Construction method of ultraviolet-induced mouse lip squamous cell carcinoma model and fixing device thereof

Abstract

The invention belongs to the technical field of animal models, and particularly discloses an ultraviolet-induced mouse lip squamous cell carcinoma model construction method and a fixing device thereof.The fixing device comprises a fixing box, a cover plate and an iron gauze, the cover plate is inserted into the fixing box, baffles are arranged at the front end and the rear end of the fixing box respectively, and clamping plates are arranged on the inner sides of the baffles; the bottom of the clamping plate and the side edge of the fixing box form an inserting groove, the top of the clamping plate and the inner side wall face of the baffle form an adjusting groove, the two ends of the cover plate are inserted into the inserting groove respectively, and the fixing box is divided into two fixing chambers through a partition plate. According to the construction method of the ultraviolet-induced mouse lip squamous cell carcinoma model and the fixing device thereof, the fixing device can effectively fix the mouse in a waking state, anesthesia is not needed, and therefore mouse death or experimental result deviation possibly caused by general anesthesia is avoided; the construction method is high in feasibility and effectiveness.

Description

Technical Field

[0001] The invention relates to the technical field of animal models, in particular to a construction method of an ultraviolet-induced mouse lip squamous cell carcinoma model and a fixing device thereof. Background Art

[0002] Lip squamous cell carcinoma (LSCC) is a highly invasive malignant tumor that occurs in the red mucosa of the lips. It has the ability to spread to the oral mucosa and the skin outside the lips, causing serious effects on the patient's basic physiological functions, such as chewing, swallowing, and language expression. Ultraviolet radiation (UV) is the main risk factor for lip squamous cell carcinoma, especially in areas with long-term exposure to sunlight. The incidence of lip squamous cell carcinoma accounts for as high as 50%-70% of oral malignancies. Clinical data show that the prognosis of patients with early (stage I and II) lip squamous cell carcinoma is relatively good, while the 10-year survival rate of patients in the late stage (stage III and IV) is only 11%-28%, which greatly reduces the treatment effect and the quality of life of patients.

[0003] At present, the study of the occurrence, development, and prevention and treatment mechanisms of human tumors usually relies on animal models with high reproducibility and biological relevance. However, the field of lip squamous cell carcinoma still lacks a highly reproducible and highly replicable animal model suitable for local treatment research, which hinders the in-depth study of this type of tumor. Experimental animals play an irreplaceable role in life science research. They are not only important subjects for basic theoretical research and clinical trials, but also play a key supporting role in the development, production and testing of new drugs and biological products. Therefore, it is urgent to develop an animal model that can simulate the occurrence and development of lip squamous cell carcinoma to promote basic research on the disease and the exploration of treatment methods.

[0004] Among the commonly used experimental animals, mice, rats, rabbits and dogs are widely used, but mice have become the most commonly used animal model in experimental research due to their biological characteristics and easy operation. At present, ultraviolet (UV)-induced and dimethylbenzanthracene (7,2-dimethy1-1,2-benz[a]anthracene, DMBA) chemically induced mouse squamous cell carcinoma (SCC) model has been widely used in the study of the occurrence and development mechanism of SCC and has become an internationally recognized classic mouse SCC animal model. Although mice are docile in nature, they often resist during experimental operations, especially when being grabbed, which may sometimes cause the experimenter to be scratched or bitten. In order to ensure the safety of the experimenter and improve the success rate of the experimental operation, it is usually necessary to use a special fixation device to effectively fix the mouse.

[0005] At present, there is no fixture that can specifically expose the lips of mice, which brings great challenges to the ultraviolet irradiation experiment of the lips. Therefore, it is urgent to develop a mouse lip irradiation fixture that meets the above needs. Summary of the invention

[0006] The purpose of the present invention is to provide a method for constructing an ultraviolet-induced mouse lip squamous cell carcinoma model and a fixing device thereof, wherein the fixing device can achieve effective fixation when the mouse is awake without the aid of anesthesia, thereby avoiding the death of the mouse or deviation of the experimental results that may be caused by general anesthesia; the construction method has high feasibility and effectiveness.

[0007] To achieve the above-mentioned purpose, the present invention provides a fixing device for an ultraviolet-induced mouse lip squamous cell carcinoma model, comprising a fixing box, a cover plate, and a wire mesh, wherein the cover plate is plugged into the fixing box, baffles are respectively provided at the front and rear ends of the fixing box, a card plate is provided inside the baffle, a slot is formed between the bottom of the card plate and the side of the fixing box, an adjustment slot is formed between the top of the card plate and the inner wall of the baffle, the two ends of the cover plate are respectively inserted into the slots, and the fixing box is divided into two fixing chambers by a partition.

[0008] Preferably, an observation window is provided at one end of the bottom of the fixed chamber, and a wire mesh is provided on the observation window. The mesh size of the wire mesh is 6mm×6mm, and the diameter of the wire is 0.6mm.

[0009] Preferably, the fixed chamber is composed of a lower supporting plate, a side plate, a partition, two front and rear baffles and an observation window. The lower supporting plate is used to support the wire mesh and the experimental mice. An ultraviolet irradiation source is provided on the outer side of the bottom of the observation window.

[0010] Preferably, the height of the fixing box is 1.5 cm.

[0011] Preferably, the fixing device is directly manufactured by 3D printing technology, and the printing material includes polymer resin, nylon or metal.

[0012] The method for constructing a mouse lip squamous cell carcinoma model induced by ultraviolet light comprises the following steps:

[0013] S1. Determine the minimum dose of the mouse lips to ultraviolet light. Use a depilatory cream to remove hair from the mouse lips. After waiting for 1 week, use a fixing device to fix the mouse and irradiate the mouse lips with ultraviolet light through an ultraviolet irradiation source. Use an irradiator to measure the intensity of ultraviolet light and calculate the ultraviolet irradiation dose at different irradiation times. Within 2 hours to 7 days after irradiation, observe whether the mouse lips show redness, desquamation, swelling and scab reaction. Select the minimum dose that causes the above reactions to determine the minimum dose of the mouse lips to ultraviolet light.

[0014] S2. Construction of a UV-induced mouse lip squamous cell carcinoma model. After one week of adaptive feeding of mice, their lips were depilated with a depilatory cream. One week later, dimethylbenzanthracene was applied to the lips. One week later, the mice were irradiated with UV light using a determined minimum UV response dose, twice a week, with an interval of 3-4 days between each exposure. One week after exposure, the UV exposure dose was gradually increased each week, twice a week, with an interval of 3-4 days between each exposure. After exposure to the highest UV dose, the exposure was maintained twice a week, with an interval of 3-4 days. When the appearance of neoplasms on the mouse lips was observed, the frequency of UV exposure was reduced to once a week. UV exposure was suspended one month after the appearance of neoplasms. The mice were killed three months after the appearance of neoplasms, and it was determined whether the neoplasms were squamous cell carcinoma tumors.

[0015] Preferably, in step S2, the ultraviolet radiation dose is gradually increased to 125% MRD, 150% MRD, 175% MRD, and 200% MRD each week.

[0016] Preferably, in step S2, the maximum dose of ultraviolet light is 200% MRD.

[0017] The advantages and beneficial effects of the method for constructing the ultraviolet-induced mouse lip squamous cell carcinoma model and the fixing device thereof in the present invention are:

[0018] 1. The present invention can achieve effective fixation in a conscious state of mice without the aid of anesthesia, thereby avoiding the death of mice or deviation of experimental results that may be caused by general anesthesia. The device design can accurately expose the lips of mice for ultraviolet irradiation, while covering and protecting other body parts of the mice to prevent ultraviolet damage to non-target areas. It can ensure that the irradiation distance between the lips of each mouse and the ultraviolet light source is consistent, thereby ensuring the repeatability of the experiment and the reliability of the results.

[0019] 2. The construction method of the present invention is highly feasible and effective. With the assistance of a fixation device, after 6 to 9 months of ultraviolet irradiation, 32% of the mice successfully induced lip tumors, reflecting the efficiency and reliability of the model in the study of lip squamous cell carcinoma.

[0020] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a schematic diagram of the fixing device of the present invention;

[0022] Figure 2 It is a schematic diagram of the interior of a fixing box in the fixing device of the present invention;

[0023] Figure 3 is a schematic diagram of a cover plate in the fixing device of the present invention;

[0024] Figure 4 is a schematic diagram of a wire mesh in a fixing device of the present invention;

[0025] Figure 5 3. It is a diagram of the construction result of the mouse lip squamous cell carcinoma model induced by ultraviolet irradiation of the present invention, wherein A is a schematic diagram of the fixation device of the mouse lip squamous cell carcinoma model and the mouse fixation effect, B is the reaction of the lips of C57 mice under different ultraviolet irradiation doses within 2 hours to 7 days, C is a schematic diagram of the formation of neoplasms on the upper and lower lips of mice, and DF are the H&E staining results of the mouse lip tissue, and the scale is 1 mm. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] 1. Fixed box; 11. Lower bearing plate; 12. Side plate; 13. Partition plate; 14. Baffle plate; 15. Observation window; 16. Slot; 17. Adjustment slot; 2. Cover plate; 3. Wire mesh; 4. Experimental mice. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0029] Unless otherwise defined, the technical terms or scientific terms used in the present invention should be understood by people with ordinary skills in the field to which the present invention belongs. The words "first", "second" and similar words used in the present invention do not indicate any order, quantity or importance, but are only used to distinguish different components. "Include" or "comprise" and similar words mean that the elements or objects appearing before the word include the elements or objects listed after the word and their equivalents, without excluding other elements or objects. "Connect" or "connected" and similar words are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect. "Up", "down", "left", "right" and the like are only used to indicate relative positional relationships. When the absolute position of the described object changes, the relative positional relationship may also change accordingly.

[0030] Example

[0031] The fixing device of the ultraviolet-induced mouse lip squamous cell carcinoma model includes a fixing box 1, a cover plate 2, and a wire mesh 3. The fixing device is directly manufactured by 3D printing technology, and the printing material includes polymer resin, nylon or metal. The height of the fixing box 1 is 1.5 cm, and this height is suitable for the cover plate 2 to suppress the mouse so that the mouse cannot move in the fixing box 1. The cover plate 2 is plugged into the fixing box 1, and baffles 14 are respectively provided at the front and rear ends of the fixing box 1, and a card plate is provided inside the baffle plate 14. The bottom of the card plate and the side of the fixing box 1 form a slot 16, and the top of the card plate and the inner wall of the baffle plate 14 form an adjustment slot 17. The two ends of the cover plate 2 are respectively inserted into the slot 16.

[0032] The fixing box 1 is divided into two fixing chambers by a partition 13. An observation window 15 is provided at one end of the bottom of the fixing chamber, and a wire mesh 3 is provided on the observation window 15. Each fixing chamber is composed of a lower bearing plate 11, a side plate 12, a partition 13, two front and rear baffles 14, and an observation window 15. The lower bearing plate 11 is used to carry the wire mesh 3 and the experimental mouse 4.

[0033] The mesh size of the wire mesh 3 is 6mm×6mm, and the diameter of the wire is 0.6mm. This specification design ensures good lip exposure and ventilation, while providing effective fixation support for the mice, avoiding excessive restraint or damage to the experimental mice 4, and ensuring the safety and comfort of the mice during the experiment. An ultraviolet irradiation source is provided on the outer side of the bottom of the observation window 15.

[0034] Working principle of the fixing device of the ultraviolet-induced mouse lip squamous cell carcinoma model: First, cut the wire mesh 3 to a size of 90 mm × 35 mm and set it on the observation window 15 on the lower bearing plate 11 of the mouse fixing chamber. Then put the experimental mouse 4 into the fixing chamber with its head facing the observation window 15. For the experimental mouse 4 with a smaller volume, insert the plug-in cover plate 2 into the lower slot 16 to fix the mouse firmly in the fixing chamber. When the experimental mouse 4 is larger in volume, insert the plug-in cover plate 2 into the upper adjustment slot 17, and fix the plug-in cover plate 2 with tape or heavy objects to ensure that the mouse is stably and safely fixed in the fixing chamber during the experiment.

[0035] The fixing device of the present invention can effectively fix the mouse when it is awake, without the aid of anesthesia, thereby avoiding the death of the mouse or deviation of the experimental results that may be caused by general anesthesia. Accurately expose the lips of the mouse and protect the rest of the body: The design of the device can accurately expose the lips of the mouse for ultraviolet irradiation, while covering and protecting other body parts of the mouse to prevent ultraviolet damage to non-target areas. Ensure consistency of irradiation distance: The irradiation distance between the lips of each mouse and the ultraviolet light source can be guaranteed to be consistent, thereby ensuring the repeatability of the experiment and the reliability of the results. Adjustable fixing height to avoid excessive restraint: The fixing chamber in the device has a height-adjustable function, which ensures that the mouse's head is fixed and prevented from rotating without causing excessive restraint to it, avoiding the risk of suffocation or death.

[0036] The method for constructing a mouse lip squamous cell carcinoma model induced by ultraviolet light comprises the following steps:

[0037] S1. Determine the minimum reactive dose of the mouse lips to ultraviolet light. Use a depilatory cream to remove hair from the mouse lips. After waiting for 1 week, use a fixing device to fix the mouse and irradiate the mouse lips with ultraviolet light through an ultraviolet irradiation source. Use an irradiator to measure the intensity of ultraviolet light and calculate the ultraviolet irradiation dose at different irradiation times. Within 2 hours to 7 days after irradiation, observe whether the mouse lips show redness, desquamation, swelling and scab reaction. Select the minimum dose that causes the above reaction and determine the minimum reactive dose (MRD) of the mouse lips to ultraviolet light.

[0038] S2. Construction of a UV-induced mouse lip squamous cell carcinoma model. After one week of adaptive feeding of mice, their lips were depilated with a depilatory cream. One week later, dimethylbenzanthracene was applied to the lips, with 100 μg applied to the lips of each mouse. After another week, the lips of the mice were irradiated with UV light using the determined minimum UV response dose, twice a week, with an interval of 3-4 days between each exposure. After one week of irradiation, the UV irradiation dose was gradually increased each week, with UV doses of 125% MRD, 150% MRD, 175% MRD, and 200% MRD used in sequence, twice a week, with an interval of 3-4 days between each exposure. After irradiation to the highest UV dose of 200% MRD, irradiation was maintained twice a week, with an interval of 3-4 days. During the experiment, if the vital signs of the mice were not good, the UV dose could be reduced to 100%-150% MRD for irradiation or the number of irradiations per week could be reduced. After tumors were observed on the lips of mice, the frequency of ultraviolet irradiation was reduced to once a week. Ultraviolet irradiation was suspended one month after the tumors appeared. The mice were killed three months after the tumors appeared, and the tumors were dissected, dehydrated, paraffin-embedded, sectioned, and HE-stained to determine whether they were squamous cell carcinoma tumors.

[0039] After one week of adaptive feeding of C57 mice, the lips were depilated with a depilatory cream, and one week later, 100 μg of dimethylbenzanthracene (DMBA) was applied to the lips. After another week, the mice were fixed with a fixing device and their lips were exposed (such as Figure 5 Then 250 mJ / cm 2 、500mJ / cm 2 、1000mJ / cm 2 、2000mJ / cm 2 The mouse lips were exposed to ultraviolet radiation at a dose of Figure 5 The experimental results show that at 250mJ / cm 2 There was no significant change in the mouse lips under the UV dose of 500mJ / cm 2 After irradiation with a dose of 1000mJ / cm, obvious desquamation was observed on the lips of mice within 2 days, and returned to normal after 7 days. 2After irradiation with a dose of 2000mJ / cm 2 After irradiation with the dose, the mouse lips showed obvious redness and swelling within 2 hours, scabs formed after 2 days, and obvious scabs were still present on the lips after 7 days. The minimum response dose (MRD) of C57 mouse lips to ultraviolet light was finally determined to be 500mJ / cm 2 When using 500mJ / cm 2 After 1 week of ultraviolet radiation, the dose was gradually increased every week, using 625 mJ / cm 2 、750mJ / cm 2 、875mJ / cm 2 、1000mJ / cm 2 The mice were irradiated with a dose of ultraviolet light twice a week, with an interval of 3-4 days between each exposure. After 6 to 9 months of irradiation, 32% (22 / 68) of the C57 mice developed neoplasms on their lips (such as Figure 5 The incidence of neoplasms in the upper and lower lips was 7:15, and the ratio of male to female was 1:1. The mouse lips were dissected and dehydrated, paraffin-embedded, sectioned, and HE-stained (as shown in Figure 2A). Figure 5 Middle D, Figure 5 Middle E, Figure 5 As shown in F, the normal lip mucosa was observed to have significant adverse hyperplasia and tumor formation. The modeling scheme of the present invention is highly feasible and effective. With the assistance of a fixation device, after 6 to 9 months of ultraviolet irradiation, 32% of the mice successfully induced lip tumors, reflecting the efficiency and reliability of the model in the study of lip squamous cell carcinoma.

[0040] Therefore, the present invention adopts the above-mentioned UV-induced mouse lip squamous cell carcinoma model construction method and its fixing device, which can achieve effective fixation when the mouse is awake without the aid of anesthesia, thereby avoiding the death of mice or deviation of experimental results that may be caused by general anesthesia; the construction method has high feasibility and effectiveness.

[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A fixture for ultraviolet-induced mouse lip squamous cell carcinoma model, characterized in that: It includes a fixing box, a cover plate, and a wire mesh. The cover plate is plugged into the fixing box. The front and rear ends of the fixing box are respectively provided with baffles. The inner side of the baffle is provided with a card plate. The bottom of the card plate and the side of the fixing box form a slot. The top of the card plate and the inner wall of the baffle form an adjustment slot. The two ends of the cover plate are respectively inserted into the slots. The fixing box is divided into two fixing chambers by a partition.

2. The fixing device for the ultraviolet-induced mouse lip squamous cell carcinoma model according to claim 1, characterized in that: An observation window is arranged at one end of the bottom of the fixed chamber, and a wire mesh is arranged on the observation window. The mesh size of the wire mesh is 6mm×6mm, and the diameter of the wire is 0.6mm.

3. The fixing device for the ultraviolet-induced mouse lip squamous cell carcinoma model according to claim 2, characterized in that: The fixed chamber is composed of a lower bearing plate, a side plate, a partition plate, two front and rear baffles and an observation window. The lower bearing plate is used to carry the wire mesh and the experimental mice. An ultraviolet irradiation source is arranged on the outer side of the bottom of the observation window.

4. The fixing device for the ultraviolet-induced mouse lip squamous cell carcinoma model according to claim 1, characterized in that: The height of the fixing box is 1.5 cm.

5. The fixing device for the ultraviolet-induced mouse lip squamous cell carcinoma model according to claim 1, characterized in that: The fixing device is directly manufactured by 3D printing technology, and the printing material includes polymer resin, nylon or metal.

6. A method for constructing a UV-induced mouse lip squamous cell carcinoma model, characterized in that: The steps include: S1. Determine the minimum reaction dose of the mouse lips to ultraviolet rays, use a depilatory cream to remove hair from the mouse lips, wait for 1 week, fix the mouse using the fixing device as described in any one of claims 1 to 5, and irradiate the mouse lips with ultraviolet rays through an ultraviolet irradiation source, use an irradiator to measure the ultraviolet intensity, and calculate the ultraviolet irradiation dose at different irradiation times, observe whether the mouse lips show redness, desquamation, swelling and scab reactions within 2 hours to 7 days after irradiation, select the minimum dose that causes the above reactions, and determine the minimum reaction dose of the mouse lips to ultraviolet rays; S2. Construction of a UV-induced mouse lip squamous cell carcinoma model. After one week of adaptive feeding of mice, their lips were depilated with a depilatory cream. One week later, dimethylbenzanthracene was applied to the lips. One week later, the mice were irradiated with UV light using a determined minimum UV response dose, twice a week, with an interval of 3-4 days between each exposure. One week after exposure, the UV exposure dose was gradually increased each week, twice a week, with an interval of 3-4 days between each exposure. After exposure to the highest UV dose, the exposure was maintained twice a week, with an interval of 3-4 days. When the appearance of neoplasms on the mouse lips was observed, the frequency of UV exposure was reduced to once a week. UV exposure was suspended one month after the appearance of neoplasms. The mice were killed three months after the appearance of neoplasms, and it was determined whether the neoplasms were squamous cell carcinoma tumors.

7. The method for constructing a UV-induced mouse lip squamous cell carcinoma model according to claim 6, characterized in that: In step S2, the ultraviolet radiation dose is gradually increased to 125% MRD, 150% MRD, 175% MRD, and 200% MRD each week.

8. The method for constructing a UV-induced mouse lip squamous cell carcinoma model according to claim 6, characterized in that: In step S2, the highest dose of ultraviolet light is 200% MRD.

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