A method for constructing a mouse model of solar dermatitis

Abstract

The application discloses a kind of construction methods of sunlight dermatitis mouse model, specifically belongs to animal experiment model experimental technical field, the construction method includes the following steps: S01, ultraviolet lamp intensity measurement and selection;S02, the establishment of sunlight dermatitis model;S03, fresh tissue paraffin embedding section;S04, wax tissue section respectively by H-E staining experiment, Masson staining experiment, immunohistochemical staining experiment, transmission electron microscopy sheet observation, obtain the structure characteristic change of material organization.This construction method is by controlling the consistency of keeping radiation dose, to restore actual situation to a greater extent, provides real and reliable basis for developing sunlight dermatitis drug.

Description

Technical Field

[0001] This invention belongs to the field of animal experimental model technology, specifically relating to a method for constructing a mouse model of photodermatitis. Background Technology

[0002] Solar dermatitis, also known as sunburn, sunspots, or ultraviolet erythema, is an acute phototoxic reaction occurring on localized skin. It is primarily caused by UVB (medium-wave ultraviolet radiation) with wavelengths of 290–320 nm from sunlight. Its main clinical manifestation is erythema, with UVB being the primary inducing spectrum, causing bright red patches.

[0003] Solar ultraviolet (UV) radiation has wavelengths ranging from 100 to 400 nm, and is generally divided into long-wave ultraviolet (UVA) with wavelengths of 320–400 nm, medium-wave ultraviolet (UVB) with wavelengths of 290–320 nm, and short-wave ultraviolet (UVC) with wavelengths of 100–290 nm. Because the highest-energy UVC is blocked and absorbed by the atmosphere, 95% of the ultraviolet radiation reaching the Earth's surface is lower-energy UVA, and 5% is higher-energy UVB. UVA causes much less damage to the skin than UVB; only UVA radiation doses 1000 times higher than UVB can cause the same level of damage to the skin.

[0004] The skin is the body's first line of defense against external damage. Ultraviolet radiation (UVR) is a major environmental factor causing skin damage. The longer the wavelength of ultraviolet radiation, the stronger its penetrating power. UVB has a shorter wavelength and limited penetrating power; the epidermis is the primary target tissue for UVB radiation. High doses of acute UVB radiation mainly cause acute inflammatory reactions in the skin, leading to erythema, edema, and even erosion and exudation, resulting in photodermatitis. Photodermatitis is common in late spring and early summer, and is more prevalent among residents in high-altitude areas of my country and military personnel stationed there. The prevalence of photodermatitis is also high abroad; a report in the United States states that 57.2% of adolescents have a history of sunburn more than once a year. With increasing environmental pollution and ozone layer depletion, the incidence of photodermatitis is rising year by year.

[0005] Ideal animal models are excellent vehicles for studying the pathological mechanisms of photodermatitis and evaluating drug efficacy. Currently, mouse models of photodermatitis are commonly constructed using UVB radiation to mouse skin. However, inconsistent irradiation doses lead to a lack of reproducibility. If inconsistent UVB radiation doses are used to simulate photodermatitis, results from different experiments may not be directly comparable. Due to dose inconsistencies, experimental results may vary due to differences in radiation intensity, making the research findings difficult to reproduce. Furthermore, it may introduce uncertainty and misleading information, as the symptoms and severity of photodermatitis are closely related to the UVB radiation dose received. If the radiation dose in the model is inconsistent or significantly different from reality, the research results may be inaccurate, potentially leading to erroneous conclusions or misleading inferences. It may also make it difficult to accurately assess treatment efficacy; inconsistent UVB radiation doses can hinder accurate evaluation of treatment effects. In research on drugs for treating photodermatitis, it is crucial to explore how to determine the effective therapeutic dose based on different UVB radiation doses received. Summary of the Invention

[0006] The purpose of this invention is to provide a method for constructing a mouse model of photodermatitis. This method, by controlling and maintaining the consistency of radiation dose, can better replicate the actual situation and provides a real and reliable basis for the development and research of drugs for photodermatitis.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A method for constructing a mouse model of photodermatitis, comprising the following steps:

[0009] S01. Measurement and Selection of Ultraviolet Lamp Intensity

[0010] The intensity of the ultraviolet lamp was measured at 10cm, 15cm and 20cm respectively, and 20cm was selected as the ultraviolet irradiation intensity for this experiment. The UVB dose (mJ / cm2) = UVB intensity (mW / cm2) × irradiation time (s).

[0011] S02. Establishment of a solar dermatitis model

[0012] S02.1. C57BL / 6 mice were randomly divided into 5 groups using a random number table: 0 min group, 15 min group, 30 min group, 45 min group and 60 min group, with 8 animals in each group.

[0013] S02.2 After anesthetizing all five groups of mice, use an electric shaver to shave the hair from the tail to the back of the mice. Then use depilatory cream to remove the hair from the back of the mice to obtain the experimental area.

[0014] S02.3 After waiting for 24 hours, the experimental area of ​​the mice was exposed to a UV lamp at a distance of 20 cm with a UV intensity of 0.45 mW / cm2. The irradiation time for the 0 min, 15 min, 30 min, 45 min and 60 min groups were 0 min, 15 min, 30 min, 45 min and 60 min, respectively.

[0015] S02.4 After observing changes in the appearance of the back skin at 24h, 48h, and 72h, skin biopsies were performed on each mouse 72h after UVB exposure. A 1.0cm × 0.5cm piece of skin tissue was taken from the back and fixed in 4% paraformaldehyde and 3% glutaraldehyde, respectively, to obtain processed fresh tissue. The purpose of using different fixatives was: 4% paraformaldehyde was used to fix the tissue specimen for subsequent HE, masson, and immunohistochemical staining; 3% glutaraldehyde was used to fix the tissue specimen for subsequent electron microscopy sections.

[0016] S03, fresh tissue paraffin-embedded sections

[0017] S03.1 Fix the obtained fresh tissue by immersing it in 4% paraformaldehyde for at least 24 hours. After immersion, remove the fresh tissue, trim the tissue at the target site, attach the corresponding label to the trimmed tissue, and place it in a dehydration box.

[0018] S03.2. Place the dehydration box into the basket and add reagents to the dehydrator in sequence according to the dehydration steps. The specific dehydration steps are as follows:

[0019] The tissue was sequentially soaked in 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, benzene for 5-10 minutes, xylene I for 5-10 minutes, xylene II for 5-10 minutes, wax I for 1 hour, wax II for 1 hour, and wax III for 1 hour to obtain a wax tissue.

[0020] S03.3. Embed the paraffin tissue in the embedding machine. First, put the melted paraffin into the embedding frame. Before the paraffin solidifies, take the tissue out of the dehydration box and put it into the embedding frame according to the requirements of the embedding surface. Affix the corresponding label and cool it on a -20℃ freezing stage. After the paraffin solidifies, take the paraffin block out of the embedding frame and trim the paraffin block. The specific requirements for the embedding surface are: cross-section of the full skin layer, skin side facing left, clear skin layers, and consistent embedding horizontal plane.

[0021] S03.4 Place the trimmed wax block on a paraffin microtome and slice it to a thickness of 4μm. Float the slice on 40℃ warm water in a slide spreader to flatten the tissue. Use a glass slide to pick up the tissue and place it in a 60℃ oven to bake. After the water dries and the wax melts, remove the slice to obtain a wax tissue section.

[0022] S04 and paraffin tissue sections were subjected to HE staining, Masson staining, immunohistochemical staining, and transmission electron microscopy to observe changes in the structural characteristics of the tissue samples.

[0023] Preferably, the HE staining experiment in step S04 is as follows:

[0024] The waxed tissue sections were sequentially immersed in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 10 min, anhydrous ethanol II for 10 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, and 70% ethanol for 5 min, and then washed with distilled water to obtain dewaxed tissue sections.

[0025] Dewaxed tissue sections were stained with Harris hematoxylin for 3-8 minutes, washed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, rinsed with tap water, blued with 0.6% ammonia water, and rinsed with running water to obtain hematoxylin-stained cell nuclear sections.

[0026] Hematoxylin-stained cell nuclear sections were stained in eosin staining solution for 1-3 minutes to obtain eosin-stained cytoplasmic sections.

[0027] Eosin-stained cytoplasmic sections were sequentially immersed in 95% ethanol I for 5 min, 95% ethanol II for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, xylene I for 5 min, and xylene II for 5 min to dehydrate and clear. The eosin-stained cytoplasmic sections were then removed from the xylene and air-dried. They were then mounted with neutral resin, examined under a microscope, and the images were acquired and analyzed.

[0028] Preferably, in step S04, the Masson staining procedure is as follows:

[0029] Dewaxing paraffin sections to water: Place the paraffin tissue sections in xylene I for 15 min, xylene II for 15 min, xylene III for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 95% ethanol for 5 min, 85% ethanol for 5 min, and then wash with tap water to obtain dewaxed sections.

[0030] Potassium dichromate staining: Dewaxed sections were soaked in potassium dichromate overnight and then washed with tap water.

[0031] Iron hematoxylin staining: Iron hematoxylin A solution and iron hematoxylin B solution are mixed in equal proportions to form iron hematoxylin staining solution. The sections are immersed in iron hematoxylin for 3 minutes, washed with tap water, differentiated with differentiation solution, washed with tap water, blued with blue solution, and rinsed with running water. Among them, iron hematoxylin A solution is hematoxylin staining solution, and iron hematoxylin B solution is ferric chloride solution.

[0032] Ponceau Acid Fuchsia Staining: Immerse dewaxed sections in Ponceau Acid Fuchsia for 5-10 minutes, then rinse with tap water.

[0033] Phosphomolybdic acid staining: Immerse in phosphomolybdic acid aqueous solution for 1-3 minutes.

[0034] Aniline blue staining: After phosphomolybdic acid, do not wash with water, directly immerse in aniline blue staining solution for 3-6 minutes.

[0035] Differentiation: The sections were differentiated with 1% glacial acetic acid and dehydrated with two tanks of anhydrous ethanol.

[0036] Clearing and mounting: The sections were placed in the third container of anhydrous ethanol for 5 minutes, xylene for 5 minutes for clearing, and then mounted with neutral resin; microscopic examination and image acquisition and analysis were performed.

[0037] Preferably, in step S04, the immunohistochemical staining procedure is as follows:

[0038] Dewaxing paraffin sections to water: Place the sections in xylene I for 15 min, xylene II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and then wash with distilled water.

[0039] Antigen retrieval: Place the paraffin sections in a retrieval box filled with citrate antigen retrieval buffer and microwave on medium heat for 8 minutes, then turn off for 8 minutes and microwave on medium-low heat for 7 minutes. During this process, prevent excessive evaporation of the buffer and do not dry the sections.

[0040] After natural cooling, place the slide in PBS buffer and wash it three times on a decolorizing shaker for 5 minutes each time.

[0041] Draw circles: After slightly drying the slices, use a histology pen to draw circles around the tissue.

[0042] Serum blocking: Add 3% BSA to the circle and incubate at room temperature for 30 min.

[0043] Add primary antibody: Gently shake off the blocking solution, add the primary antibody prepared in PBS at a certain ratio to the slide, and incubate the slide flat in a humidified chamber at 4°C overnight.

[0044] Add secondary antibody: Place the slide in PBS buffer and wash three times on a decolorizing shaker for 5 minutes each time. After slightly drying the section, add the secondary antibody corresponding to the species of the primary antibody to the inner circle to cover the tissue, and incubate at room temperature in the dark for 50 minutes.

[0045] DAB staining and counterstaining of cell nuclei: Place the slide in PBS buffer and wash it three times on a decolorizing shaker for 5 minutes each time. After slightly drying the slide, add DAB staining agent to the circle. Control the staining under a microscope. After complete staining, rinse with distilled water or tap water, counterstain with hematoxylin, differentiate with 1% hydrochloric acid alcohol, rinse with tap water, return to blue with ammonia, and rinse with running water.

[0046] Dehydration and mounting: Immerse the sections in 75% alcohol for 5 min, 85% alcohol for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and xylene I for 5 min in sequence to dehydrate until transparent. Remove the sections from the xylene, air dry, and mount with neutral resin.

[0047] Furthermore, the pH of the citrate antigen retrieval buffer is 6.0.

[0048] Furthermore, the pH of the PBS buffer is 7.4.

[0049] Furthermore, the differentiation time of 1% hydrochloric acid alcohol is 1 second.

[0050] Furthermore, the preparation steps for transmission electron microscopy (TEM) slides are as follows:

[0051] Sampling and fixation: Determine the sampling site for fresh tissue, with a tissue volume not exceeding 1mm×1mm×1mm, immerse it in electron microscopy fixative, and fix it at 4℃ for 2-4 hours.

[0052] After centrifuging the cells to the bottom of the tube, cell clumps the size of mung beans can be seen. Discard the culture medium and add electron microscopy fixative at 4°C for 2-4 hours.

[0053] Rinse three times with 0.1M phosphate-buffered saline (PBS), 15 min each time.

[0054] Post-fixation: Fixation with 1% osmium tetroxide in 0.1M phosphate buffered PBS at room temperature for 2 hours; rinse three times with 0.1M phosphate buffered PBS, 15 minutes each time.

[0055] Dehydration: The tissue was sequentially immersed in 50%, 70%, 80%, 90%, 95%, 100%, and 100% alcohol solutions, each for 15 minutes.

[0056] Infiltration: A 1:1 mixture of acetone and 812 embedding agent was infiltrated overnight, and pure 812 embedding agent was infiltrated overnight.

[0057] Encapsulation: Polymerize at 60℃ for 48 hours.

[0058] Sectioning: The slicing machine cuts ultrathin sections of 60-80nm.

[0059] Staining: 2% uranium acetate saturated aqueous solution, lead citrate, stain for 15 min each, and let sections dry at room temperature overnight.

[0060] Observe under a transmission electron microscope and collect and analyze images.

[0061] The pH of the phosphate-buffered saline (PBS) is 7.4.

[0062] Compared with the prior art, the beneficial effects of the present invention are:

[0063] 1. This invention utilizes ultraviolet (UVB) radiation for modeling. Specifically, C57BL / 6J mice are subjected to continuous UVB irradiation at different doses after initial exposure, simulating the irradiation state of mice. Controllable application of different radiation doses to the mice facilitates the representation of symptoms resulting from varying radiation levels in the model, thereby aiding in the development of therapeutic drugs for treating humans exposed to different degrees of radiation.

[0064] 2. After successful establishment of the photodermatitis mouse model constructed using this invention, the mice exhibited erythema; sunburned cells appeared in the epidermis, and neutrophils and macrophages infiltrated the dermis; furthermore, the expression of inflammatory factors IL-1β, IL-6, and TNF-α gradually increased. Similar to human photodermatitis, the photodermatitis model constructed using UVB-irradiated C57BL / 6J mice shows high similarity and can reflect the skin lesion process that occurs in humans under sunlight exposure. Technicians can use this model to evaluate the development of anti-inflammatory, antioxidant, and immunomodulatory drugs.

[0065] 3. In this construction method, the irradiation dose can be accurately controlled by controlling the UVB radiation time to meet experimental requirements, and it is convenient for researchers to adjust and operate the model. The operation process is very simple. Attached Figure Description

[0066] Figure 1 The addition of a primary antibody statistical table is provided for this invention;

[0067] Figure 2 The statistical table for adding secondary antibodies provided by this invention;

[0068] Figure 3 This is a schematic diagram of the intensity of an ultraviolet lamp;

[0069] Figure 4 The image shows the changes in appearance of the mouse experimental area after UVB irradiation (the right side is a magnified image at the corresponding time).

[0070] Figure 5 Image showing the effect of HE and Masson staining on fresh tissue;

[0071] Figure 6 Image showing the results of immunohistochemical staining;

[0072] Figure 7 It is the electron microscope observation effect diagram of tissue cells;

[0073] Figure 8 It is the result of fluorescence quantitative PCR experiment;

[0074] Figure 9 It is the pathological diagram of first-degree sunburn and second-degree sunburn. Specific implementation manners

[0075] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0076] Embodiment:

[0077] 1 Materials and methods

[0078] 1.1 Experimental animals

[0079] A total of 40 SPF-grade C57BL / 6 mice, male, 6-8 weeks old, weighing 18-22 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. [SCXK (Beijing) 2021-0006]. The experimental environment was the animal house of the Academy of Military Medical Sciences. The animals were allowed to freely eat and drink water. The environmental temperature of the animal house was controlled at 20-26 °C, the relative humidity was 40%-70%, the minimum air change rate was 15 times / h, and the light / dark cycle was 12 h / 12 h.

[0080] 1.2 Main reagents and instruments

[0081] Ultraviolet lamp (PHILIPS, TL20W / 01 Germany, power 20 W, peak wavelength 311 nm); ultraviolet intensity detector (OHSP-350UV, 230-850 nm, Hangzhou Hongpu Optoelectronic Color Technology Co., Ltd.); pet hair clipper (Jingdiao Yuzhuo) razor; depilatory cream (Veet, Veeting, mild type); isoflurane (Jiangsu Hengfengqiang Biotechnology Co., Ltd., batch number 20230301); MatrxVMR small animal anesthesia machine (Midmark Corporation). Nikon camera (D7500)

[0082] 1.3 Experimental methods

[0083] 1.3.1 Measurement and selection of ultraviolet lamp intensity

[0084] The intensity of the ultraviolet lamp was measured at 10cm, 15cm, and 20cm, and 20cm was selected as the ultraviolet irradiation intensity for this experiment. UVB dose (mJ / cm²) = UVB intensity (mW / cm²) × irradiation time (s)

[0085] 1.3.2 Establishment of a solar dermatitis model

[0086] C57BL / 6 mice were randomly divided into 5 groups using a random number table: 0 min, 15 min, 30 min, 45 min, and 60 min groups, with 8 animals in each group. After anesthetizing the mice, the fur on the dorsal side (from the tail to the back) was shaved off using an electric shaver, followed by hair removal cream. 24 hours later, the mice were irradiated at 20 cm under a UV lamp (UV intensity 0.45 mW / cm²) for 15 min, 30 min, 45 min, and 60 min, respectively. The 0 min group received no UV irradiation. The 15 min, 30 min, 45 min, and 60 min irradiations corresponded to 405 mJ / cm², 810 mJ / cm², 1215 mJ / cm², and 1620 mJ / cm², respectively. After irradiation, mice were scored and photographed at 24h, 48h, and 72h. They were sacrificed and samples were collected after 72h. Each mouse underwent a skin biopsy, and a small piece of skin tissue measuring 1.0cm × 0.5cm was taken from the back. The tissue was placed in 4% formaldehyde and 3% glutaraldehyde, respectively, and routinely stained with hematoxylin and eosin (HE), masson staining, immunostaining, and electron microscopy. In addition, 50mg of fresh tissue was taken for the detection of inflammatory factors.

[0087] 1.3.3 HE staining experimental procedure

[0088] 1.3.3.1 Procedure for paraffin-embedded tissue sections:

[0089] Material collection: Fresh tissue was fixed in 4% paraformaldehyde for at least 24 hours. The tissue was removed from the fixative and trimmed in a fume hood using a scalpel. The trimmed tissue and corresponding labels were then placed in a dehydration box.

[0090] Dehydration: Place the dehydration box into the basket and then into the dehydrator for sequential dehydration with alcohol in a gradient. 75% alcohol 4h - 85% alcohol 2h - 90% alcohol 2h - 95% alcohol 1h - anhydrous ethanol I 30min - anhydrous ethanol II 30min - benzene 5-10min - xylene I 5-10min - xylene II 5-10min - wax I 1h - wax II 1h - wax III 1h.

[0091] Embedding: The paraffin-impregnated tissue is embedded in an embedding machine. First, molten paraffin is placed into the embedding frame. Before the paraffin solidifies, the tissue is removed from the dehydration box and placed into the embedding frame according to the required embedding surface, and labeled accordingly. The tissue is then cooled on a -20°C freezing stage. After the paraffin solidifies, the paraffin block is removed from the embedding frame and trimmed.

[0092] Sectioning: Place the trimmed wax block on a paraffin microtome and section it to a thickness of 4μm. Float the sections on 40℃ warm water in a slide spreader to flatten the tissue. Use a glass slide to lift the tissue and place it in a 60℃ oven to bake. After the water has dried and the wax has melted, remove it and store it at room temperature for later use.

[0093] 1.3.3.2 HE staining experimental procedure

[0094] Dewaxing paraffin sections to water: The sections are placed in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 10 min, anhydrous ethanol II for 10 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, and then washed with distilled water.

[0095] Hematoxylin staining of cell nuclei: Slices are stained with Harris hematoxylin for 3-8 minutes, washed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, rinsed with tap water, blued with 0.6% ammonia water, and rinsed with running water.

[0096] Eosin staining of cytoplasm: Immerse the sections in eosin staining solution for 1-3 minutes.

[0097] Dehydration and mounting: Immerse the sections in 95% ethanol I for 5 min, 95% ethanol II for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, xylene I for 5 min, and xylene II for 5 min in sequence to dehydrate and clear the sections. Remove the sections from the xylene and let them air dry slightly before mounting with neutral resin.

[0098] Microscopic examination, image acquisition and analysis.

[0099] Staining results: cell nuclei are blue, cytoplasm is red.

[0100] 1.3.4 Masson staining experimental procedure

[0101] Dewaxing paraffin sections to water: Place the sections in xylene I for 15 min, xylene II for 15 min, xylene III for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 95% ethanol for 5 min, and 85% ethanol for 5 min, then wash with tap water.

[0102] Potassium dichromate staining: Soak the sections in potassium dichromate overnight, then wash with tap water.

[0103] Iron hematoxylin staining: Iron hematoxylin solution A and solution B are mixed in equal proportions to prepare iron hematoxylin staining solution. The sections are immersed in the iron hematoxylin solution for 3 minutes, then...

[0104] Wash with tap water, use differentiating solution to differentiate, wash with tap water, use blueing solution to blue, and rinse with running water.

[0105] Ponceau Sin Acid Fuchsia Staining: Immerse the sections in Ponceau Sin Acid Fuchsia for 5-10 minutes, then rinse with tap water.

[0106] Phosphomolybdic acid staining: Immerse in phosphomolybdic acid aqueous solution for 1-3 minutes.

[0107] Aniline blue staining: After phosphomolybdic acid, do not wash with water, directly immerse in aniline blue staining solution for 3-6 minutes.

[0108] Differentiation: The sections were differentiated with 1% glacial acetic acid and dehydrated with two tanks of anhydrous ethanol.

[0109] Clearing and mounting: Place the sections in the third container of anhydrous ethanol for 5 minutes, xylene for 5 minutes to clear, and then mount with neutral resin.

[0110] Microscopic examination, image acquisition and analysis.

[0111] Result interpretation:

[0112] Collagen fibers are blue; muscle fibers, cellulose, and red blood cells are red.

[0113] 1.3.4 Immunohistochemical staining

[0114] Dewaxing paraffin sections to water: The sections are placed in xylene I for 15 min, xylene II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and then washed with distilled water.

[0115] Antigen retrieval: Tissue sections were placed in a retrieval container filled with citrate antigen retrieval buffer (pH 6.0) and microwaved for antigen retrieval. Microwave on medium heat for 8 minutes, then turn off for 8 minutes, and then microwave on medium-low for 7 minutes. During this process, excessive evaporation of the buffer should be prevented; the slides should not be allowed to dry. After natural cooling, the slides were placed in PBS (pH 7.4) and washed three times on a destaining shaker for 5 minutes each time. (The retrieval solution and conditions are determined based on the specific tissue.)

[0116] Draw circles: After slightly drying the slices, use a histochemical pen to draw circles around the tissue (to prevent antibody migration).

[0117] Serum blocking: Add 3% BSA to the circle and incubate at room temperature for 30 min.

[0118] Add primary antibody: Gently shake off the blocking solution, add the prepared primary antibody (prepared with PBS in a specific ratio) to the slide, and incubate the slide flat in a humidified chamber at 4°C overnight. (Add a small amount of water to the humidified chamber to prevent antibody evaporation.) See [link to humidified chamber]. Figure 1 The column highlighted in yellow.

[0119] Add secondary antibody: Place the slide in PBS (pH 7.4) and wash three times on a destaining shaker for 5 minutes each time. After slightly drying the sections, add the secondary antibody corresponding to the species of the primary antibody to the inner circle to cover the tissue, and incubate at room temperature in the dark for 50 minutes. See Figure 2 The second column of the last column.

[0120] DAB staining and counterstaining of cell nuclei: Slides were placed in PBS (pH 7.4) and washed three times on a destaining shaker for 5 minutes each time. After slightly drying the sections, DAB chromogenic reagent was added to the inner circle, and staining was controlled under a microscope. After complete staining, the sections were rinsed with distilled or tap water, counterstained with hematoxylin, and differentiated with 1% hydrochloric acid alcohol (approximately 1 second).

[0121] Rinse with tap water, ammonia turns blue, rinse with running water.

[0122] Dehydration and mounting: Immerse the sections in 75% alcohol for 5 min, 85% alcohol for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and xylene I for 5 min in sequence to dehydrate and clear them. Remove the sections from the xylene and let them dry slightly before mounting them with neutral resin.

[0123] Interpretation of immunohistochemical results of paraffin sections: Cell nuclei stained with hematoxylin appear blue under a white light microscope, while positive expression appears as corresponding brown or dark brown.

[0124] 1.3.5 Transmission Electron Microscopy (TEM) Slide Preparation Procedure

[0125] Tissue sampling and fixation: Determine the sampling site for fresh tissue, minimizing mechanical damage such as traction, contusion, and compression. The tissue volume should generally not exceed 1mm × 1mm × 1mm. Quickly immerse the tissue in electron microscopy fixation solution and fix at 4°C for 2-4 hours. Centrifuge the cells until pea-sized cell clumps are visible at the bottom of the tube. Discard the culture medium and add electron microscopy fixation solution, fixing at 4°C for 2-4 hours. Wash three times with 0.1M phosphate-buffered saline (PBS, pH 7.4), 15 minutes each time.

[0126] Post-fixation: Fixation was performed for 2 hours at room temperature (20°C) using 1% osmium tetroxide in 0.1M phosphate buffered PBS (pH 7.4). The sample was then rinsed three times with 0.1M phosphate buffered PBS (pH 7.4), each time for 15 minutes.

[0127] Dehydration: Tissues are sequentially immersed in 50%-70%-80%-90%-95%-100%-100% alcohol for upward dehydration, each time for 15 minutes.

[0128] Infiltration: A 1:1 mixture of acetone and 812 embedding agent was infiltrated overnight, and pure 812 embedding agent was infiltrated overnight.

[0129] Encapsulation: Polymerize at 60℃ for 48 hours.

[0130] Sectioning: The slicing machine cuts ultrathin sections of 60-80nm.

[0131] Staining: Uranium-lead double staining (2% uranium acetate saturated aqueous solution, lead citrate, each staining for 15 min), sections dried at room temperature overnight.

[0132] Observe under a transmission electron microscope and collect and analyze images.

[0133] 1.3.6 Quantitative Real-Time PCR Experimental Procedure

[0134] Total RNA extraction:

[0135] 1) Take a homogenizing tube, pre-cool it on ice, add 1 ml of Trizol Reagent, and grind until no visible precipitate is visible.

[0136] 2) Centrifuge at 12000 rpm for 15 min and collect the supernatant.

[0137] 3) Add 200 μl of chloroform, invert the centrifuge tube for 30 seconds, mix thoroughly, and let stand for 5 minutes.

[0138] 4) Centrifuge at 12000 rpm for 15 min at 4℃.

[0139] 5) Transfer the supernatant to a new centrifuge tube, add 0.8 times the volume of isopropanol, and mix by inverting.

[0140] 6) Place at -20℃ for 20 minutes.

[0141] 7) Centrifuge at 12000 rpm for 10 min at 4℃. The white precipitate at the bottom of the tube is RNA.

[0142] 8) Remove the liquid, add 1 ml of 75% ethanol to wash the precipitate, and centrifuge at 12000 rpm for 5 min at 4℃.

[0143] 9) Wash once more.

[0144] 10) Remove all liquid and place the centrifuge tube on a clean bench and blow it for 1 minute.

[0145] 11) Add 20 μl of RNase-free water to dissolve the RNA.

[0146] 12) Incubate at 55℃ for 5 minutes.

[0147] Reverse transcription

[0148] Reverse transcription program settings

[0149] Quantitative PCR

[0150] 1) Take a 0.2ml PCR tube and prepare the following reaction system. Prepare 3 tubes for each reverse transcription product.

[0151] 2) PCR amplification was performed on a PCR instrument.

[0152] Result Processing

[0153] CT scan:

[0154] A = CT(target gene, test sample) - CT(internal control gene GAPDH, test sample) B = CT(target gene, control sample) - CT(internal control gene GAPDH, control sample)

[0155] K = AB

[0156] Expression multiple = 2 - K

[0157] 2. Results

[0158] 2.1 UV lamp intensity (e.g.) Figure 3 )

[0159] 2.2 Appearance changes (e.g.) Figure 4 )

[0160] 2.3 HE and Masson staining (e.g.) Figure 5 )

[0161] 2.4 Immunohistochemistry (MPO, F4 / 80) (e.g.) Figure 6 )

[0162] 2.5 Electron microscopy observation (e.g.) Figure 7 )

[0163] 2.6 Results of quantitative real-time PCR (e.g.) Figure 8 )

[0164] 3. Conclusion

[0165] In this study, c57BL / 6 mice in the 15-minute (405 mJ / cm²) UVB radiation group showed moderate abnormalities in skin tissue structure, with thickened stratum corneum and hyperkeratosis within the visual field. Figure 9 The white arrow A indicates the layer of spinous processes; some sunburned cells are visible, with eosinophilic cytoplasm and pyknotous or absent nuclei. Figure 9 As indicated by the black arrow A; the dermis is rich in collagen fibers, which are neatly and tightly arranged; numerous skin appendages are visible. Figure 9 The blue arrow A in the diagram represents a hair follicle. Figure 9 The green arrow A in the diagram represents a sebaceous gland; a small amount of inflammatory cell infiltration is visible in the tissue, such as... Figure 9 The yellow arrow in the diagram indicates point A.

[0166] In the 30-minute group, c57BL / 6 mice irradiated with 810 mJ / cm² UVB showed severe abnormalities in skin tissue structure, with thickened stratum corneum and hyperkeratosis within the visual field. Figure 9 The white arrow at point B indicates the layer of spinous processes; numerous sunburned cells are visible, with eosinophilic cytoplasm and pyknotous or absent nuclei. Figure 9 The black arrow B indicates localized loss of collagen fibers in the dermis, cell necrosis, nuclear fragmentation, condensation, and deep staining, as shown in the image. Figure 9 The red arrow in section B indicates a reduction in the number of skin appendages. Figure 9 The blue arrow in B indicates a hair follicle; the tissue shows extensive infiltration of inflammatory cells, such as... Figure 9 As indicated by the yellow arrow B in the image, the sunburn reaches the papillary layer of the dermis.

[0167] First-degree sunburn: 3-5 hours after excessive ultraviolet radiation exposure, the skin develops erythema and swelling, accompanied by burning, pain, and itching. The local skin is more sensitive to heat and mechanical stimulation. First-degree sunburn only affects the epidermis, presenting as localized redness and swelling, pain and burning sensation, and slightly elevated skin temperature. It manifests as bright red patches, swelling, and a feeling of burning or stinging. It usually heals completely in 3-5 days, with desquamation but no scarring.

[0168] Second-degree sunburn: In addition to redness and swelling, severe sunburn can also cause skin blisters and bullae, accompanied by intense burning pain, reaching the level of superficial second-degree burns. Second-degree sunburn penetrates to the dermis and causes localized blisters.

[0169] The photodermatitis mouse model provided by this invention effectively simulated the skin inflammation response of patients with first-degree sunburn in the 15-min group (405 mJ / cm2) and the skin inflammation response of patients with second-degree sunburn in the 30-min group (810 mJ / cm2). This model can be used to explore the pathogenesis of photodermatitis progression and the development of therapeutic drugs, which is of great significance for accelerating the translation of scientific research into clinical practice. Figure 9 As shown.

Claims

1. A method for constructing a mouse model of photodermatitis, characterized in that, Includes the following steps: S01. Measurement and Selection of Ultraviolet Lamp Intensity The intensity of the ultraviolet lamp was measured at 10cm, 15cm and 20cm respectively, and 20cm was selected as the ultraviolet irradiation intensity for this experiment. UVB dose (mJ / cm2) = UVB intensity (mW / cm2) × irradiation time (s); S02. Establishment of a solar dermatitis model S02.

1. C57BL / 6 mice were randomly divided into 5 groups using a random number table: 0 min group, 15 min group, 30 min group, 45 min group and 60 min group, with 8 animals in each group. S02.2 After anesthetizing all five groups of mice, use an electric shaver to shave the hair above the tail and below the back of the mice, and then use depilatory cream to remove the hair on the back of the mice to obtain the experimental area. S02.3 After waiting for 24 hours, the experimental area of ​​the mice was exposed to a UV lamp at a distance of 20 cm with a UV intensity of 0.45 mW / cm2. The irradiation time for the 0 min group, 15 min group, 30 min group, 45 min group and 60 min group were 0 min, 15 min, 30 min, 45 min and 60 min respectively. S02.4 After observing the changes in the appearance of the back skin by taking pictures at 24h, 48h and 72h, skin biopsy was performed on each mouse 72h after exposure to UVB. A 1.0cm×0.5cm skin tissue was taken from the back and fixed in 4% paraformaldehyde and 3% glutaraldehyde, respectively. S03, fresh tissue paraffin-embedded sections S03.1 Fix the obtained fresh tissue by immersing it in 4% paraformaldehyde for at least 24 hours. After immersion, remove the fresh tissue, trim the tissue at the target site, attach the corresponding label to the trimmed tissue, and place it in a dehydration box. S03.

2. Place the dehydration box into the basket and add reagents to the dehydrator in sequence according to the dehydration steps. The specific dehydration steps are as follows: The tissue was sequentially soaked in 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, benzene for 5-10 minutes, xylene I for 5-10 minutes, xylene II for 5-10 minutes, wax I for 1 hour, wax II for 1 hour, and wax III for 1 hour to obtain a wax tissue. S03.

3. Embed the wax tissue in the embedding machine. First, put the melted wax into the embedding frame. Before the wax solidifies, take the tissue out of the dehydration box and put it into the embedding frame according to the requirements of the embedding surface. Then attach the corresponding label and cool it on the -20℃ freezing stage. After the wax solidifies, take the wax block out of the embedding frame and trim the wax block. S03.4 Place the trimmed wax block on a paraffin microtome and slice it to a thickness of 4μm. Float the slice on 40℃ warm water in a slide spreader to flatten the tissue. Use a glass slide to pick up the tissue and place it in a 60℃ oven to bake. After the water dries and the wax melts, remove the slice to obtain a wax tissue section. S04 and paraffin tissue sections were subjected to HE staining, Masson staining, immunohistochemical staining, and transmission electron microscopy to observe changes in the structural characteristics of the tissue samples. In S04, the immunohistochemical staining procedure is as follows: Dewaxing paraffin sections to water: Place the sections in xylene I for 15 min, xylene II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and then wash with distilled water. Antigen retrieval: Place the paraffin sections in a retrieval box filled with citrate antigen retrieval buffer and microwave on medium for 8 minutes, turn off for 8 minutes, and then microwave on medium-low for 7 minutes. During this process, prevent excessive evaporation of the buffer and do not dry the sections. After natural cooling, place the slide in PBS buffer and wash it three times on a decolorizing shaker for 5 minutes each time. Draw circles: After slightly drying the slices, use a histological pen to draw circles around the tissue; Serum blocking: Add 3% BSA to the circle and incubate at room temperature for 30 min; Add primary antibody: Gently shake off the blocking solution, add the primary antibody prepared in PBS at a certain ratio to the slide, and incubate the slide flat in a humidified chamber at 4°C overnight; Add secondary antibody: Place the slide in PBS buffer and wash it three times on a decolorizing shaker for 5 minutes each time; after slightly drying the section, add the secondary antibody of the corresponding species to the primary antibody in the circle to cover the tissue, and incubate at room temperature in the dark for 50 minutes. DAB staining and counterstaining of cell nuclei: Place the slide in PBS buffer and wash it three times on a decolorizing shaker for 5 minutes each time. After slightly drying the slide, add DAB staining agent to the circle. Control the staining under a microscope. After complete staining, rinse with distilled water or tap water, counterstain with hematoxylin, differentiate with 1% hydrochloric acid alcohol, rinse with tap water, return to blue with ammonia, and rinse with running water. Dehydration and mounting: Immerse the sections in 75% alcohol for 5 min, 85% alcohol for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and xylene I for 5 min in sequence to dehydrate until transparent. Remove the sections from the xylene, air dry, and mount with neutral resin.

2. The method for constructing a mouse model of photodermatitis according to claim 1, characterized in that, In step S04, the HE staining experiment steps are as follows: The waxed tissue sections were sequentially immersed in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 10 min, anhydrous ethanol II for 10 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, and 70% ethanol for 5 min, and then washed with distilled water to obtain dewaxed tissue sections. Dewaxed tissue sections were stained with Harris hematoxylin for 3-8 minutes, washed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, rinsed with tap water, blued with 0.6% ammonia water, and rinsed with running water to obtain hematoxylin-stained cell nuclear sections. Hematoxylin-stained cell nuclear sections were stained in eosin staining solution for 1-3 minutes to obtain eosin-stained cytoplasmic sections. Eosin-stained cytoplasmic sections were sequentially immersed in 95% ethanol I for 5 min, 95% ethanol II for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, xylene I for 5 min, and xylene II for 5 min to dehydrate and clear. The eosin-stained cytoplasmic sections were then removed from the xylene and air-dried. They were then mounted with neutral resin, examined under a microscope, and the images were acquired and analyzed.

3. The method for constructing a mouse model of photodermatitis according to claim 1, characterized in that, In step S04, the Masson staining experiment steps are as follows: Dewaxing paraffin sections to water: Place the paraffin tissue sections in xylene I for 15 min, xylene II for 15 min, xylene III for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 95% ethanol for 5 min, 85% ethanol for 5 min, and then wash with tap water to obtain dewaxed sections. Potassium dichromate staining: Dewaxed sections were soaked in potassium dichromate overnight and then washed with tap water; Iron hematoxylin staining: Iron hematoxylin solution A and solution B are mixed in equal proportions to form iron hematoxylin staining solution. The sections are immersed in iron hematoxylin for 3 minutes, washed with tap water, differentiated with differentiation solution, washed with tap water, blued with blue solution, and rinsed with running water. Ponceau Sin Acid Fuchsia Staining: Dewaxed sections are immersed in Ponceau Sin Acid Fuchsia for 5-10 minutes, then rinsed with tap water; Phosphomolybdic acid staining: Immerse in phosphomolybdic acid aqueous solution for 1-3 minutes; Aniline blue staining: After phosphomolybdic acid, do not wash with water, directly immerse in aniline blue staining solution for 3-6 minutes; Differentiation: The sections were differentiated with 1% glacial acetic acid and dehydrated with two tanks of anhydrous ethanol; Clearing and mounting: The sections were placed in the third container of anhydrous ethanol for 5 minutes, xylene for 5 minutes for clearing, and then mounted with neutral resin; microscopic examination and image acquisition and analysis were performed.

4. The method for constructing a mouse model of photodermatitis according to claim 3, characterized in that, The pH of the citrate antigen retrieval buffer is 6.

0.

5. The method for constructing a mouse model of photodermatitis according to claim 3, characterized in that, The pH of the PBS buffer is 7.

4.

6. The method for constructing a mouse model of photodermatitis according to claim 3, characterized in that, The differentiation time for 1% hydrochloric acid alcohol is 1 second.

7. The method for constructing a mouse model of photodermatitis according to claim 3, characterized in that, The preparation steps for transmission electron microscopy (TEM) slides are as follows: Sampling and fixation: Determine the sampling site for fresh tissue, with a tissue volume not exceeding 1mm×1mm×1mm, immerse it in electron microscopy fixative, and fix it at 4℃ for 2-4 hours; After centrifuging the cells to the bottom of the tube, cell clumps the size of mung beans can be seen. Discard the culture medium and add electron microscopy fixation solution to fix the cells at 4°C for 2-4 hours. Wash three times with 0.1M phosphate-buffered saline (PBS), 15 min each time; Post-fixation: Fixation with 1% osmium tetroxide in 0.1M phosphate buffered PBS at room temperature for 2 hours; wash 3 times with 0.1M phosphate buffered PBS, 15 minutes each time; Dehydration: The tissue was sequentially immersed in 50%, 70%, 80%, 90%, 95%, 100%, and 100% alcohol solutions, each for 15 minutes. Infiltration: A 1:1 mixture of acetone and 812 embedding agent was infiltrated overnight, and pure 812 embedding agent was infiltrated overnight; Encapsulation: Polymerize at 60℃ for 48 hours; Sectioning: Microtome to slice 60-80nm ultrathin sections; Staining: 2% uranium acetate saturated aqueous solution, lead citrate, stain for 15 min each, then section dry at room temperature overnight; Observation under a transmission electron microscope, image acquisition and analysis; The pH of the phosphate-buffered saline (PBS) is 7.4.

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