A method for distinguishing local irradiation based on the number of γ-H2AX foci in hair follicle cells
By detecting the γ-H2AX focus number in hair follicle cells, the dose effect curve of systemic and local radiation is established, and the problem of judging local radiation in the existing technology is solved, and rapid and accurate radiation dose evaluation is achieved, which is suitable for large-scale sample collection and Acute radiation assessment.
Patent Information
- Application Number
- CN202410024037.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-01-08
- Publication Date
- 2024-12-10
- Estimated Expiration
- 2044-01-08
AI Technical Summary
It is difficult to quickly and accurately judge local radiation accidents in the prior art, and the existing methods are time-consuming and require professional skills, which cannot meet the needs of rapid classification diagnosis and dose estimation of injured people in large-scale radiation accidents.
By detecting the γ-H2AX focus number in hair follicle cells after local irradiation and whole body irradiation at different doses, the dose effect curves for systemic and local irradiation were established, and the illuminated dose was estimated and whether there was a significant difference was determined to determine local irradiation.
It provides a fast method that does not require cell culture, which can directly detect γ-H2AX focus number in hair follicle cells, is suitable for large-scale sample collection, is suitable for non-clinical personnel operation, can quickly judge the locality and dose intensity of radiation accidents, satisfying the Need for acute ionizing radiation assessment.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of radiation damage detection, and in particular relates to a method for distinguishing local irradiation based on the number of γ-H2AX foci of hair follicle cells. Background Art
[0002] Ionizing radiation has been widely used in the fields of industry, agriculture, health, etc., which has greatly promoted the development of society. At the same time, ionizing radiation accidents occur from time to time, and most of them are local irradiation. In the process of responding to large-scale radiation accidents, rapid classification diagnosis and rapid dose estimation of the injured are one of the important issues in emergency response to radiation accidents.
[0003] According to the recommendations of the 2011 International Atomic Energy Agency (IAEA) recommendations, cytogenetic indicators such as chromosome aberrations are used to determine whether local irradiation has occurred. Specifically, the mitotic metaphase dicentric indicators of chromosomes prepared from patient blood samples are observed to show a non-Poisson distribution. The above judgment method is to use the U test. If the data conforms to the Poisson distribution, it is recommended to estimate the dose value according to the whole body uniform irradiation; if the data conforms to the non-Poisson distribution, it is recommended to use the Dolphin and Qdr methods to obtain the estimated value of the local irradiation dose, rather than simply citing the whole body uniform irradiation for dose estimation. The U test is recommended for evaluating whether the data conforms to the Poisson distribution method, but many people believe that the U test is not completely reliable, especially when the metaphase division phases counted are small. Therefore, the routine practice of many laboratories is to use either the Dolphin and Qdr methods to compare the whole body irradiation dose estimate with the local dose estimate. If there is a significant difference between the whole-body dose estimate and the local irradiation dose estimate, the laboratory should treat the situation as a potential local irradiation event; if it is uncertain whether there is a significant difference between the whole-body irradiation and local irradiation dose estimates, it is recommended to use the recommended Dolphin and Qdr methods only when the data significantly present a non-Poisson distribution.
[0004] The biological dose estimation method represented by chromosome aberration requires a long cell culture time and professional technical ability, and cannot meet the needs of rapid classification diagnosis and rapid dose estimation of casualties in large-scale radiation accidents. In recent years, the research on radiation emergency response methods has been striving to improve the detection speed of radiation biological dosimeters. The number of γ-H2AX foci or protein expression level can also be used to evaluate local irradiation. Ionizing radiation causes DNA double-strand breaks (DSBs), and phosphatidylinositol 3-kinases such as ataxia telangiectasia-mutated kinase (ATM) and ataxia telangiectasia and Rad3-related kinase (ATR) phosphorylate the 139th serine on H2AX to form phosphorylated H2AX, namely γ-H2AX foci. Using γ-H2AX antibody to stain γ-H2AX foci and counting γ-H2AX foci in many tissues including lymphocytes, hair follicles and skin biopsies is the most sensitive method for quantitatively estimating the radiation dose. In 2010, Redon et al. found that the number of γ-H2AX foci and flow cytometry can be used to estimate uniform and non-uniform radiation exposure doses (Redon CE, Nakamura AJ, Gouliaeva K, et al. The use of gamma-H2AX as a biodosimeter for total-body radiation exposure in non-human primates [J]. PLoS One, 2010, 5 (11): e15544. DOI: 10.1371 / journal.pone.0015544.). In 2011, Redon et al. verified this result in rhesus monkeys and extracted hair from rhesus monkeys to detect the number of γ-H2AX foci. They found that this method is applicable to local irradiation of all parts of the body, but did not mention the method of distinguishing local irradiation (Redon CE, Nakamura AJ, Gouliaeva K, et al. Q (gamma-H2AX), an analysis method for partial-body radiation exposure using gamma-H2AX in nonhuman primate lymphocytes [J]. Radiat Meas, 2011, 46 (9): 877-881. DOI: 10.1016 / j.radmeas.2011.02.017.).Compared with chromosomal aberrations, dose estimation using γ-H2AX foci has the advantage of not requiring cell culture and can be used as an alternative and rapid method to assess dose intensity and heterogeneity after acute ionizing radiation. Summary of the invention
[0005] In order to solve the problems existing in the prior art that local irradiation cannot be accurately judged, or the local irradiation judgment method is time-consuming and requires professional skills, the present invention detects the number of γ-H2AX foci in hair follicle cells after local irradiation and whole-body irradiation at different doses for a certain period of time, and respectively establishes a whole-body irradiation dose-effect curve fitted by the average number of γ-H2AX foci of hair follicle cells after whole-body irradiation of 0-8Gy X-rays and a local irradiation dose-effect curve fitted by the average number of γ-H2AX foci of hair follicle cells after local irradiation of 0-8Gy X-rays. The average number of γ-H2AX foci of hair follicle cells of irradiated individuals is respectively brought into the above two effect curves to obtain a whole-body dose estimation value and a local irradiation dose estimation value, respectively. When the whole-body dose estimation value is significantly different from the local irradiation dose estimation value, it is determined that local irradiation has occurred.
[0006] In order to solve the above technical problems and achieve corresponding technical effects, the present invention provides the following technical solutions:
[0007] The first object of the present invention is to provide a method for distinguishing local irradiation based on the number of γ-H2AX foci in hair follicle cells, which specifically comprises the following steps:
[0008] S1. Whole-body irradiation and local irradiation were performed on individuals using different doses of X-rays ranging from 0 to 8 Gy, and the number of γ-H2AX foci in the hair follicle cells of the individuals after irradiation was detected to obtain the average number of γ-H2AX foci in the hair follicle cells to fit the whole-body irradiation dose-effect curve and the average number of γ-H2AX foci in the hair follicle cells to fit the local irradiation dose-effect curve;
[0009] S2, detecting the number of γ-H2AX foci in the hair follicle cells of the irradiated individuals, and inserting the average number of γ-H2AX foci into the two curves in S1 to obtain the estimated value of whole body dose and the estimated value of local irradiation dose;
[0010] S3. Analyze the difference between the estimated whole-body dose and the estimated local irradiation dose. If the difference is significant, it is judged that local irradiation has occurred.
[0011] In one embodiment of the present invention, S1 detects the number of γ-H2AX foci in individual hair follicle cells 6 hours after irradiation.
[0012] In one embodiment of the present invention, the method for detecting the number of γ-H2AX foci in S1 hair follicle cells is to take hair with hair follicle cells as samples, transfer the samples to 2 mL centrifuge tubes, and use 1 mL The samples were fixed with 2% paraformaldehyde solution (PFA), and then PFA was removed and washed once with 1× phosphate buffered saline (PBS); pre-cooled 70% ethanol was added and the samples were permeabilized at 4°C for one week, then the samples were taken out and washed with PBS for 5 minutes; the samples were blocked with 5% bovine serum albumin solution (BSA); after washing the samples with PBS once, the samples were incubated with rabbit monoclonal anti-γ-H2AX antibody (primary antibody), and then incubated with goat F(ab')2 anti-rabbit Alexa-594 labeled IgG (H+L) antibody (secondary antibody) (protected from light); the samples were washed twice with PBS, placed on a glass slide, one end containing hair follicle cells was placed in a hydrophobic circle drawn with an immunohistochemical pen (PAP pen), and the other end of the sample was fixed on a glass slide; anti-fading and anti-fading 4',6-diamidino-2-phenylindole solution (DAPI) was added and incubated at 37°C; photos were taken using a confocal microscope to observe the number of γ-H2AX foci in the cells.
[0013] In one embodiment of the present invention, the PFA fixation time is 30 min, and the BSA blocking time is 30 min.
[0014] In one embodiment of the present invention, the rabbit monoclonal anti-γ-H2AX antibody is diluted 1:400 and incubated for 3 hours, and the goat F(ab')2 anti-rabbit Alexa-594 labeled IgG (H+L) antibody is diluted 1:500 and incubated for 1.5 hours.
[0015] In one embodiment of the present invention, DAPI was added and incubated for 30 min.
[0016] In one embodiment of the present invention, the average number of γ-H2AX foci in hair follicle cells in S1 is the average number of γ-H2AX foci obtained by detecting 200 hair follicle cells in each sample.
[0017] In one embodiment of the present invention, S2 detects the number of γ-H2AX foci in hair follicle cells of an irradiated individual 6 hours after irradiation.
[0018] In one embodiment of the present invention, the analysis of S3 differences was performed using an independent sample t-test, and P<0.05 indicated a significant difference.
[0019] The second object of the present invention is to provide application of the above method in local irradiation dose assessment.
[0020] Beneficial effects of the present invention:
[0021] The present invention provides a method for distinguishing local irradiation based on the number of γ-H2AX foci in hair follicle cells. The method detects the number of γ-H2AX foci in hair follicle cells after local irradiation at different doses and whole body irradiation for a certain period of time, respectively establishes a whole body irradiation dose-effect curve fitted by the average number of γ-H2AX foci in hair follicle cells after whole body irradiation of 0-8Gy X-rays and a local irradiation dose-effect curve fitted by the average number of γ-H2AX foci in hair follicle cells after local irradiation of 0-8Gy X-rays, and respectively brings the average number of γ-H2AX foci in hair follicle cells of the irradiated individual into the above two effect curves, respectively obtains a whole body dose estimation value and a local irradiation dose estimation value, and when the whole body dose estimation value and the local irradiation dose estimation value have a significant difference, it is judged that local irradiation has occurred. The method for distinguishing local irradiation using the number of γ-H2AX foci provided by the present invention has the advantage of not requiring cell culture, because the formation of the number of γ-H2AX foci can be directly detected in hair follicle cells, and the method can be applied in the local ionizing radiation assessment practice of various organisms, and can also be used as an alternative rapid method for assessing the dose intensity and heterogeneity after acute ionizing radiation. At the same time, the study showed that the number of γ-H2AX foci in hair follicle cells after radiation had a good dose response. This suggests that hair follicle damage may be developed as a powerful tool for local irradiation dose assessment. In addition, since non-clinical personnel can also pluck hair, this method will be very suitable for large-scale sample collection. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 The whole-body irradiation dose-effect curve was fitted to the average number of γ-H2AX foci in hair follicle cells after whole-body irradiation with 0-8Gy X-rays;
[0023] Figure 2 The local irradiation dose-effect curve was fitted with the average number of γ-H2AX foci in hair follicle cells after local irradiation with 0-8Gy X-rays. DETAILED DESCRIPTION
[0024] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the spirit of the contents disclosed in the present invention will be described in detail below. After understanding the embodiments of the contents of the present invention, any technician in the relevant technical field can change and modify the contents of the present invention by the techniques taught by the contents of the present invention without departing from the spirit and scope of the contents of the present invention.
[0025] The exemplary embodiments of the present invention and the description thereof are used to explain the present invention but are not intended to limit the present invention.
[0026] The experimental methods involved in the following examples are all conventional methods unless otherwise specified, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.
[0027] The strain of mice used in the present invention is C57BL / 6J, the age is 8 weeks, and the weight is 20 g±1.5 g.
[0028] The primary antibody used in the present invention was purchased from Cell Signaling Technology, product number: 9718s, product name: Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb; the secondary antibody used was purchased from Cell Signaling Technology, product number: 8889s, product name: Anti-rabbit IgG (H + L), F (ab') 2 Fragment (Alexa 594Conjugate); DAPI was purchased from Vector Laboratories, Spain.
[0029] The biological irradiator used in the present invention is KUBTEC XCELL 225 (purchased from KUBTEC Corporation of the United States), and the laser confocal microscope is ZEISS LSM700 (purchased from Carl ZEISS Corporation of Germany).
[0030] The statistical analysis method of the present invention is as follows: SPSS26.0 software was used for statistical analysis, and the average number of γ-H2AX foci was expressed as mean ± standard deviation. The independent sample t test was used to compare the data differences between two groups. The one-way analysis of variance was used to compare the data differences between multiple groups, and the LSD test was used for subsequent pairwise comparisons. P<0.05 indicated that the difference was statistically significant.
[0031] C57BL / 6J mice were irradiated with 0Gy, 2Gy, 4Gy, 6Gy, and 8Gy of X-ray whole body irradiation and partial abdominal irradiation, respectively, with an absorbed dose rate of 1Gy / min and a source-target distance of 50cm; 6h after irradiation, 3 whiskers with hair follicle cells were plucked from the whiskers of the mice as samples; the samples were transferred to 2mL centrifuge tubes, fixed with 1mL of 2% paraformaldehyde solution (PFA) for 30min at room temperature, and PFA was removed after fixation, and the samples were washed once with 1× phosphate buffered saline (PBS); pre-cooled 70% ethanol was added, and the samples were permeabilized at 4℃ for one week, then the samples were taken out and washed with PBS for 5min; the samples were blocked with 5% bovine serum albumin solution (BSA) for 30min; after washing the samples once with PBS, the samples were incubated with rabbit monoclonal anti-γ-H2AX antibody (primary antibody, 1:400 dilution) for 3h, and then incubated with goat F(ab')2 anti-rabbit Alexa-594 labeled I gG (H+L) antibody (secondary antibody, 1:500 dilution) was incubated for 1.5h (protected from light); the sample was washed twice with PBS, placed on a glass slide, one end containing hair follicle cells was placed in a hydrophobic circle drawn with an immunohistochemistry pen (PAP pen), and the other end of the sample was fixed on a glass slide; anti-fading and anti-fading 4',6-diamidino-2-phenylindole solution (DAPI) was added, and incubated at 37°C for 20min; a confocal microscope was used to take pictures and observe, and the average number of γ-H2AX foci in the sample was counted, with about 200 hair follicle cell nuclei recorded for each sample.
[0032] The average number of γ-H2AX foci in mouse whisker hair follicle cells 6 h after different doses of whole body irradiation and local abdominal irradiation are shown in Tables 1 and 2, respectively.
[0033] Table 1 Average number of γ-H2AX foci in mouse whisker hair follicle cells 6 hours after whole-body irradiation at different doses
[0034]
[0035] Note: * indicates P < 0.05 compared with 0 Gy group.
[0036] Table 2 Average number of γ-H2AX foci in mouse whisker hair follicle cells 6 h after local irradiation at different doses
[0037]
[0038] Note: * indicates P < 0.05 compared with 0 Gy group.
[0039] As can be seen from Tables 1 and 2, the average number of γ-H2AX foci increases with the increase of irradiation dose, which indicates that the number of γ-H2AX foci in hair follicle cells of individuals exposed to ionizing radiation is dose-dependent.
[0040] The whole-body irradiation dose-effect curve was established using the average number of focal spots after whole-body irradiation. Figure 1 The curve equation fitted 6 hours after the treatment is Y = 2.203D, R 2 =0.927; Y is the average number of γ-H2AX focal spots, and D is the irradiation dose (in Gy). P To establish a local irradiation dose-effect curve, see Figure 2 The curve equation fitted 6 hours after the test is Y P =2.764D+3.156, R 2 =0.923; Y P is the average number of local irradiation γ-H2AX foci; D is the irradiation dose (in Gy).
[0041] C57BL / 6J mice were randomly divided into two groups, 3 mice in each group, and the abdomen of the mice was irradiated with X-rays. The irradiation doses of the two groups of mice were 2Gy and 6Gy, respectively. The whisker hair follicle cells of the mice were collected for detection 6 hours after irradiation. The irradiation conditions, sample preparation and analysis methods were the same as described above. The dose was estimated using the fitted whole-body irradiation dose-effect curve and the local irradiation dose-effect curve. The estimation results are shown in Tables 3 and 4. The dose estimation results show that when the local irradiation dose-effect curve is used to estimate the irradiation dose, the relative deviation between the estimated irradiation dose and the actual irradiation dose does not exceed 30%.
[0042] Table 3 Results of estimating local irradiation dose using whole body irradiation dose-effect curve
[0043]
[0044] Table 4 Results of estimating local irradiation dose using local irradiation dose-effect curve
[0045]
[0046] The doses after 2Gy and 6Gy local irradiation were estimated using the fitted whole body irradiation dose-effect curve and local irradiation dose-effect curve, and the difference in the dose estimates of the two curves was analyzed for significance (Table 5). The results of the significance analysis showed that the difference between the dose estimates of whole body irradiation and local irradiation was significant (P < 0.05), which could be used to determine the occurrence of potential local irradiation events.
[0047] Table 5 Results of significance analysis of the difference between the estimated values of whole body irradiation dose and the estimated values of local irradiation dose
[0048]
[0049] Note: aIt indicates that the estimated value of local irradiation of 2 Gy was compared with the estimated value of whole body irradiation, P < 0.05; b It indicates that compared with the estimated value of local irradiation of 6 Gy and the estimated value of whole body irradiation, P < 0.05.
[0050] Therefore, we concluded that after whole-body irradiation and local irradiation of mice with 0-8GyX-rays, a whole-body irradiation dose-effect curve and a local irradiation dose-effect curve were established based on the average number of γ-H2AX foci in hair follicle cells, respectively. After substituting the average number of γ-H2AX foci estimated after the radiation accident into the two curves, if there is a significant difference between the estimated value of the whole-body dose and the estimated value of the local irradiation dose, it is considered that a potential local irradiation event has occurred and the irradiation dose can be estimated.
[0051] Although the present invention has been disclosed as above in the form of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.
Claims
1. A method for distinguishing local irradiation based on the number of γ-H2AX foci in hair follicle cells, It is characterized in that The details are as follows: S1. Whole-body irradiation and local irradiation were performed on individuals using different doses of X-rays ranging from 0 to 8 Gy, and the number of γ-H2AX foci in the hair follicle cells of the individuals after irradiation was detected to obtain the average number of γ-H2AX foci in the hair follicle cells to fit the whole-body irradiation dose-effect curve and the average number of γ-H2AX foci in the hair follicle cells to fit the local irradiation dose-effect curve; the method for detecting the number of γ-H2AX foci in the hair follicle cells was to take hair with hair follicle cells as samples, fix them with 2% PFA, remove PFA, and wash once with 1×PBS; Add pre-cooled 70% ethanol, place at 4°C for one week, wash with PBS; block the sample with 5% BSA; wash with PBS, incubate the sample with rabbit monoclonal anti-γ-H2AX antibody, and then incubate with goat F(ab')2 anti-rabbit Alexa-594 labeled IgG (H+L) antibody in the dark; wash with PBS twice, place on a glass slide, place one end containing hair follicle cells in a hydrophobic circle made with PAP strokes, and fix the other end of the sample on a glass slide; add DAPI and incubate at 37°C; take pictures using a confocal microscope to observe the number of γ-H2AX foci in the cells; S2, detecting the number of γ-H2AX foci in the hair follicle cells of the irradiated individuals, and inserting the average number of γ-H2AX foci into the two curves in S1 to obtain the estimated value of whole body dose and the estimated value of local irradiation dose; S3. Analyze the difference between the estimated whole-body dose and the estimated local irradiation dose. If the difference is significant, it is judged that local irradiation has occurred.
2. The method according to claim 1, It is characterized in that S1 detected the number of γ-H2AX foci in individual hair follicle cells 6 h after irradiation.
3. The method according to claim 1, It is characterized in that In S1, the PFA fixation time was 30 min, and the BSA blocking time was 30 min.
4. The method according to claim 1, It is characterized in that In S1, the rabbit monoclonal anti-γ-H2AX antibody was diluted 1:400 and incubated for 3 h. The goat F(ab')2 anti-rabbit Alexa-594 labeled IgG (H+L) antibody was diluted 1:500 and incubated for 1.5 h.
5. The method according to claim 1, It is characterized in that DAPI was added to S1 and incubated for 30 min.
6. The method according to claim 1, It is characterized in that The average number of γ-H2AX foci in hair follicle cells in S1 was obtained by detecting 200 hair follicle cells in each sample.
7. The method according to claim 1, It is characterized in that S2 detected the number of γ-H2AX foci in hair follicle cells of irradiated individuals 6 hours after irradiation.
8. The method according to claim 1, It is characterized in that The differences in S3 were analyzed using the independent sample t-test, and P < 0.05 indicated a significant difference.
9. Use of any one of the methods of claims 1 to 8 in local irradiation dose assessment.