A method for estimating radiation biological dose using acrocentric chromosome synapsis

Abstract

The invention discloses a method for estimating radiation biological dose by using acrocentric chromosome synapsis, comprising: 1) irradiating a sample, comprising irradiating a human peripheral blood sample in a container by using radiation; 2) culturing the sample, comprising culturing the irradiated sample, comprising adding a culture solution and culturing in a 37°C incubator for 48-56 hours; 3) harvesting a cell suspension, comprising performing a harvesting operation after culturing to obtain a cell suspension; 4) dropping a slide, comprising dropping the cell suspension on a precooled glass slide and burning the suspension for 1-2 seconds with an alcohol lamp; 5) staining, staining the glass slide with a Giemsa staining solution; 6) reading and counting, comprising counting the incidence of chromosome synapsis by using a microscope; and 7) fitting a dose-effect curve based on the correlation between the incidence of chromosome synapsis and the irradiation dose, and estimating the radiation dose by using the dose-effect curve.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for estimating radiation biological dose by utilizing acrocentric chromosome synapsis. Background Art

[0002] With the rapid development of nuclear technology, nuclear energy has been widely used in the fields of military, energy, industry, agriculture, medicine, scientific research and environmental protection. However, while nuclear energy benefits mankind, it can also have potential hazards. Radiation hazard assessment is based on radiation dose and quantitatively analyzes different types of biological effects that occur after irradiation. Physical dose estimation and biological dose estimation are generally used to estimate the dose of exposed personnel. Among them, biological dose estimation often uses chromosome aberration analysis methods. Human chromosomes are very sensitive to ionizing radiation, and chromosome aberrations can be induced at lower doses, and there is a good dose-effect relationship between different doses. Chromosome aberrations are divided into two types: stable aberrations and unstable aberrations. Among them, stable chromosome aberration analysis mainly includes: chromosome fluorescence in situ hybridization (FISH), premature condensed chromosomes (PCC), G banding, etc. (G-band); unstable chromosome aberration analysis is mainly dicentric chromosome + centromere ring (dicentrics+ring, dic+r) analysis.

[0003] As a mature technology, chromosome aberration analysis has been listed in the technical report series by the International Atomic Energy Agency (IAEA). [1] Bender and Gooch [2] Firstly, the DIC+R method was used to establish a standard curve for the relationship between the chromosome aberration rate of peripheral blood lymphocytes and the irradiation dose, which was applied to the dose estimation of personnel exposed in nuclear accidents. The dose-effect relationship between ionizing radiation, influencing factors and the influence of some use conditions were explained through a large amount of experimental data. [3,4] DIC+R has a special morphology and is easy to identify. It has a low spontaneous rate and is not affected by gender and age. It is radiation-specific. Statistics of its incidence in the metaphase of chromosomes can be used to estimate the dose of local and whole-body irradiated subjects. [5-8] .

[0004] FISH analysis technology uses chemically labeled nucleic acid probes to hybridize with DNA in interphase cells or metaphase chromosomes after radiation exposure, thereby measuring the chromosome aberration translocation rate (t). After hybridization, the fluorescent signal can only be seen through immunofluorescence antibody detection. When the signal is weak or small, it can be amplified by antigen-antibody reaction. Using FISH analysis technology to detect translocation rate can also accurately estimate biological doses. It can be used to estimate the cumulative dose of low-dose or long-term chronic radiation recipients and to reconstruct the exposure dose of victims in earlier accidents. [9-11] .

[0005] PCC analysis can directly detect chromosome damage in interphase cells shortly after irradiation. This technique is to fuse metaphase cells and interphase cells, so that the interphase nucleus enters the mitotic phase early. The interphase cell nuclear membrane dissolves, and the extremely dispersed chromatin in the nucleus condenses into a chromosome-like structure. This fine fibrous chromosome is called PCC. This method can estimate the dose to more than 20Gy, and is a method suitable for estimating large doses. [12,13] .

[0006] However, the above methods have some shortcomings. For example, the analysis of unstable chromosome aberrations is not suitable for internal irradiation, fractionated irradiation and low-dose multiple irradiation; the frequency of dic+r during acute irradiation will decrease with time, resulting in inaccurate dose estimation in the later stage; dic+r analysis has high professional and technical requirements for analysts, and it is necessary to group and analyze chromosomes in the division phase when necessary. FISH analysis technology has many operating steps and is cumbersome to operate. It is necessary to use fluorescent probes and analyze under a fluorescent microscope. The instruments and reagents are expensive and the scope of promotion is narrow. The time-consuming cell preparation, high technical requirements, low PCC yield and instability limit its application in estimating radiation biological doses.

[0007] Therefore, new technologies and methods are needed to address the deficiencies in the prior art.

[0008] References:

[0009] 1. IAEA.BioIogicaIDosimetry, ChromosomaI Abrration Analysis for DoseAssessment. IAEA TechnicaI Reports, Series No. 260, Vienna: IAEA, 1986.

[0010] 2. Bender MA, Gooch P C.Types and Rates of X-ray-induced

[0011] Chromosome Aberrations in Human BIood Irradiated invitro.Proc.NatI.Acad.Sci., 1962, 4(8 4): 522.

[0012] 3. Bender MA, Gooch PC. Persistent chromosome aberrations in irradiated human subjects. Radiat Res, 1962, 16: 44–53.

[0013] 4. IAEA. EPR-biodosimetry. Cytogenetic dosimetry: applications inpreparedness for and response to radiation emergencies. IAEA, Vienna, Austria, 2011.

[0014] 5. Jin Cuizhen. Chromosome aberration. Wang Jixian (chief editor). Radiation biological dosimetry. Beijing: Atomic Energy Press, 1997: 58.

[0015] 6. Lü Yumin, Fu Baohua, Han Lin, et al. Estimation of biological dose (chromosome aberration) of victims in the “4.26” 60Co source radiation accident in Henan. Chinese Journal of Radiological Medicine and Protection, 2001, 2(13): 153.

[0016] 7. RamaIho AT, Nascimento ACH, Natarajan A T. Does Assessment by Cytogenetic Analysis in the Goiania (BraziI) Radiation Accident. Rad. Protect. Dosimetry, 1988, 25(2): 97.

[0017] 8. LittIefieId LC, Jomer EF, CoIyer S P.The 1989San SaIvadon 60CoRadiation Accident Cytogenetic Dosimetry and FoIIow-up Evaluation in ThreeAccident Victims.Rad.Protect.Dosimetry, 1991.3(5 2): 115.

[0018] 9. UNSCEAR.1988Report on the United Nations Scientific Committee on the Effects of Atomic Radiation to the GeneraI AssembIy.Annex 7, CernobyI'sAccident: 616.

[0019] 10. Bauchinger M, BraseImann H, Savage JRK, et al.

[0020] 11. Zhang Zeyun, Liu Xiulin, Cheng Xin, et al. Detection of chromosomal aberrations in victims of the 6.25 accident in Shanghai by fluorescence in situ hybridization. Chinese Journal of Radiological Medicine and Protection, 2000, 2(05): 338.

[0021] 12. Kanda R, Hayata I, Lloyd DC.Easy biodosimetry for high-doseradiation exposures using drug-induced, prematurely condensed chromosomes

[0022] [J].Int J Radiat Biol, 1999, 75(4):441-446.

[0023] 13. Yao Bo, Li Yufang, Liu Guangxian, et al. Dose-effect curve of premature agglutinated chromosome rings in human blood after ultra-high dose irradiation with 60Co γ-rays[J]. Chinese Journal of Radiological Medicine and Protection, 2009, 29(1): 54-56 Summary of the invention

[0024] Acrocentric chromosome associations (ACA) represent early chromosomal instability damage. The satellite fibers or short arm connections on acrocentric chromosomes are composed of nucleoprotein B, and studies have found that these chromosomal regions contain DNA complementary to rRNA, i.e., ribosomal DNA. The connection between rDNA on heterologous chromosomes was found using super-resolution microscopy, indicating that dynamic DNA sites contribute to the network between somatic chromosomes and become an essential and persistent feature of the genome. The inventors studied the incidence of ACA in the metaphase phase of human peripheral blood chromosomes induced by ionizing radiation and constructed a dose-response curve; it was found that it has a good dose-effect relationship for different types of rays and smaller doses of ionizing radiation, and thus ACA has the potential to be a candidate cytogenetic biomarker for radiation biodosimetry. Based on this discovery, the present invention provides a method for estimating radiation biological dose using acrocentric chromosome associations.

[0025] More specifically, according to one aspect of the present invention, a method for estimating radiation biological dose using acrocentric chromosome synapsis is provided, comprising:

[0026] 1) irradiating the sample, including irradiating the human peripheral blood sample in the container with radiation;

[0027] 2) culturing the sample, including culturing the irradiated sample, including adding culture medium and culturing in a 37° C. incubator for 48-56 hours;

[0028] 3) harvesting the cell suspension, including performing a harvesting operation after culturing to obtain a cell suspension;

[0029] 4) Dropping, including dropping the cell suspension on a pre-cooled glass slide, burning the suspension with an alcohol lamp for 1 to 2 seconds,

[0030] 5) staining: staining the slide with Giemsa staining solution;

[0031] 6) Reading and counting, including the use of microscopy to identify and count the incidence of acrocentric chromosome synapses; and

[0032] 7) Based on the correlation between chromosome synapsis frequency and radiation dose, the dose-effect curve was fitted and used to estimate the radiation dose.

[0033] According to an embodiment of the present invention, in step 1), the radiation includes gamma rays and X-rays, the dose rate of the gamma rays does not exceed 2 Gy / min, and the dose does not exceed 5 Gy; the dose rate of the X-rays does not exceed 2 Gy / min, and the dose does not exceed 2 Gy.

[0034] According to an embodiment of the present invention, step 2) further includes repairing the irradiated sample before culturing.

[0035] According to an embodiment of the present invention, in step 2), the culture medium is RPMI1640 culture medium containing 20% ​​fetal bovine serum, 0.2% PHA, 1% penicillin-streptomycin and a final concentration of 0.04 μg / ml colchicine.

[0036] According to an embodiment of the present invention, step 3) includes centrifugation, hypotonicity and fixation operations.

[0037] According to an embodiment of the present invention, step 6) includes observing the chromosome metaphase at a magnification of 200 times under a microscope, and switching the metaphase with well-dispersed chromosomes and clear morphology to be analyzed under a 1000-fold oil microscope to identify and count the incidence of acrocentric chromosome synapsis.

[0038] According to an embodiment of the present invention, the identification criteria for acrocentric chromosome association include: the distance between chromosome centromeres is less than the length of the long arm of the largest G group chromosome; when the short arms of the chromosomes are connected by a visible line, the distance between the chromosome centromeres is less than 1.2 times the length of the long arm of the largest G group chromosome; when the associated chromosomes are close to each other in a longitudinal cluster, the distance between the chromosome centromeres does not exceed the length of the long arm of the largest D group chromosome.

[0039] According to an embodiment of the present invention, the gamma rays are 60 After Coγ-ray irradiation, the dose-effect equation of the acrocentric chromosome synapsis rate is: Y = (0.03960 ± 0.00409) * X + (0.2344 ± 0.01), R 2 =0.9968; after X-ray irradiation, the dose-effect equation of the acrocentric chromosome synapsis rate is Y=(0.2022±0.12772)*X+(0.3165±0.0731), R 2 =0.9587.

[0040] According to an embodiment of the present invention, step 7) further comprises: before using the dose-effect curve to estimate the radiation dose, verifying the accuracy of the fitted dose-effect curve.

[0041] The present invention uses a conventional chromosome preparation method familiar to analysts to prepare ACA samples, but there is no need to group chromosomes in the metaphase division phase. As long as the two groups of acrocentric chromosome morphology are familiar and the number of chromosomes in the division phase is ≥ 44, analysis can be performed. The problem that professionally trained personnel are required to perform group analysis of chromosome aberrations is solved, and the frequency of ACA induced by ionizing radiation is higher than that of DIC+R, which is convenient for observation and statistical analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Flow chart of a method for estimating radiation biological dose for acrocentric chromosome synapsis according to an embodiment of the present invention;

[0043] Figure 2 A picture of a sample observed by a microscope in a method for estimating radiation biological dose according to an embodiment of the present invention for acrocentric chromosome synapsis;

[0044] Figure 3 For different doses 60 The frequency of chromosome synapsis during metaphase after Coγ-ray irradiation;

[0045] Figure 4 is the frequency of chromosome synapsis in metaphase after different doses of X-ray irradiation. DETAILED DESCRIPTION

[0046] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] Figure 1 Flow chart of a method for estimating radiation biological dose by acrocentric chromosome synapsis according to an embodiment of the present invention. As shown in the figure, the method for estimating radiation biological dose by acrocentric chromosome synapsis according to an embodiment of the present invention can be performed in the following steps.

[0048] First, samples were collected and irradiated. Peripheral blood was collected from healthy subjects through the vein and placed in a heparin anticoagulation negative pressure blood collection tube. The samples were divided into two groups, 5 in each group, totaling 10 initial samples, and irradiated with gamma rays and X-rays respectively.

[0049] More specifically, the first group of human peripheral blood samples were treated with a dose rate of 0.27 Gy / min and doses of 0, 1, 2, 3, and 4 Gy. 60 Coγ-ray irradiation, and the second group of human peripheral blood samples were irradiated with X-rays with an energy of 160 keV, a dose rate of 1 Gy / min, and doses of 0, 0.25, 0.5, 0.75, and 1 Gy.

[0050] After irradiation, culture was performed. All samples were placed in a 37°C incubator for two hours. After that, 700 μl of peripheral blood was drawn from each sample in a clean bench and added to the prepared RPMI1640 culture medium containing 20% ​​fetal bovine serum, 0.2% PHA, 1% penicillin-streptomycin and a final concentration of 0.04 μg / ml colchicine. After mixing, the samples were placed in a 37°C incubator for 48 to 56 hours. Two parallel samples were made for each sample.

[0051] For each sample, the culture was dispersed with a pipette, centrifuged at 1000 rpm for 10 min at room temperature, the supernatant was discarded, and then 8 ml of hypotonic solution (0.075 mol / L KCI) preheated at 37°C was added to rupture the red blood cells; then gently pipette to mix, and place at 37°C.

[0052] Incubate in a hypotonic incubator for 10-15 minutes. Then add 1 ml of freshly prepared pre-cooled fixative (methanol: glacial acetic acid = 3:1, v / v) for pre-fixation, mix by blowing; centrifuge at 1000 rpm for 10 minutes, discard the supernatant; then add 8 ml of pre-cooled fixative (methanol: glacial acetic acid = 3:1, v / v) for fixation, mix by blowing, and let stand for 20 minutes; centrifuge again at 1000 rpm for 10 minutes, and discard the supernatant.

[0053] Repeat the above steps 1 to 2 times.

[0054] For each sample obtained from the above treatment, add about 300 μl of fixative solution (methanol:

[0055] Mix by blowing to suspend the precipitate, then drop 400 μl of the cell suspension on a pre-cooled glass slide and burn the suspension with an alcohol lamp for 1 to 2 seconds.

[0056] After the samples treated in the above steps are dried at room temperature, they are stained with freshly prepared 8% Giemsa stain for about 8 minutes (8-10 min), rinsed with water, dried and stored in a slide box.

[0057] Then the slides were read and counted using a microscope.

[0058] The metaphase of chromosomes was observed under a microscope at a magnification of 200 times. The metaphase with well-dispersed chromosomes and clear morphology was analyzed under an oil microscope at a magnification of 1000 times to identify and count the incidence of acrocentric chromosome association (ACA).

[0059] The identification criteria for acrocentric chromosome synapsis include: 1. The distance between chromosome centromeres is less than the length of the long arm of the largest G group chromosome. 2. When a line connecting the chromosome short arms is visible, a longer distance can be accepted, for example, the distance between chromosome centromeres is less than 1.2 times the length of the long arm of the largest G group chromosome. 3. If the associated chromosomes are close to each other in a longitudinal cluster, a larger distance is accepted, for example, not exceeding the long arm length of the largest D group chromosome (see Figure 2 The image of the sample observed under a microscope shows that the short arms of the acrocentric chromosomes are close to each other, as shown by the arrows). A normal person has 46 (23 pairs) chromosomes, for example, group D chromosomes include chromosomes 13, 14, and 15, group G chromosomes include chromosomes 21 and 22, etc.

[0060] Then, based on the correlation between the frequency of acrocentric chromosome synapsis and irradiation dose, a dose-effect curve was fitted, and the radiation dose was estimated using the dose-effect curve.

[0061] The results showed that after irradiating human peripheral blood samples with different doses of different radiation, the frequency of chromosome synapsis in metaphase increased with increasing dose, but there was no statistical difference among the dose groups (see Table 1).

[0062] Table 1. Chromosome synapsis frequency in metaphase after irradiation with different radiation doses (ACA / cell):

[0063]

[0064] The relationship between chromosome synapsis rate and dose was fitted according to different ray types. 60 After Coγ-ray irradiation, the dose-effect equation of chromosome synapsis rate is: Y = (0.03960 ±

[0065] 0.00409)*X+(0.2344±0.01)(R 2 =0.9968)(See Figure 3 ); After X-ray irradiation, the chromosome synapsis rate dose effect equation is Y = (0.2022 ± 0.12772) * X +

[0066] (0.3165±0.0731)(R 2 =0.9587)(See Figure 4 ).

[0067] Verification: Exploitation 60 A normal human peripheral blood sample was blindly irradiated with Coγ-rays. The ACA incidence was counted and analyzed after preparation according to the method of this study. The ACA incidence was (0.298±0.0244) ACA / cell. 60The fitting curve of Coγ-ray irradiation was estimated to be 1.6 Gy, with a 95% confidence interval of (1.2,

[0068] 2.07Gy), which is close to the actual irradiation dose of 0.8Gy.

[0069] The results show that: 60 The frequency of acrocentric chromosome synapsis in metaphase increased with the increase of Coγ-ray irradiation and X-ray irradiation, and there was a statistically significant difference (χ 2 The values ​​were 28.015 and 83.410, respectively, p < 0.01), and there was a good dose-effect relationship. The acrocentric chromosome synapsis indicator can be used as a candidate radiation biodosimeter.

[0070] The method of the present invention reduces the time cost of personnel training, and the ACA has a high frequency of occurrence, which can improve the efficiency and accuracy of statistical analysis.

[0071] The above description of the embodiments is to facilitate the understanding and application of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the embodiments herein, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for estimating radiation biological dose using acrocentric chromosome synapsis, comprising: 1) irradiating the sample, including irradiating the human peripheral blood sample in the container with radiation; 2) culturing the sample, including culturing the irradiated sample, including adding culture medium and culturing in a 37° C. incubator for 48-56 hours; 3) harvesting the cell suspension, including performing a harvesting operation after culturing to obtain a cell suspension; 4) Dropping, including dropping the cell suspension on a pre-cooled glass slide, burning the suspension with an alcohol lamp for 1 to 2 seconds, 5) staining: staining the slide with Giemsa staining solution; 6) Reading and counting, including the use of a microscope to identify and count the frequency of acrocentric chromosome synapses; and 7) Based on the correlation between chromosome synapsis frequency and radiation dose, the dose-effect curve was fitted and used to estimate the radiation dose; Wherein, in step 1), the radiation includes gamma rays and X-rays, the dose rate of the gamma rays does not exceed 2 Gy / min, and the dose does not exceed 5 Gy; The X-ray dose rate shall not exceed 2 Gy / min and the dose shall not exceed 2 Gy.

2. A method for estimating radiation biological dose using acrocentric chromosome synapsis as claimed in claim 1, characterized in that: Step 2) also includes repairing the irradiated sample at 37° C. for 2 hours before culturing.

3. A method for estimating radiation biological dose using acrocentric chromosome synapsis as claimed in claim 1, characterized in that: In step 2), the culture medium is RPMI1640 culture medium containing 20% ​​fetal bovine serum, 1% penicillin-streptomycin, 0.2% PHA and colchicine with a final concentration of 0.04 μg / ml.

4. The method for estimating radiation biological dose using acrocentric chromosome synapsis according to claim 1, characterized in that: Step 3) includes centrifugation, hypotonicity and fixation operations.

5. The method for estimating radiation biological dose using acrocentric chromosome synapsis according to claim 1, characterized in that: Step 6) includes observing the metaphase of chromosomes under a microscope at a magnification of 200 times, and switching the metaphase with well-dispersed chromosomes and clear morphology to be analyzed under a 1000-fold oil microscope to identify and count the incidence of acrocentric chromosome synapsis.

6. The method for estimating radiation biological dose using acrocentric chromosome synapsis according to claim 1, characterized in that: The identification criteria for acrocentric chromosome synapsis include: the distance between chromosome centromeres is less than the length of the long arm of the largest G group chromosome; when a line is visible between the short arms of the chromosomes, the distance between chromosome centromeres is less than 1.2 times the length of the long arm of the largest G group chromosome; when the associated chromosomes are close to each other in longitudinal aggregation, the distance between chromosome centromeres does not exceed the length of the long arm of the largest D group chromosome.

7. A method for estimating radiation biological dose using acrocentric chromosome synapsis as claimed in claim 1, characterized in that: Gamma rays are 60 After Coγ-ray irradiation, the dose-effect equation of the acrocentric chromosome synapsis rate is: Y = (0.03960 ± 0.00409) * X + (0.2344 ± 0.01), R 2 =0.9968; after X-ray irradiation, the dose-effect equation of the acrocentric chromosome synapsis rate is Y=(0.2022±0.12772)*X+(0.3165±0.0731), R 2 =0.9587.

8. The method for estimating radiation biological dose using acrocentric chromosome synapsis according to claim 5, characterized in that: The number of chromosomes in the mitotic phase is ≥44.

9. The method for estimating radiation biological dose using acrocentric chromosome synapsis as claimed in claim 1, characterized in that: Step 7) also includes: before using the dose-effect curve to estimate the radiation dose, verifying the accuracy of the fitted dose-effect curve.

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