CN102508283B - Biochemical method for detecting ionizing radiation dose - Google Patents
Biochemical method for detecting ionizing radiation dose Download PDFInfo
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- CN102508283B CN102508283B CN 201110333811 CN201110333811A CN102508283B CN 102508283 B CN102508283 B CN 102508283B CN 201110333811 CN201110333811 CN 201110333811 CN 201110333811 A CN201110333811 A CN 201110333811A CN 102508283 B CN102508283 B CN 102508283B
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Abstract
The invention belongs to the technical field of biological detection, in particular to a biochemical method for detecting the ionizing radiation dose. According to the principle that a cell cycle can be interfered by ionizing radiation and cytochalasin B can retard cell division but cannot retard cell nucleus division, after different doses of gamma rays are used for irradiating lymphocyte, a certain quantity of cytochalasin B is added for processing so as to detect the four-core rate and the double-core rate of the lymphocyte irradiated with different doses of gamma rays. An experiment result shows that the four-core rate of the irradiated lymphocyte and the irradiated dose are in a linear dose-effect relationship within a low-dose range of 0-6.4cGY, a relevant coefficient is 0.957, and the radiation excitability effect is presented; and within a high-dose range of 0.4-20Gy, the double-core rate of the irradiated lymphocyte and the irradiated dose are in an exponential equation dose-effect relationship, the relevant coefficient is 0.978, and the radiation damage effect can be favorably reflected. The biochemical method for detecting the ionizing radiation dose is visual in detection indexes, is simple to operate, is quick and economic and is accurate in detection result.
Description
Technical field
The invention belongs to technical field of biological, be specifically related to a kind of biological detection method of radiation dose.
Background technology
Assessment ionising radiation healthhazard degree, the diagnosis of acute and chronic radiation damage and treatment etc. all need to understand and definite radioactive dose.Body cell, DNA, protein etc. exist certain dose-effect relationship between the variation that occurs after the ionising radiation and radiation dose, can estimate the size of radiation dose by these detectable biological indicators.Compare with physical detection, the sharpest edges that biological dose detects are directly to have reflected the reaction of body to ionising radiation.
The method of biological detection of carrying out at present radiation dose is a lot, and commonly used has: the micronucleus method and the chromosome karyotype analysis method that propose by detecting the indexs such as the caused chromosome fragment of irradiation, two centromere, exchange or transposition; Study single cell gel electrophoresis (Using Comet Assay) method of predicting radiation dosage by the relation between the comet core of shining comet tail that rear dna damage forms and the performance of int cell.Along with molecular biological fast development, increasing molecular indexes is as the sign of dose measurement, can cause the quick phosphorylation of histone γ-H2AX such as ionising radiation, after its specific antibody reaction, can form visible foci, γ-H2AX is the most important dna damage induction molecule of studying so far.Thereby the damage of the hematopoietic cell of ionization radiation induction can cause the gene point mutation of some coding maker albumen of cell to produce unusual encoding proteins or protein delation.These albumen unusual or disappearance can be used as the sign of dose monitoring, common are the Hb(haemoglobin), the GPA(glycophorin A), the HLA(human leucocyte antigen (HLA)), TCR (T cell antigen receptor), HPRT (hypoxanthine phosphoribosyltransferase) etc.But the common shortcoming of these methods be have complicated operation, subjective factor is more, testing cost is higher, can only detect the radioactive ray of high dose and tolerance etc.To biological detection and the tolerance of low dosage of radiation, also there is not a kind of method of accurate sensitivity at present.Along with the application in clinical diagnosis and treatment and worker, agricultural production of nuclear power development and radiotechnology, contacting in effect of low dose radiation and the people's lives is increasing, and the biological study of low dose exposure is seemed that realistic meaning is more arranged.Therefore current in the urgent need to have a kind of easy to use, degree of accuracy is high, can carry out to the radiation of high and low dose the method for biological detection simultaneously.
Summary of the invention
The object of the present invention is to provide a kind of easy to use, degree of accuracy is high, can carry out to the radiation of high and low dose simultaneously the method for biological detection.
The present invention is based on following principle: the cell cycle is that the cell of continuously division finishes to finish this continuous process that experiences to next time mitosis from last mitosis, comprises G1 phase, S phase, G2 phase, M phase four-stage.Radiation can be disturbed the normal cell cycle, and low dose radiation may be accelerated the cell cycle, makes the time shorten of whole cell cycle; On the contrary, high dose radiation can cause the cell cycle prolongation, makes time lengthening even the retardance of whole cell cycle; The medicine Cytochalasin B can block the integral body division of cell and not block nuclear division.The cell institute's dikaryotic number that forms after Cytochalasin B is processed that is subject to like this various dose irradiation is just closely related with exposure dose.
The biological detection method of radiation dose provided by the invention, concrete steps are as follows:
1, get the radiosusceptibility cell line, be divided into low dosage and high-dose irradiation group, exposure dose is the low dose exposure group less than 10cGy's, and exposure dose is the high-dose irradiation group greater than 40cGy;
2, Cytochalasin B is processed: after the irradiation, add Cytochalasin B immediately in cell culture fluid, final concentration is 2-6 μ g/ml, and 37 ℃ of incubators continue to cultivate 24-36h;
3, hypotonic processing: after cell centrifugation was abandoned supernatant, every solencyte added 5-10ml 0.075M KCl mixing, the hypotonic 10-20min of room temperature;
4, immobile liquid is fixed: adopt two step fixations, and hypotonic rear adding immobile liquid, in mass ratio, and immobile liquid: hypotonic medium=1:5-8), and centrifugal immediately behind the dropper mixing, abandon the every pipe of supernatant and add fixedly 20-30min of 5-8ml immobile liquid; Described immobile liquid is methyl alcohol and glacial acetic acid mixed solution, is methyl alcohol: glacial acetic acid=7-9:1 in mass ratio;
5, the judgement of double-core and quadrinucleate cell: because after cell shone, the mitosis meeting of cell is interfered, the nucleus atypia is obvious, Cytochalasin B is processed later on except producing normal dikaryocyte, two nucleus that small one and large one also can occur, if it is nuclear 1/3 that small nut size surpasses, this type of cell also is designated as dikaryocyte so; For the criterion of quadrinucleate cell, all cells greater than 3 nuclears all are designated as quadrinucleate cell.Each sample 1000 cells that count at least;
6, by statistics cell four nuclear rates, low dose radiation is carried out quantitatively, by statistics cell double-core rate, high dose radiation is carried out quantitatively.Double-core rate=dikaryocyte number/N, four nuclears rate=quadrinucleate cell number/N.N is the statistics cell number.N is no less than 1000.
Advantage of the present invention:
The present invention can solve the various problems of existing radiation dose bioassay method.As: can improve the degree of accuracy of dose measurement, the complicated operation of existing single cell gel electrophoresis (Using Comet Assay) method and chromosome karyotype analysis, subjective factor is more during detection, and is too high to technical requirement, and the parameter of detection is a lot, is difficult to accurate quantitative analysis; And postradiation cell of the present invention cell nuclei changes after Cytochalasin B is processed what just can be accurately and carry out intuitively quantitative and qualitative analysis.Secondly, it is all right that single cell gel electrophoresis (Using Comet Assay) method and chromosome karyotype analysis etc. carry out quantitative susceptibility to high dose radiation, but can not low dose radiation be carried out quantitatively; And four nuclear rates of cell changed and can well low dose radiation be carried out quantitatively after postradiation cell was processed with Cytochalasin B among the present invention, can carry out quantitatively it for the double-core rate of high dose radiation cell.Therefore, the present invention can carry out accurate quantitative analysis to low, high dose radiation respectively by these two of four nuclear rates and double-core rates index very intuitively.
The present invention can be used for the biological detection of high and low radioactive dosage, is a kind of new method of Biological Detection of radiation dose.This technology also can be used for the impact of various medicine or poisonous substance on cell proliferation in addition, also can be used for the size of estimation medicine or poisonous substance dosage, therefore can be used for the toxicologic study of medicine or poisonous substance.
Embodiment
1) illuminate condition and experiment grouping: the HMy2.CIR people B lymphoblast (1 * 10 in growth period of taking the logarithm
6Cell/ware) is inoculated in the 60mm double dish, cultivates 24h, at room temperature cell is imposed 1.6,3.2,4.8,6.4,8cGy low dose exposure or 0.4,1,2,4,8,12,16,20Gy high-dose irradiation arranges the blank group without irradiation simultaneously, and each group is established three repetitions (n=3).
2) behind the cell irradiation, cell is hatched in the fresh medium that contains 3 μ g/ml Cytochalasin Bs (Sigma company), continue to cultivate 24h immediately.
3) cell transfer is entered in the 15ml centrifuge tube, the centrifugal 10min of 300g removes nutrient solution, and every solencyte adds 7ml 0.075M KCl mixing, the hypotonic 10min of room temperature.
4) add the 1ml immobile liquid and (count in mass ratio methyl alcohol: glacial acetic acid 9:1) pre-fix cell.
5) the centrifugal 10min of 300g removes hypotonic medium, and every pipe adds fixedly 30min of 5ml immobile liquid.
6) the centrifugal 10min of 300g keeps about 0.5ml immobile liquid, drips sheet, drying.
7) with Giemsa dye liquor dyeing 30min, 40 power microscopes are observed.
8) data are processed: calculate respectively double-core rate and the four nuclear rates of cell, double-core rate=dikaryocyte number/1000, four nuclears rate=quadrinucleate cell number/1000 according to dikaryocyte number and quadrinucleate cell number.1000 cell number for statistics.
Experiment effect:
In 0 to 6.4cGy the low dosage scope of using, be subjected to be linear dose-effect relationship Y according to lymphocytic four nuclear rates and radioactive dose
4N=a+bD, related coefficient is 0.957, can significantly show the radiation excitability effect; In 0.4 to 20Gy dosage range, be subjected to be secondary exponential decay dependence Y between the double-core rate of photo cell and the radioactive dose
BN=exp (aD-bD
2), related coefficient is 0.978, can reflect well the radiation damage effect.
This new method is simple to operate, quick, economical, the detection index is directly perceived, testing result is accurate.
Claims (1)
1. biological method that detects ionizing radiation dose is characterized in that concrete steps are as follows:
(1), get the radiosusceptibility cell line, be divided into low dosage and high-dose irradiation group, exposure dose is the low dose exposure group less than 10cGy's, exposure dose is the high-dose irradiation group greater than 40cGy;
(2), Cytochalasin B is processed: after the irradiation, add Cytochalasin B immediately in cell culture fluid, final concentration is 2-6 μ g/ml, and 37 ℃ of incubators continue to cultivate 24-36h;
(3), hypotonic processing: after cell centrifugation was abandoned supernatant, every solencyte added 5-10ml 0.075M KCl mixing, the hypotonic 10-20min of room temperature;
(4), immobile liquid fixes: adopt two step fixations, hypotonic rear adding immobile liquid, in mass ratio, immobile liquid: hypotonic medium=1:5-8, centrifugal immediately behind the dropper mixing, abandon the every pipe of supernatant and add fixedly 20-30min of 5-8ml immobile liquid; Described immobile liquid is methyl alcohol and glacial acetic acid mixed solution, is methyl alcohol: glacial acetic acid=7-9:1 in mass ratio;
(5), the judgement of double-core and quadrinucleate cell: nuclear 1/3 if the size of small nut surpasses for small one and large one two nucleus except producing normal dikaryocyte, this type of cell also is designated as dikaryocyte; Criterion for quadrinucleate cell is that all cells greater than 3 nuclears all are designated as quadrinucleate cell; Each sample, the statistics cell number is no less than 1000;
(6), by statistics cell four nuclear rates, low dose radiation is carried out quantitatively, by adding up cell double-core rate, high dose radiation is carried out quantitatively; Double-core rate=dikaryocyte number/N, four nuclears rate=quadrinucleate cell number/N, N is the statistics cell number, N is no less than 1000.
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| CN110672501A (en) * | 2013-12-17 | 2020-01-10 | 阿兰蒂克微科学股份有限公司 | Dosimeter comprising a lensless imaging system |
| CN104017876A (en) * | 2014-06-10 | 2014-09-03 | 复旦大学 | Method for detecting lymphopoiesis by utilizing medicament cytochalasin B |
| CN109407133B (en) | 2017-08-18 | 2023-09-22 | 南京中硼联康医疗科技有限公司 | Biological dosimeter and neutron capture treatment system with same |
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