Method for constructing mouse radiation-induced oral mucositis model based on fractionated irradiation

Abstract

This invention discloses a method for constructing a mouse model of radiation-induced oral mucositis (RIOM) based on fractionated irradiation, belonging to the field of biomedical technology. Male BALB / c mice undergo localized head and neck irradiation, including fractionated irradiation and a final irradiation. For fractionated irradiation, a cycle consists of 5 consecutive days of irradiation at a dose of 3 Gy / day, followed by 2 days of irradiation-free treatment. After two cycles of fractionated irradiation, a final irradiation at a dose of 14 Gy is performed, thus obtaining the mouse model of radiation-induced oral mucositis. This invention is the first to successfully establish a RIOM mouse model by periodically stimulating mucosal sensitivity with low-dose irradiation followed by high-dose irradiation to stimulate ulceration on the dorsum of the tongue. Methylene blue staining showed that the RIOM mouse model constructed by this invention exhibited significant ulceration on the dorsum of the tongue, and the mice maintained a relatively good survival status before the end of the experiment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for constructing a mouse model of radiation-induced oral mucositis based on fractionated irradiation. Background Technology

[0002] Radiotherapy-induced oral mucositis (RIOM), also known as radiation-induced oral mucositis or radiation stomatitis, is an acute or chronic injury to the oral mucosa caused by ionizing radiation. It is clinically common in patients receiving radiotherapy for head and neck tumors and is one of the most frequent and serious complications of tumor radiotherapy.

[0003] To study and explore the pathogenesis and prevention of recurrent intraepithelial neoplasia (RIOM), it is necessary to construct stable RIOM animal models. This is also an important preliminary research foundation for the clinical prevention and treatment of RIOM, serving as an in vivo experimental validation of the efficacy and safety of different prevention and treatment methods, and is a crucial part of RIOM research. Currently, the construction of RIOM animal models using radiation irradiation is often categorized according to the irradiation protocol, including single-dose irradiation, multiple low-dose irradiation, and irradiation combined with local stimulation, to promote the development of RIOM. Among these, the local stimulation method is often difficult to quantify and standardize, and has a low degree of clinical simulation, and is generally not recommended.

[0004] In existing technologies, Tao et al. used C57 / BL6 mice to irradiate their head and neck with a single high dose of 25 Gy and recommended 25 Gy as the optimal dose for rapidly inducing RIOM (see miR-200c Modulates the Pathogenesis of Radiation-Induced Oral Mucositis [J]. Oxid Med Cell Longev, 2019, 2019:2352079.); Maria et al. used BALB / c mice to model the disease and believed that 18 Gy could ensure the persistence of ulceration while maintaining a high survival rate of mice, and therefore recommended 18 Gy as the optimal dose for a single irradiation (see Single-Dose Radiation-Induced Oral Mucositis Mouse Model [J]. Front Oncol, 2016, 6: 154.). Recent studies have found that when using high-dose single-shot irradiation technology for modeling, the irradiation dose for mice is 16 Gy to 25 Gy, while the irradiation dose for rats is 15 Gy to 20 Gy. Different studies recommend different optimal irradiation doses for modeling.

[0005] Han et al. irradiated mice with 8 Gy / day for three consecutive days to simulate clinical radiotherapy with multiple small doses. Their study recommended this as the minimum irradiation dose to induce RIOM. The experimental mice began to show oral mucosal inflammatory lesions on the 9th day after the last irradiation (see Preventive and therapeutic effects of Smad7 on radiation-induced oral mucositis [J]. Nat Med, 2013, 19(4): 421-8.). Kowaliuk et al. irradiated mice for 5 consecutive days at 3 Gy / day for 2 consecutive weeks and successfully constructed a mouse model of RIOM (see Heparin treatmentmitigates radiation-induced oral mucositis in mice by interplaying with repopulation processes [J]. Strahlenther Onkol, 2019, 195(6): 534-43.). Chao et al. compared a single dose of 16.5 Gy to 8 Gy / day of irradiation over three consecutive days in C57BL / 6J mice. The results showed that a single dose of 16.5 Gy irradiation induced ulceration of the tongue mucosa in mice, but the mice's body weight decreased by more than 45% compared to the initial dose, and they died within 15 days after irradiation. The experiment confirmed that the mortality rate of mice irradiated with a single high dose was high (see DimethylSulfoxide Prevents Radiation-Induced Oral Mucositis Through Facilitating DNA Double-Strand Break Repair in Epithelial Stem Cells [J]. Int J Radiat OncolBiol Phys, 2018,102(5): 1577-89.).

[0006] Based on the above research, while multiple low-dose irradiations are more similar to clinical radiotherapy protocols, they require multiple anesthesias of experimental animals, often leading to high mortality rates due to resuscitation failure. Single-dose irradiation is more convenient and avoids the drawbacks of multiple anesthesias, and it induces RIOM effectively. However, it often results in the death of experimental animals shortly after radiation exposure or difficulty in forming obvious ulcers due to the difficulty in controlling the appropriate irradiation dose, and it does not conform to the actual radiotherapy process in clinical practice.

[0007] To ensure the constructed animal model accurately reflects the radiation-induced oral mucositis experienced by human malignant tumor patients undergoing comprehensive radiotherapy, Chinese patent CN110226566A employed allogeneic transplantation of B16-F10 mouse melanoma cells and used a biological X-ray small animal irradiation device to irradiate the head and neck of the mice to create a mouse model of radiation-induced oral mucositis. The patent confirmed that a single dose of 16 Gy was the appropriate dose for inducing oral mucositis, achieving a success rate of 83.3% and a survival rate of 90.5%, with the mice surviving for more than 6 months. However, this patent still uses injection anesthesia to immobilize the irradiated mice, increasing the risk of accidental death during anesthesia. Furthermore, the patent's method of modeling mice using a single irradiation session lasting 4 minutes is inconsistent with the actual situation of clinical cancer patients undergoing comprehensive radiotherapy. Additionally, the patent does not clearly specify how to ensure that only the melanoma cells are irradiated, and it is unclear whether other parts of the mouse require protection.

[0008] Therefore, it is important to develop and study a new RIOM animal model that can better simulate the clinical radiotherapy process in patients with head and neck tumors. Summary of the Invention

[0009] The present invention aims to provide a method for constructing a mouse model of radiation-induced oral mucositis by fractionated irradiation. This method combines fractionated low-dose irradiation of the head and neck of mice with a single high-dose irradiation to successfully construct a stable RIOM mouse model, ensuring that the mice are still in good condition and have obvious ulcers on the tongue before the end of the experiment.

[0010] This invention is achieved through the following technical solution: a method for constructing a mouse radiation-induced oral mucositis model based on fractionated irradiation. Male BALB / c mice are subjected to localized head and neck irradiation, which includes fractionated irradiation and a final irradiation. For fractionated irradiation, a cycle consists of 5 consecutive days of irradiation at a dose of 3 Gy / day, followed by 2 days of no irradiation. After two cycles of fractionated irradiation, a final irradiation with a dose of 14 Gy is performed, thus obtaining the mouse radiation-induced oral mucositis model.

[0011] The steps for performing localized head and neck irradiation are as follows: S1. Cut off the bottom of the pointed centrifuge tube so that the diameter of the cut surface is controlled between 5 and 10 mm; S2. Insert the mouse into the opening of the conical centrifuge tube and secure the mouse's neck at the cut surface to ensure that the mouse is fixed in place without affecting its breathing. S3. Place the mouse in a supine position, cover the body parts of the mouse except for the mouth and jaw with a lead shield, and expose the head and neck area of ​​the mouse in the radiation area for local irradiation.

[0012] In step S2, after placing the mouse into a conical centrifuge tube, the opening of the conical centrifuge tube is blocked with a foam strip.

[0013] The irradiation was performed using a biological X-ray irradiator RS2000.

[0014] When the irradiation dose is 3 Gy, the irradiation time is 1.142 Gy / min for 158 s.

[0015] When the irradiation dose is 14 Gy, the irradiation time is 1.142 Gy / min for 735 s.

[0016] (1) In the prior art, to construct a RIOM mouse model, it is necessary to observe whether the mouse's weight loss is too rapid or too low, whether the conjunctival inflammation is too severe, whether the fecal texture is too loose, whether the mouse's movement is excessively slowed or weak, whether the pseudomembrane covering the tongue ulcer is too large, or whether the oral bleeding is too severe after irradiation. Based on the manifestation of these symptoms, it is determined whether the RIOM mouse model has been successfully constructed. However, due to the many problems with the existing RIOM mouse model construction methods, it may lead to unexpected death of the mice after irradiation, or it may have poor consistency with the actual situation. Therefore, this invention is the first to successfully establish a RIOM mouse model by periodically irradiating the mucosa with small doses and then irradiating the tongue with large doses to stimulate the formation of tongue ulcers. Methylene blue staining showed that the RIOM mouse model constructed in this invention had obvious tongue ulcers, and the mice were still in good condition before the end of the experiment.

[0017] Secondly, during the construction of the RIOM mouse model in this invention, the mouse body weight showed a stable decrease, avoiding the sudden weight loss that might occur after a single high-dose irradiation. HE staining analysis showed that this invention can confirm the successful induction of RIOM in mice at the histopathological level. After radiation irradiation, the continuity of the tongue dorsal mucosa in the experimental mice was significantly disrupted, with inflammatory cell infiltration. The mucosal thickness and basal cell density gradually decreased with increasing irradiation dose, showing a certain degree of radiation dose dependence.

[0018] (2) The method of the present invention can successfully construct a stable RIOM mouse model by using fractionated irradiation, specifically in the following ways: Stable mouse weight: No sudden weight loss or death due to low weight occurred, and the overall survival rate of mice was high; The mice were in stable physiological condition: there was no excessive exudation or hyperplasia of conjunctival inflammation, the stool texture was relatively normal, and there was no obvious sluggishness in movement; Ulcer formation in mice was stable: oral mucosal bleeding was moderate and did not affect the collection of oral mucosal swab samples. Ulcers on the dorsum of the tongue did not show excessive pseudomembrane coverage, and the overall ulcer area accounted for approximately 40% to 60% of the dorsum of the tongue, facilitating subsequent observation and analysis. The model is stable: the model construction has good repeatability and can satisfy the stability conditions mentioned above to the greatest extent.

[0019] (3) This invention is the first to use a self-made mouse tube to fix BALB / c mice in a non-anesthetic manner, realizing the fixation of the irradiation process in a non-anesthetic state. By reasonably controlling the size of the opening at the tip of the mouse tube, the size and thickness of the foam strip, and the packing method, it can minimize the obstruction of the normal breathing of the mice, avoid excessive compression of the mice's bodies, and achieve a good fixation effect. It also reduces the impact of anesthetic drug injection and anesthesia recovery on the mice's life status to a certain extent, reduces the accidental death of mice caused by anesthesia and recovery during irradiation, and the required instruments and materials are inexpensive and readily available, can be recycled, are easy to operate, and have high experimental efficiency.

[0020] (4) The present invention uses lead shielding plates to cover the mice after fixing them, which not only facilitates the irradiation of multiple experimental mice at one time, but also protects the parts of the mice other than the head and neck to the greatest extent, avoids radiation to other parts of the mice's body, and reduces the mortality rate of the mice.

[0021] (5) By appropriately selecting specific radiation times and specific radiation doses, the present invention makes the constructed RIOM mouse model consistent with the actual situation of clinical cancer patients receiving comprehensive radiotherapy. At the same time, it overcomes the significant weight loss, sudden weight loss or even sudden death that mice may experience after receiving radiation. By irradiating with appropriate small doses in the early stage, sufficient radiation dose is accumulated while stimulating mucosal sensitivity, so as not to cause mice to experience significant weight loss, deterioration of other physiological conditions or accidental death in advance. In addition, by setting specific irradiation gradients, the mice can develop ulcers after irradiation without affecting their survival and health, so as not to cause mice to experience sudden weight loss or even sudden death after receiving irradiation. Attached Figure Description

[0022] Figure 1 This is a flowchart of a fractional irradiation experiment on mice.

[0023] Figure 2 A schematic diagram of irradiating mice with radiation.

[0024] Figure 3 The trend of mouse body weight change.

[0025] Figure 4 For comparative analysis of weight loss in mice.

[0026] Figure 5 Image showing ulcers on the dorsal surface of a mouse tongue sample as observed by macroscopic examination and methylene blue staining.

[0027] Figure 6 Statistical analysis of the area of ​​ulcers on the dorsal surface of the tongue in mice (***)P <0.001).

[0028] Figure 7 Histopathological analysis of the dorsal mucosa of the mouse tongue, where a) is a paraffin section of the mouse tongue stained with HE (scale bar 50 μm), and b) is a statistical analysis of the thickness of the dorsal mucosa of the mouse tongue (***). P <0.001), c represents the statistical analysis of the basal cell density of the mouse tongue dorsum (*** P <0.001). Detailed Implementation

[0029] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0030] Example 1: This embodiment describes the process of constructing a RIOM mouse model by subjecting male BALB / c mice to multiple localized low-dose irradiations of the head and neck, followed by a final high-dose irradiation. The specific experimental procedure can be summarized as follows: The main instruments and reagents included: Rad Source 2000 biological X-ray irradiator (RS2000, USA), custom lead shield, syringes (1 mL), centrifuge tubes (15 mL, 50 mL, NEST, USA), 4% paraformaldehyde (Biosharp, China), acetic acid (Jinshan, Chengdu), methylene blue staining solution (Solarbio, Beijing), PBS buffer (Zhongshan, Beijing), xylene (Sinopharm, China), anhydrous ethanol (Sinopharm, China), neutral resin (Solarbio, Beijing), positively charged adhesive slides (Shitai, China), paraffin tissue sectioner (Leica, Germany), hematoxylin staining solution (Beyotime, China), eosin staining solution (Beyotime, China), and microscope (Olympus, Japan).

[0031] See the experimental procedure. Figure 1 As shown, the specific experimental method is as follows: 1. Experimental grouping and methods Thirty 7-week-old male BALB / c mice weighing approximately 20 g were randomly divided into 5 groups: control group, 12 Gy group, 14 Gy group, 16 Gy group, and 18 Gy group, with 6 mice in each group. The mice in the experimental groups first received fractional small-dose radiation to their head and neck (3 Gy / day, continuous irradiation for 5 days, with a 2-day break between irradiation periods, constituting one cycle; this process was repeated, resulting in a cumulative radiation dose of 30 Gy for two cycles). Then, according to their respective groups, they received a single large-dose irradiation (irradiation doses of 12 Gy, 14 Gy, 16 Gy, and 18 Gy, respectively). The mice's weight was measured and recorded daily.

[0032] 2. Establishing the RIOM mouse model Mice in the 12 Gy, 14 Gy, 16 Gy, and 18 Gy groups underwent localized head and neck irradiation, as detailed below: (1) Weigh the mice and record their initial weight.

[0033] (2) Cut the 50 mL centrifuge tube at about 1 mL mark with scissors. The diameter of the cut surface should be about 10 mm. Grab the mouse and guide it into the centrifuge tube opening by means of the stress. Position the mouse's neck at the bottom of the tube without obstructing its airway and breathing. Bend a pre-cut foam strip about 5 mm thick and gently rotate it into the tube to block the opening and gently fix the mouse in the centrifuge tube.

[0034] (3) Place the centrifuge tube containing the mice on the inner surface of the irradiator. The mice should be in a supine position. Use a custom-made lead shield to cover the body parts except for the head and neck, leaving the head and neck area exposed. See [link to relevant documentation]. Figure 2 As shown; (4) Start the local irradiation mode of the irradiator (160 KV, 25 mA, 0.3 mm copper alloy filter, no reflector), administer a single dose of 3 Gy (1.142 Gy / min, 158 s) of radiation, remove the foam strip, and catch the mouse and put it back in the cage.

[0035] (5) On the day of the final high-dose irradiation, repeat steps (1) to (3) above. After irradiating the mice with doses of 12 Gy (1.142 Gy / min, 630 s), 14 Gy (1.142 Gy / min, 735 s), 16 Gy (1.142 Gy / min, 840 s), and 18 Gy (1.142 Gy / min, 945 s) respectively according to the group, remove the foam strips and put the mice back into the cage.

[0036] Example 2: This embodiment involves the analysis and evaluation of different RIOM mouse models constructed in Example 1.

[0037] The body weight of the model mice in Example 1 was analyzed at different time points. The mice in Example 1 were sacrificed on the 8th day after high-dose irradiation. The mice's tongues were taken for methylene blue staining to observe the ulcer area on the dorsal surface of the tongue. HE staining was used to analyze the changes in the thickness of the dorsal mucosa and the density of basal cells on the dorsal surface of the tongue.

[0038] (a) Weight changes See Figure 3 and Figure 4The figures show the trends in body weight change and the amount of body weight loss in mice from the control group, the 12 Gy group, the 14 Gy group, the 16 Gy group, and the 18 Gy group, respectively. There was no statistically significant difference in body weight loss among the different experimental groups. P >0.05). At the end of the experiment, compared to their initial body weight, the control group had an increased body weight, while the experimental group had a decreased body weight. There was no statistically significant difference in the amount of body weight loss among the different experimental groups. P >0.05).

[0039] Specifically, by Figure 3 As can be seen, compared with the control group, the experimental group mice gradually lost weight, indicating that the use of fractionated local irradiation (3 Gy per dose) and a final high-dose irradiation (12 Gy, 14 Gy, 16 Gy, 18 Gy) in this experiment can gradually reduce the weight of mice, overcoming the possibility of significant weight loss in mice after radiation exposure. Figure 4 It is evident that the fractionated local irradiation and final high-dose irradiation in this experiment resulted in significant weight loss in mice during the experimental period. The weight loss at the end of the experiment, compared to the initial weight, was correlated with the radiation dose of the final high-dose irradiation, a stark contrast to the overall weight gain observed in the control group mice that did not receive radiation. This suggests that the radiation dose of the final high-dose irradiation is related to the amount of weight loss in the experimental mice.

[0040] (II) Analysis of tongue ulcers Tongue sample collection: On the 8th day after the final high-dose irradiation (the 23rd day of the total animal experiment), mice were euthanized by cervical dislocation. The entire tongue sample of the mouse was carefully separated by animal surgical instruments and methylene blue staining experiment was performed.

[0041] The methylene blue staining analysis procedure is as follows: (1) Rinse the surface of the mouse tongue sample with physiological saline to remove bloodstains and other contaminants; (2) Use a cotton swab dipped in 1% acetic acid to wipe the mucus on the surface of the mouse's tongue; (3) Apply 1% methylene blue staining solution evenly to the surface of the mouse tongue; (4) After staining for 1 minute, wipe the back of the mouse tongue with a 1% acetic acid cotton swab until there is no staining residue on the normal mucosa of the mouse and the shape and edge of the ulcer on the back of the mouse tongue are clear; (5) After staining, place the mouse tongue sample flat on a clean white paper, take a picture with a camera, and save it for subsequent analysis.

[0042] See the experimental results. Figure 5 and Figure 6 As shown.

[0043] Figure 5This image shows the ulceration on the dorsum of the tongue as observed by macroscopic examination and methylene blue staining in a mouse tongue sample. Figure 5 As can be seen from the methylene blue staining, the experimental group of mice had obvious ulcers on the back of their tongues, indicating that the fractionated local irradiation (3 Gy per dose) and the final high-dose irradiation (12 Gy, 14 Gy, 16 Gy, 18 Gy) used in this experiment can stimulate the formation of ulcers on the back of the tongue.

[0044] Figure 6 Statistical analysis of the area of ​​ulcers on the dorsal surface of the tongue in mice, by Figure 6 It can be seen that as the final high-dose irradiation dose increases, the area of ​​ulceration on the dorsal surface of the mouse tongue also gradually increases. P <0.001), indicating that increased irradiation dose can aggravate the severity of RIOM lesions in mice, and has a certain irradiation dose dependence.

[0045] (III) Histopathological examination of the dorsal mucosa of the tongue The specific steps for preparing tongue tissue sections are as follows: (1) Fixation: After cleaning the surface blood stains of the removed mouse tongue sample with physiological saline, immerse it in a centrifuge tube containing 4% paraformaldehyde and store it in a refrigerator at 4°C for 24 hours to fix the sample. Then take it out and rinse it with PBS to remove excess fixative. (2) Dehydration: The samples were soaked in alcohol of varying concentrations (75%, 85%, 95%, 100%) for 2 hours at each concentration; (3) Transparency treatment: Immerse in xylene solution twice, 2 hours each time; (4) Wax impregnation: Immerse in paraffin at 60℃ twice, the first time for 1 hour and the second time for 2 hours, overnight; (5) Embedding: The embedding direction is the sagittal plane of the mouse tongue; (6) Sectioning: Section the paraffin wax using a microtome to a thickness of 5 μm and load the slides; (7) Baking: Dry the slices in a baking machine and store them in a refrigerator at 4°C for later use.

[0046] The prepared tongue tissue sections were subjected to HE staining analysis. The specific HE staining process is as follows: (1) Baking slices: Bake at 55-65℃ for 2 hours; (2) Dewaxing: Immerse twice in xylene solution, 10 minutes each time; (3) Hydrate with gradient concentrations of alcohol (100%, 95%, 85%), 5 minutes per concentration; (4) Wash with distilled water for 2 minutes, then stain with hematoxylin for 30 seconds; (5) After separating the colors with 0.7% hydrochloric acid alcohol for 3 seconds, rinse with tap water for 3 minutes, and then stain with eosin for 1 minute; (6) After washing with distilled water for 3 minutes, dehydrate with a gradient of alcohols (75%, 85%, 95%, 100%) for 5 minutes at each concentration; (7) Transparency treatment: Immerse twice in xylene solution, 10 minutes each time; (8) Mount the slide with neutral resin, observe and collect images under an inverted microscope.

[0047] Histopathological changes of the dorsal tongue mucosa in mice of the control group, 12 Gy group, 14 Gy group, 16 Gy group, and 18 Gy group were obtained in the manner described above. See [link to relevant documentation]. Figure 7 As shown.

[0048] Depend on Figure 7 As can be seen, HE staining results showed that, compared with the control group, the experimental group mice exhibited significant disruption of the continuity of the lingual dorsal mucosa after radiation irradiation, inflammatory cell infiltration, and a gradual decrease in mucosal thickness and basal cell density with increasing irradiation dose, showing a certain dose-dependent effect. The 12 Gy group and the 14 Gy group ( P <0.001), 14 Gy group and 18 Gy group ( P Results with values ​​<0.001 were statistically significant.

[0049] The present invention selects the RIOM mouse model based on the characteristics of obvious tongue ulcers generated by the RIOM mouse model, which can ensure the experimental mice maintain a good survival status before the end of the experiment, and the overall experimental effect is the best. The model construction is stable and highly reproducible. Finally, the optimal final irradiation dose of 14 Gy was selected to construct the RIOM mouse model.

[0050] In the above experimental analysis, the area of ​​ulcers on the dorsum of the tongue and the total area of ​​the dorsum of the tongue were measured using ImageJ software. For histological staining images, six non-overlapping fields of view were randomly selected from tongue samples of at least three different mice for subsequent statistical analysis. The thickness of the dorsum of the tongue mucosa in HE-stained images was measured and analyzed at three random sites. Basal cells were counted using a one-to-one definition method. Results are expressed as mean ± standard deviation. One-way ANOVA was used to determine whether differences between groups were statistically significant. P A value <0.05 is considered statistically significant. GraphPad Prism 8.0 software is used for statistical analysis and graphing.

[0051] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications or equivalent changes made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for constructing a mouse radiation-induced oral mucositis model based on fractionated irradiation, characterized in that: Male BALB / c mice were used to implement head and neck local irradiation, which included fractionated irradiation and final irradiation; in the fractionated irradiation, continuous irradiation was performed for 5 days, and the irradiation dose was 3 Gy / day, and no irradiation was performed for 2 days thereafter as a cycle; after two cycles of fractionated irradiation, final irradiation was performed once, and the irradiation dose was 14 Gy, thereby obtaining a mouse radiation-induced oral mucositis model.

2. The method according to claim 1, characterized in that: The steps of implementing head and neck local irradiation are as follows: S1. The bottom of a sharp-bottomed centrifuge tube is cut off, and the aperture diameter of the cross section is controlled to be 5-10 mm; S2. The mouse is placed into the tube opening of the sharp-bottomed centrifuge tube, and the neck of the mouse is clamped at the cross section, so that the mouse is fixed while the breathing of the mouse is not affected; S3. The mouse is placed in a supine position, the body parts other than the mouth and jaw of the mouse are covered with a lead shield, the head and neck region of the mouse is exposed in the radiation region, and local irradiation is performed.

3. The method according to claim 2, characterized in that: In the step S2, after the mouse is placed into the sharp-bottomed centrifuge tube, a foam strip is used to block the tube opening of the sharp-bottomed centrifuge tube.

4. The method of claim 1, characterized by: In the irradiation, a biological X-ray irradiator RS2000 is used.

5. The method according to claim 4, characterized in that: When the irradiation dose is 3 Gy, the irradiation is performed at 1.142 Gy / min for 158 s.

6. The method of claim 4, characterized in that: When the irradiation dose is 14 Gy, the irradiation is performed at 1.142 Gy / min for 735 s.

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