Construction method of animal model based on X-ray induced radiation oral ulceration

Abstract

The invention provides a method for constructing an animal model based on X-ray induced radiation oral ulceration, which comprises the following steps: selecting SPF-level SD (Sprague Dawley) rats, adaptively feeding the rats for 7 days, and randomly grouping the rats; the method comprises the following steps: locally irradiating oral mucosa by adopting X rays for 15 Gy single time, taking oral mucosa tissues 14 days after irradiation, and carrying out HE staining to observe epithelial exfoliation and inflammatory cell infiltration conditions, TUNEL apoptosis detection and inflammatory factor detection. Through local X-ray irradiation, clinical characteristics such as mucous membrane erosion and inflammatory factor increase caused by radiotherapy are reproduced, the drug curative effect is comprehensively evaluated in combination with multiple indexes (behavioristics, histology and molecular biology), and compared with a small animal precise radiotherapy instrument (SARRP), the method is low in equipment requirement and suitable for laboratory popularization.

Description

Technical Field

[0001] This invention relates to the field of biomedical experimental technology, and in particular to a method for constructing an animal model of radioactive oral ulcers induced by X-rays. Background Technology

[0002] Radiation-induced oral ulcers are a common complication of radiotherapy for head and neck tumors. Existing models mostly employ chemical corrosion or mechanical trauma methods, which are difficult to simulate the characteristics of tissue damage and numerous complications caused by radiation. Research on patented traditional Chinese medicine prescriptions shows that drugs such as licorice and rehmannia can alleviate radiation damage by inhibiting inflammatory factors such as IL-6 and TNF-α, but a standardized model is lacking to verify its mechanism.

[0003] The above problems urgently need to be addressed. Summary of the Invention

[0004] This invention discloses a method for constructing an animal model of radioactive oral ulcers based on X-ray induction, aiming to solve the technical problems existing in the prior art.

[0005] The present invention adopts the following technical solution: This invention provides a method for constructing an animal model of radioactive oral ulcers induced by X-rays, comprising the following steps: SPF-grade SD rats were selected and randomly grouped after 7 days of acclimatization. The oral mucosa was irradiated locally with X-rays at a single dose of 15 Gy. Fourteen days after irradiation, oral mucosal tissue was collected for HE staining to observe epithelial shedding, inflammatory cell infiltration, TUNEL apoptosis detection, and inflammatory factor detection.

[0006] In one possible implementation, the focal distance of the X-ray irradiation is 30 cm.

[0007] In one possible implementation, the dose rate of the X-ray irradiation is 1.2 Gy / min.

[0008] In one possible implementation, the detection is compared with the gold peptide treatment group (5000 IU / kg) to verify the sensitivity to drug efficacy.

[0009] In one possible implementation, SPSS 26.0 is used for inter-group comparisons.

[0010] In one possible implementation, changes in body weight and food intake, as well as oral mucosal scores, are recorded daily after X-ray irradiation.

[0011] In one possible implementation, the inflammatory factor detection includes the measurement of IL-6, TNF-α, and IL-1β levels.

[0012] The technical solution adopted in this invention can achieve the following beneficial effects: This invention provides a method for constructing an animal model of radiation-induced oral ulcers based on X-rays. The method includes the following steps: SPF-grade SD rats are selected and randomly grouped after 7 days of acclimatization; the oral mucosa is locally irradiated with X-rays at a single dose of 15 Gy; 14 days after irradiation, oral mucosal tissue is collected for HE staining to observe epithelial shedding, inflammatory cell infiltration, and TUNEL apoptosis detection; inflammatory factors are detected: IL-6, TNF-α, and IL-1β levels are measured using ELISA, and the model is validated. Through local X-ray irradiation, the clinical characteristics of radiotherapy-induced mucosal erosion and elevated inflammatory factors are reproduced. Combined with multiple indicators (behavioral, histological, and molecular biological), the efficacy of drugs is comprehensively evaluated. Compared with small animal precision radiotherapy (SARRP), this method has lower equipment requirements and is suitable for laboratory application. Attached Figure Description

[0013] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below, which constitute a part of the present invention. The illustrative embodiments of the present invention and their descriptions explain the present invention and do not constitute an improper limitation of the present invention.

[0014] In the attached diagram: Figure 1 HE staining image of oral mucosal tissue after X-ray irradiation, provided in an embodiment of the present invention. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments and corresponding drawings. In the description of this invention, it should be noted that the term "or" is generally used to include the meaning of "and / or," unless otherwise expressly indicated.

[0016] Radiation-induced oral ulcers are a common complication of radiotherapy for head and neck tumors. Existing models mostly use chemical corrosion or mechanical trauma methods, which are difficult to simulate the tissue damage characteristics and numerous complications caused by radiation.

[0017] 1. Significant differences between animal models and clinical settings. 1) Dose-volume parameter mismatch: Existing models mostly use single high-dose irradiation (e.g., 15-30 Gy), while clinical radiotherapy uses fractionated cumulative doses (usually 40-70 Gy), making it impossible for models to simulate the dynamic process of mucosal repair and re-injury induced by fractionated irradiation. 2) Anatomical limitations: The oral mucosa area of ​​rodents is small, making it difficult to distinguish the damage specificity of different areas such as the cheek and tongue margin, and it is impossible to simulate the effect of protective devices such as human oral stents.

[0018] 2. Insufficient standardization of the evaluation system 1) Subjective scoring bias: Existing mucosal scoring (0-4 grades) relies on the observer's subjective judgment and lacks objective quantitative tools (such as optical coherence tomography) for dynamic monitoring of ulcer depth and microvascular changes. 2) Incomplete coverage of molecular mechanisms: Most models only detect inflammatory factors such as IL-6 and TNF-α, ignoring the simulation of key aspects such as the effects of radiation on oral flora imbalance and salivary gland function.

[0019] 3. Limitations of drug evaluation 1) Limited positive control: Traditional drugs such as Jin Yintai only verify analgesic effects and cannot assess the physical barrier effect of novel biomaterials (such as piezoelectric films) or the repair-promoting ability of stem cell therapy. 2) Cross-species conversion barrier: Mouse models have significantly higher radiation tolerance than humans (ED50 difference of 3-5 times), resulting in a lack of reliable basis for drug dosage conversion.

[0020] 4. Technical repeatability defects 1) Insufficient irradiation precision: Localized X-ray irradiation is prone to uneven dose distribution due to animal movement, while the high cost of small animal precision radiotherapy (SARRP) restricts its widespread adoption in laboratories. 2) Interference from complications: Fractionated irradiation requires multiple anesthesias, increasing animal mortality; and it cannot simultaneously simulate the synergistic damage effect of radiotherapy combined with chemotherapy (such as 5-FU).

[0021] This invention provides a method for constructing an animal model of radiation-induced oral ulcers based on X-rays, the method comprising: Experimental animal selection: 40 SPF-grade SD rats (weighing 180-220g), half male and half female, were randomly divided into groups after 7 days of acclimatization.

[0022] Irradiation parameters: The oral mucosa was irradiated with a single 15 Gy X-ray, with a focal distance of 30 cm and a dose rate of 1.2 Gy / min.

[0023] Clinical observation: Daily records of changes in body weight and food intake, and oral mucosal scores (0-4 grades, including ulcer area and degree of congestion).

[0024] Histological examination: Mice were euthanized 14 days after irradiation, and oral mucosal tissue was taken for HE staining to observe epithelial shedding and inflammatory cell infiltration. Figure 1 HE staining image of oral mucosal tissue after X-ray irradiation provided by the present invention, such as Figure 1 As shown, Figure 1 In case A, no X-ray irradiation was performed; the oral mucosa tissue was normal and had not sloughed off. Figure 1 B Figure 1 C Figure 1 D、 Figure 1 E and Figure 1 F underwent X-ray irradiation, which clearly showed lesions in the oral mucosa.

[0025] TUNEL apoptosis assay: Model material selection: SD rats that had undergone local X-ray irradiation of the oral mucosa and were 14 days after a single 15 Gy irradiation were selected. The peak period of ulcer formation was 14 days after irradiation. The ulcer tissue of the left buccal mucosa and tongue was aseptically excised within 5 minutes after euthanasia. The tissue was quickly rinsed with pre-cooled PBS to remove blood and prevent DNA degradation.

[0026] Section preparation: The ulcer tissue was routinely dehydrated, paraffin-embedded, serially sectioned (5 μm thick), attached to a glass slide, and baked at 60°C for 2 hours; Dewaxing and rehydration: Dewaxing was performed twice with xylene (10 minutes each), followed by 5 minutes with anhydrous ethanol, 2 minutes with 90% ethanol, 2 minutes with 70% ethanol, and 2 minutes with distilled water to complete dewaxing and rehydration; 20 μg / ml of DNase-free proteinase K was added, and the mixture was incubated at 37°C for 20 minutes, followed by washing with PBS three times (5 minutes each time).

[0027] Fixation and strengthening: After dewaxing and rehydration of paraffin sections, fix them directly with 4% paraformaldehyde at room temperature for 40 minutes.

[0028] Permeabilization: Discard the fixative, wash twice with PBS (5 minutes each time), add PBS solution containing 0.1% Triton X-100, and incubate on ice for 3 minutes.

[0029] Labeling reaction: After permeabilization, wash twice with PBS (5 minutes each time), aspirate excess liquid from the slide, and prepare fresh TUNEL detection solution (2 μl TdT enzyme + 48 μl fluorescent labeling solution) at a ratio of 50 μl per slide. Add the prepared TUNEL detection solution evenly to the ulcer tissue section, place it in a humidified chamber (with filter paper soaked in water around it), and incubate at 37°C in the dark for 60 minutes.

[0030] Mounting and observation: After incubation, gently rinse three times with PBS (5 minutes each time), add 5 μl of anti-fluorescence quenching mounting solution, quickly cover with a coverslip, and observe using a fluorescence microscope (excitation wavelength 450-500 nm, emission wavelength 515-565 nm).

[0031] Apoptotic cell counting: Under a high-power field (400×) fluorescence microscope, 5-10 high-power fields were randomly selected from the ulcer center, ulcer edge, and normal mucosal area away from the ulcer (control group) to count the number of TUNEL positive cells (green fluorescent cells) and the average value was used to assess the degree of apoptosis.

[0032] Apoptosis index (AI): Image analysis software was used to assist in counting, and the number of positive cells and the total number of cells in each field of view were counted separately. The AI ​​value of each group was calculated according to the formula AI = number of apoptotic cells / total number of cells × 100%. The AI ​​in the ulcer center area of ​​the model group was usually significantly higher than that in the normal mucosa area, which can quantify the degree of radiation-induced apoptosis.

[0033] Distribution pattern analysis: Combined with HE staining results (referencing the histopathological characteristics of the model tissue), observe whether positive cells are concentrated in the submucosal inflammatory cell infiltration area (the site where a large number of inflammatory cells accumulate after radiation) and the edge of epithelial damage; if positive cells are mainly distributed in the inflammatory infiltration area and the edge of the ulcer, it suggests that in radiation-induced oral ulcers, cell apoptosis is directly related to local inflammatory response (elevation of factors such as IL-1β and TNF-α) and tissue damage, which can clarify the pathological role of apoptosis in ulcer formation.

[0034] Control settings: The ipsilateral oral mucosa of unirradiated rats was used as a negative control to ensure that its AI value was extremely low (usually <5%); a cell smear with known apoptosis positivity was used as a positive control to verify the effectiveness of the detection system and exclude non-specific staining interference.

[0035] Inflammatory factor detection: ELISA was used to measure the levels of IL-6, TNF-α, and IL-1β.

[0036] Sample pretreatment: Before testing, thaw frozen samples at 4°C slowly to avoid protein denaturation caused by rapid thawing at room temperature.

[0037] Perform serial dilutions according to the ELISA kit instructions, diluting the thawed sample to 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL, and 15.625 pg / mL. Aliquot each concentration gradient into 100 μl tubes and store at 4°C (use within 2 hours).

[0038] Washing buffer: Dilute the 20× concentrated washing buffer provided with the kit to the working concentration with ultrapure water at a ratio of 1:20, stir well, and store at 4°C; if turbidity or precipitation occurs, filter before use.

[0039] TMB substrate solution: It is divided into solution A and solution B. Mix them in equal volumes at a ratio of 1:1 before use. Operate in the dark (TMB is light-sensitive). Prepare fresh solution before use.

[0040] Blocking solution: Use 5% BSA (dissolved in PBS), prepare fresh before use; or use the blocking solution provided with the kit, thaw at 4°C and use directly.

[0041] 1) Capture antibody / detection antibody: Add 100 μl of diluted capture antibody to each well, ensuring the liquid evenly covers the bottom of the well (avoiding air bubbles); seal the ELISA plate with sealing film and incubate at 4°C for 12-16 hours. Discard the liquid in the wells, wash three times with washing buffer, adding 200 μl of washing buffer to each well each time, let stand for 30 seconds, then discard. After the final wash, invert the plate onto absorbent paper to pat dry. (Skip this step if using a pre-coated plate.) 2) Blocking: Add 200 μl of blocking solution to each well, gently shake the ELISA plate to distribute the blocking solution evenly, and incubate at 37°C for 1 hour (or at 4°C overnight).

[0042] 3) Sample addition: Discard the blocking solution, wash once with washing buffer (200 μl per well), pat dry, add 100 μl of standard or sample (e.g., serum) to each well, set up replicates (2-3 wells), seal the ELISA plate with sealing film, and incubate at 37°C for 1-2 hours. Add 100 μl of standard diluent to the blank control wells.

[0043] 4) Washing: Discard the liquid in the well, add 200 μl of washing buffer to each well, soak for 30 seconds, then blot dry. Repeat washing 3-5 times and then pat dry.

[0044] 5) Antibody binding detection: Add 100 μl of diluted biotin-labeled antibody to each well, gently tap the side of the plate to mix the liquid, seal with sealing film, and incubate at 37°C for 1 hour in the dark. After incubation, wash as in step 4.

[0045] 6) Enzyme-linked reaction: Add 100 μL of streptavidin-HRP complex to each well, seal with sealing film, and incubate at 37°C in the dark for 30 minutes. After incubation, wash according to step 4.

[0046] 7) Color development: Add 100 μL of TMB substrate to each well and incubate at 37°C for 15-30 minutes (adjust the time according to the color change).

[0047] 8) Termination of reaction: Add 50 μL of 2M H2SO4 to each well, gently tap the side of the plate to mix, and the solution will change from blue to yellow.

[0048] 9) Reading: Within 10 minutes after the reaction is terminated, measure the OD value of each well at a wavelength of 450nm using an ELISA reader, and read the 570nm wavelength as a reference wavelength (to correct for background interference). The final OD value = OD450 - OD570.

[0049] Data processing Data fitting was performed using GraphPad Prism software: the standard concentration (pg / mL) was used as the x-axis (logarithmic transformation), and the corresponding OD value was used as the y-axis. A four-parameter logistic regression model (4-PL) was selected to fit the standard curve, requiring R² ≥ 0.99 (indicating good fit and suitable for quantification).

[0050] Standard curve validation: Remove abnormal replicate data that deviate from the curve (if the coefficient of variation (CV) of the replicate OD value is >10%, it needs to be retested) to ensure the consistency of replicate data for each concentration gradient.

[0051] Calculate sample concentration: Calculate the concentration by interpolation from the standard curve based on the sample OD value. If the sample OD value exceeds the range of the standard curve, it needs to be diluted and retested. Data processing for replicates: Take the average concentration of the three replicates for each sample and calculate the CV value (CV = standard deviation / average value × 100%). Data with CV < 15% is considered valid; otherwise, the experiment must be repeated.

[0052] Results analysis: The mean concentration ± standard deviation (Mean ± SD) of IL-6, TNF-α, and IL-1β in each group were calculated. The differences between groups were analyzed by independent samples t test (for comparison between two groups) or one-way ANOVA (for comparison between multiple groups). P < 0.05 was considered statistically significant.

[0053] Correlation analysis: Combining TUNEL apoptosis detection results, the correlation between inflammatory factor concentration and ulcer tissue apoptosis index (AI) was analyzed (Pearson / Spearman correlation analysis). If a positive correlation is found, it suggests that inflammatory factors may participate in ulcer formation by inducing cell apoptosis.

[0054] In radiation-induced oral ulcers, IL-6, TNF-α, and IL-1β act as pro-inflammatory factors, and elevated concentrations usually indicate an aggravated local inflammatory response. If the serum / tissue levels of inflammatory factors in the model group are significantly higher than those in the control group, and are positively correlated with ulcer area and apoptosis, it can confirm the key role of the inflammatory response in radiation-induced oral ulcers.

[0055] Model Validation Positive control: Compared with the treatment group of Jin Yintai (5000 IU / kg), to verify the sensitivity of the model to the efficacy of the drug.

[0056] 1) Ulcer healing index: If the ulcer area of ​​the positive control group was significantly smaller than that of the model group on days 3, 5 and 7 (P<0.05), and the ulcer healing time was shortened by ≥2 days compared with the model group (P<0.05), it indicates that the positive drug can significantly promote ulcer healing.

[0057] 2) Pathological and inflammatory markers: The positive control group showed better recovery of mucosal epithelial integrity, significantly fewer inflammatory cell infiltrations than the model group (P<0.05), and significantly lower levels of TNF-α, IL-6, and IL-1β (P<0.05).

[0058] 3) Judgment criteria: If at least 3 of the above indicators show significant differences between the positive control group and the model group (P<0.05), the oral ulcer model is deemed to be sensitive to drug efficacy; if there are no significant differences, the model is invalid and the modeling protocol needs to be optimized.

[0059] 4) Statistical analysis: SPSS 26.0 software was used. Quantitative data (ulcer area, body weight, inflammatory factor content) were expressed as mean ± standard deviation (x ± s), and categorical data (healing rate, infection rate) were expressed as number of cases (percentage). P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0060] 5) After the normality test (Shapiro-Wilk) and the homogeneity of variance test (Levene): the independent samples t test was used for comparisons between the two groups (model group vs. positive control group); repeated measures ANOVA was used for data at multiple time points (such as ulcer area at different days), and the LSD method was used for pairwise comparisons after the fact.

[0061] The above describes embodiments of the present invention. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of the present invention.

Claims

1. A method for constructing an X-ray-induced radioactive oral ulcer animal model, characterized by, It comprises the following steps: SPF level SD rats are selected and randomly grouped after adaptive feeding for 7 days; X-ray local irradiation is adopted to irradiate oral mucosa, and the irradiation dose is 15 Gy single time; Oral mucosa tissue is taken 14 days after irradiation to observe epithelial exfoliation, inflammatory cell infiltration, TUNEL apoptosis detection, and inflammation factor detection through HE staining.

2. The construction method according to claim 1, characterized in that, The focal distance of the X-ray irradiation is 30 cm.

3. The construction method of claim 1, wherein, The dose rate of the X-ray irradiation is 1.2 Gy / min.

4. The construction method of claim 1, wherein, After the detection, the model sensitivity to drug efficacy is verified by comparing with the kinin peptide treatment group.

5. The construction method according to claim 4, characterized in that, SPSS 26.0 is adopted for inter-group comparison.

6. The construction method of claim 1, wherein, Changes in body weight and food intake are recorded daily after X-ray irradiation, and oral mucosa scoring is performed.

7. The construction method of claim 1, wherein, The inflammation factor detection comprises determination of IL-6, TNF-α, and IL-1β levels.

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