 protease-resistant relative expression quantity up-regulation analysis.
The specific steps of protein extraction are as follows:
and pouring liquid nitrogen into the mortar for precooling, quickly taking out the lung tissue sample, cutting the tissue to be uniform, putting the tissue into the mortar, immediately adding the liquid nitrogen, and grinding. Adding LB lysate 800 μ l slowly into mortar, splitting for 30 min, shaking for 1-2 times, and fully splitting. After the cleavage, 1.5 mL of the suspension was collected in an EP tube, centrifuged at 12000 r/min at 4 ℃ for 20 min, and the supernatant was collected. Precipitating protein in the supernatant with resin, centrifuging at 12000 r/min at 4 deg.C for 20 min, removing supernatant, air drying, and storing at-80 deg.C. 200-250 mu L L3 lysate was added to the dried protein pellet and the pellet was repeatedly blown up with a lance tip until the protein was well dispersed. Ultrasonic assisted dissolving, 4 ℃, 12000 r/min, centrifuging for 20 min, sucking out supernatant, and transferring to a new EP tube.
The specific steps of the total protein concentration determination are as follows:
the 0.2 mg/ml BSA standard solution was added to the diluent and diluted to 0, 5, 10, 20, 50, 100, 150 and 200. mu.g/ml standard solutions in this order. Their absorbance values at 562 nm were then determined and a standard curve was drawn. After measuring the absorbance value of each sample solution at 562 nm, the concentration of the protein in the sample was calculated by a standard curve.
The specific steps of the protein denaturation are as follows:
adding the protein solution into 5 Xprotein loading buffer solution at a ratio of 4:1, performing boiling water bath denaturation for 15 min, and storing in a refrigerator at-20 deg.C for use.
The gel electrophoresis comprises the following specific steps:
preparing 10% separation gel and 5% concentration gel from 30% acrylamide, 1.5M TRIS-HCl, 10% SDS, 10% ammonium persulfate and TEMED according to a certain proportion; and after the separation gel is solidified, adding electrophoretic fluid into the electrophoresis tank, adding the denatured protein sample into the electrophoresis hole, and performing electrophoresis. Regulating the voltage of the concentrated gel to 75V, regulating the voltage of the separation gel to 100V, and stopping electrophoresis until bromophenol blue just runs to the bottom of the separation gel, and performing membrane conversion.
The specific steps of the film transfer are as follows:
firstly, activating the PVDF membrane by adopting methanol, then placing 5-6 pieces of 7 multiplied by 9 cm filter paper at the bottom of the membrane, then placing the filter paper and the PVDF membrane together in a membrane rotating instrument, adding membrane rotating liquid, and rotating the membrane for 30 min at a constant current of 300 mA. And in the film transferring process, the film transferring groove is placed in ice water for cooling.
The immune response comprises the following specific steps:
the transferred membrane was blocked with 5% skim milk (0.5% TBST diluted) and mixed on a decolorizing shaker for 1 h. Alpha-1 anti-protease primary antibody (Alah 3 al) was diluted 1:1000 concentration with TBST solubilized 5% skim milk, then mixed well with the blocked membrane and incubated overnight at 4 ℃. The membrane incubated overnight with the alpha-1 anti-protease primary antibody was washed with 2% TBST and 3 times 5 min each on a destaining shaker at room temperature. Finally, the secondary antibody (goat anti-rabbit) was diluted 8000 times with TBST, incubated at room temperature for 1.5 h, and then washed three times for 10 min each time on a decolorization shaker.
The chemiluminescence reaction comprises the following specific steps:
mixing ECLA and ECLB reagents in equal volume in a centrifuge tube in a dark room, enabling the protein surface of the PVDF membrane to face upwards, adding the mixed ECL solution, fully reacting for 1-2 min, and then putting the mixture into a gel imaging system for scanning analysis.
The gel imaging scanning analysis comprises the following specific steps:
and (3) photographing the protein band by using a gel imaging system, and analyzing the gray value of the target band by using Image J software. Scanning the gray value of the alpha-1 protease-resistant protein to obtain the expression quantity of the alpha-1 protease-resistant protein, and scanning the gray value of the internal reference protein GAPDH to obtain the expression quantity of the GAPDH protein; the expression level of the alpha-1 protease-resistant protein is divided by the expression level of the internal reference protein GAPDH protein to obtain the relative expression level of the alpha-1 protease-resistant protein.
The specific steps of the analysis of the relative expression quantity up-regulation rate of the alpha-1 protease are as follows:
the relative expression level upregulation rate of alpha-1 antipain (A1AT) was calculated according to formula (1):
the specific method for comprehensively evaluating the internal irradiation biological damage comprises the following steps:
and evaluating biological damage caused by irradiation in the uranium ore dust by using the relative expression quantity up-regulation rate of alpha-1 protease resistance. When the up-regulation rate of the relative expression quantity of the alpha-1 protease resistance in lung tissues of WISTAR rats is within 30 percent, the low-dose gamma ray irradiation has no biological injury effect; when the up-regulation rate of the relative expression quantity of the alpha-1 protease resistance in lung tissues of WISTAR rats is more than 30% and less than 100%, the biological damage grade caused by irradiation in uranium ore dust is I grade; when the up-regulation rate of the relative expression quantity of the alpha-1 protease resistance in lung tissues of WISTAR rats is more than 100% and less than 200%, the biological damage level caused by the internal irradiation of the uranium ore dust is II level; when the relative expression quantity of the alpha-1 protease resistance in lung tissues of WISTAR rats is up-regulated by more than 200%, the biological damage caused by irradiation in uranium ore dust is grade III. When the biological damage level caused by the internal irradiation of uranium ore dust reaches level I, the occupational staff should strengthen the irradiation protection; when the biological damage level caused by the internal irradiation of the uranium ore dust reaches level II, the career should have a rest for a period of time in time; when the biological damage level caused by the internal irradiation of the uranium ore dust reaches level III, the staff should be away from the working position and receive proper treatment.
The invention utilizes the proteomic fully quantitative iTRAQ technology to analyze differentially expressed proteins, identifies alpha-1 protease resistance as a biomarker of biological damage caused by irradiation in uranium ore dust, and then evaluates the biological damage caused by irradiation in the uranium ore dust by utilizing the dose-effect relationship between the relative expression quantity of the alpha-1 protease resistance and the biological damage caused by irradiation in the uranium ore dust. Compared with other biomarkers and evaluation methods, the method has the advantages of high sensitivity to biological damage caused by internal irradiation of uranium ore dust, strong specificity, good reliability and the like.
Detailed Description
Example 1
Adult male WISTAR rats weighing between 180-220 g were selected and divided into 4 groups at random, 6 rats were collected in each group, a control group was subjected to tracheal instillation of a suspension of normal saline and silica (particle size of silica is 200 mesh), and experimental groups were subjected to tracheal instillation of a suspension of uranium ore dust (particle size of uranium ore dust is 200 mesh) at a concentration of 1.25 mg/ml, 0.2 ml was instilled each time, 3 instillations were made a week, and the administration was continued for 5 weeks. The rats were sacrificed by cervical dislocation, lung tissues were taken after dissection, each tissue was divided into 3 equal portions, blood was removed by normal saline, water was removed, the frozen tubes were filled, and immediately stored in a refrigerator at-80 ℃. The collected lung tissues of WISTAR rats which are irradiated by low-dose ionizing radiation are subjected to protein extraction, total protein concentration determination, protein denaturation, gel electrophoresis, membrane transfer, immunoreaction, chemiluminescence reaction, gel imaging scanning analysis and finally alpha-1 protease resistance relative expression up-regulation analysis according to the formula (1).
After 5 weeks of tracheal instillation of uranium ore dust suspension with the concentration of 1.25 mg/ml in adult male WISTAR rats, the up-regulation rate of the relative expression quantity of alpha-1 protease in lung tissues is 85.6%, at the moment, the biological damage level caused by internal irradiation of the uranium ore dust is I level, and professional staff need to enhance irradiation protection.
Example 2
Adult male WISTAR rats weighing between 180 and 220 g are selected, and divided into 4 groups randomly, 6 mice in each group, a control group is subjected to tracheal instillation of a suspension of normal saline and silica (the particle size of the silica is 200 meshes), and experimental groups are subjected to tracheal instillation of a suspension of uranium ore dust (the particle size of the uranium ore dust is 200 meshes) with the concentration of 2.5 mg/ml respectively, 0.2 ml of uranium ore dust is instilled each time, 3 times are instilled in one week, and the 3 times are continuously instilled for 5 weeks. The rats were sacrificed by cervical dislocation, lung tissues were taken after dissection, each tissue was divided into 3 equal portions, blood was removed by normal saline, water was removed, the frozen tubes were filled, and immediately stored in a refrigerator at-80 ℃. The collected lung tissues of WISTAR rats which are irradiated by low-dose ionizing radiation are subjected to protein extraction, total protein concentration determination, protein denaturation, gel electrophoresis, membrane transfer, immunoreaction, chemiluminescence reaction, gel imaging scanning analysis and finally alpha-1 protease resistance relative expression up-regulation analysis according to the formula (1).
After 5 weeks of tracheal instillation of uranium ore dust suspension with the concentration of 2.5 mg/ml in adult WISTAR male rats, the up-regulation rate of the relative expression quantity of alpha-1 protease in lung tissues is 158.5%, at the moment, the biological damage level caused by internal irradiation of the uranium ore dust is level II, and professional staff should have a rest for a period of time in time.
Example 3
Adult male WISTAR rats weighing between 180 and 220 g are selected, and divided into 4 groups randomly, 6 mice in each group, a control group is subjected to tracheal instillation of a suspension of normal saline and silica (the particle size of the silica is 200 meshes), and experimental groups are subjected to tracheal instillation of a suspension of uranium ore dust (the particle size of the uranium ore dust is 200 meshes) with the concentration of 5.0 mg/ml respectively, 0.2 ml of uranium ore dust is instilled each time, 3 times are instilled in one week, and the 3 times are continuously instilled for 5 weeks. The rats were sacrificed by cervical dislocation, lung tissues were taken after dissection, each tissue was divided into 3 equal portions, blood was removed by normal saline, water was removed, the frozen tubes were filled, and immediately stored in a refrigerator at-80 ℃. The collected lung tissues of WISTAR rats irradiated by low-dose ionizing radiation are subjected to protein extraction, total protein concentration determination, protein denaturation, gel electrophoresis, membrane transfer, immunoreaction, chemiluminescence reaction, gel imaging scanning analysis and finally alpha-1 protease resistance relative expression quantity up-regulation analysis according to the formula (1).
After 5 weeks of tracheal instillation of uranium ore dust suspension with the concentration of 5.0 mg/ml in adult male WISTAR rats, the up-regulation rate of the relative expression quantity of alpha-1 protease in lung tissues is 245.6%, at the moment, the biological damage level caused by internal irradiation of the uranium ore dust is III level, and professional staff need to adjust away from the original working position and receive proper treatment.
The above is only a preferred embodiment of the present invention, and various modifications and changes can be made thereto by those skilled in the art based on the above-described concept of the present invention. For example, altering uranium ore suspension concentration, contamination mode, altering rat model type, altering radiation damage assessment system, and the like. However, these similar changes and modifications are also within the spirit of the present invention.
Claims (10)
1. A method for evaluating biological damage caused by irradiation in uranium ore dust by utilizing alpha-1 protease-resistant A1AT is characterized in that biological damage caused by irradiation in the uranium ore dust is evaluated according to the dose-effect relation between the amount of uranium ore dust suspension subjected to tracheal instillation by WISTAR rats and the up-regulation rate of relative expression amount of alpha-1 protease-resistant A1AT in lung tissues, and through the up-regulation rate of relative expression amount of alpha-1 protease-resistant A1AT in the lung tissues of the WISTAR rats, and the method is characterized by comprising the following specific steps:
(1) establishing a rat model of uranium ore suspension tracheal instillation WISTAR;
(2) collecting lung tissues;
(3) detecting the up-regulation rate of the relative expression quantity of the alpha-1 protease A1 AT;
(4) comprehensively evaluating internal irradiation biological damage;
the specific method for establishing the uranium ore suspension tracheal instillation WISTAR rat model comprises the following steps:
adult WISTAR male rats with the weight of 180-220 g are selected and divided into 4 groups, 6 mice in each group and a control group are subjected to tracheal instillation of suspensions of physiological saline and silicon dioxide, and experimental groups are respectively subjected to tracheal instillation of uranium ore dust suspensions with the concentration of 1.25, 2.5 and 5.0 mg/ml, 0.2 ml is instilled each time, 3 instillations are performed in one week for 5 weeks;
the specific method for collecting the lung tissue comprises the following steps:
dissecting dead rat, collecting lung tissue, dividing each tissue into 3 equal parts, washing with normal saline to remove blood, removing water, placing into a freezing tube, and immediately storing in a refrigerator at-80 deg.C;
the specific method for detecting the relative expression quantity up-regulation rate of the alpha-1 anti-protease A1AT comprises the following steps:
subjecting collected lung tissues of rats which are irradiated with WISTAR in uranium mine dust to protein extraction, total protein concentration determination, protein denaturation, gel electrophoresis, membrane transfer, immunoreaction, chemiluminescence reaction, gel imaging scanning analysis and finally relative expression quantity up-regulation rate analysis of alpha-1 anti-protease A1 AT;
the specific method for comprehensively evaluating the internal irradiation biological damage comprises the following steps:
when the up-regulation rate of the relative expression quantity of alpha-1 anti-protease A1AT in lung tissues of WISTAR rats is within 30 percent, the low-dose gamma ray irradiation has no damage effect on organisms; when the up-regulation rate of the relative expression quantity of alpha-1 protease A1AT in lung tissues of WISTAR rats is more than 30% and less than 100%, the biological damage level caused by irradiation in uranium ore dust is I level; when the up-regulation rate of the relative expression quantity of alpha-1 protease A1AT in lung tissues of WISTAR rats is more than 100% and less than 200%, the biological damage grade caused by irradiation in uranium ore dust is II grade; when the relative expression quantity of alpha-1 anti-protease A1AT in lung tissues of WISTAR rats is up-regulated by more than 200%, the biological damage caused by irradiation in uranium ore dust is grade III.
2. The method for evaluating the biological damage of uranium ore dust irradiated by alpha-1 protease resistance A1AT according to claim 1,
the specific steps of the protein extraction are as follows:
pouring liquid nitrogen into a mortar for precooling, quickly taking out a lung tissue sample, cutting the tissue to be uniformly placed in the mortar, immediately adding the liquid nitrogen for grinding, slowly adding 800 μ l LB lysate into mortar, cracking for 30 min, shaking for 1-2 times, fully cracking, collecting 1.5 mL suspension into EP tube after cracking, then centrifuging at the rotating speed of 12000 r/min for 20 min at the temperature of 4 ℃, collecting supernatant, precipitating protein in the supernatant with resin, centrifuging at 12000 r/min at 4 deg.C for 20 min, removing supernatant, air drying, storing at-80 deg.C for use, adding 200-250 mu L L3 lysate into the dried protein pellet, repeatedly blowing and beating by using a gun head until the protein is fully dispersed, carrying out ultrasonic assisted dissolution at 4 ℃, 12000 r/min, centrifuging for 20 min, sucking out the supernatant, and transferring to a new EP tube.
3. The method for evaluating the biological damage of uranium ore dust irradiated by alpha-1 protease resistance A1AT according to claim 1,
the specific steps for determining the total protein concentration are as follows:
adding a diluent into 0.2 mg/ml BSA standard solution, sequentially diluting into 0, 5, 10, 20, 50, 100, 150 and 200. mu.g/ml standard solution, measuring their absorbance values at 562 nm, drawing a standard curve, measuring the absorbance value at 562 nm of each sample solution, and calculating the concentration of protein in the sample through the standard curve.
4. The method for evaluating the biological damage of uranium ore dust irradiated by alpha-1 protease resistance A1AT according to claim 1,
the specific steps of the protein denaturation are as follows:
adding the protein solution into 5 Xprotein loading buffer solution at a ratio of 4:1, performing boiling water bath denaturation for 15 min, and storing in a refrigerator at-20 deg.C for use.
5. The method for evaluating the biological damage of uranium ore dust irradiated by alpha-1 protease resistance A1AT according to claim 1,
the gel electrophoresis comprises the following specific steps:
preparing 10% separation gel and 5% concentration gel from 30% acrylamide, 1.5M TRIS-HCl, 10% SDS, 10% ammonium persulfate and TEMED according to a certain proportion; and after the separation gel is solidified, adding electrophoretic fluid into an electrophoresis tank, adding the denatured protein sample into an electrophoresis hole, carrying out electrophoresis, regulating the voltage of the concentrated gel to 75V, regulating the voltage of the separation gel to 100V, carrying out electrophoresis until bromophenol blue just runs to the bottom of the separation gel, stopping electrophoresis, and carrying out membrane transfer.
6. The method for evaluating the irradiation biological damage in uranium ore dust by using alpha-1 protease resistance A1AT according to claim 1,
the specific steps of the film transfer are as follows:
activating a PVDF membrane by adopting methanol, placing 5-6 pieces of filter paper with the size of 7 multiplied by 9 cm at the bottom of the membrane, placing the filter paper and the membrane transferring instrument together, adding membrane transferring liquid, transferring the membrane for 30 min at a constant current of 300 mA, and placing a membrane transferring groove in ice water for cooling in the membrane transferring process.
7. The method for evaluating the biological damage of uranium ore dust irradiated by alpha-1 protease resistance A1AT according to claim 1,
the immune response comprises the following specific steps:
sealing the transferred membrane with 5% of skimmed milk, uniformly mixing for 1 h on a decolorizing shaker, diluting the alpha-1 protease-resistant primary anti-Alah 3al with 5% of skimmed milk dissolved by TBST at a concentration of 1:1000, uniformly mixing with the sealed membrane, incubating overnight at 4 ℃, cleaning the membrane incubated overnight with alpha-1 protease-resistant primary anti-Alah 3al with 2% TBST, cleaning 3 times on the decolorizing shaker at room temperature for 5 min each time, finally diluting the secondary anti-goat anti-rabbit with TBST 8000 times, incubating for 1.5 h at room temperature, and cleaning three times on the decolorizing shaker for 10 min each time.
8. The method for evaluating the biological damage of uranium ore dust irradiated by alpha-1 protease resistance A1AT according to claim 1,
the chemiluminescence reaction comprises the following specific steps:
mixing ECLA and ECLB reagents in equal volume in a centrifuge tube in a dark room, enabling the protein surface of the PVDF membrane to face upwards, adding the mixed ECL solution, fully reacting for 1-2 min, and then putting the mixture into a gel imaging system for scanning analysis.
9. The method for evaluating the biological damage of uranium ore dust irradiated by alpha-1 protease resistance A1AT according to claim 1,
the gel imaging scanning analysis comprises the following specific steps:
taking a picture of the protein band by using a gel imaging system, analyzing the gray value of a target band by using Image J software, scanning the gray value of alpha-1 protease-resistant A1AT protein to obtain the expression quantity of alpha-1 protease-resistant A1AT protein, and scanning the gray value of reference protein GAPDH protein to obtain the expression quantity of GAPDH protein; the relative expression level of the alpha-1 protease A1AT is obtained by dividing the expression level of the alpha-1 protease A1AT protein by the expression level of the internal reference protein GAPDH protein.
10. The method for evaluating the biological damage of uranium ore dust irradiated by alpha-1 protease resistance A1AT according to claim 1,
the specific steps of the analysis of the relative expression quantity up-regulation rate of the alpha-1 anti-protease A1AT are as follows:
the relative expression level up-regulation rate of alpha-1 anti-protease A1AT was calculated according to formula (1):
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