Construction method of mouse dry eye model

Abstract

The invention provides a construction method of a mouse dry eye model, and belongs to the technical field of animal model construction. The construction method comprises the following steps: dripping a benzalkonium chloride solution into the ocular surface of a mouse, injecting a scopolamine solution into the mouse in a subcutaneous manner, placing the mouse in a dry environment, and modeling to obtain the mouse dry eye model. The formation of the mouse dry eye model is induced through a dry environment and a compound condition of benzalkonium chloride and scopolamine. According to the mouse dry eye model, the tear secretion amount is remarkably reduced, ocular surface inflammation occurs, and the dry eye syndrome can be well simulated. The model can be used for screening medicines for treating the xerophthalmia and carrying out other researches on the xerophthalmia, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the technical field of animal model construction, specifically relating to a method for constructing a mouse dry eye model. Background Technology

[0002] Dry eye disease (DED) is a multifactorial ocular surface disease caused by abnormalities in the quality or quantity of tears, decreased tear film stability, and ocular surface inflammation. Clinical manifestations include dryness, foreign body sensation, burning pain, and fluctuating vision. In severe cases, it can damage the corneal epithelium and affect visual quality. However, the pathogenesis of dry eye is complex, involving multiple factors such as reduced tear secretion, excessive evaporation, inflammatory response, neuroregulatory imbalance, decreased sex hormone levels, and mucin abnormalities. Clinical treatment still lacks highly effective and lasting interventions. There is an urgent need for animal models that can simultaneously simulate multiple etiologies to deeply analyze the pathological process and screen for new drugs.

[0003] Existing animal models of dry eye mainly include drug-induced, surgically removed, environmentally induced, and genetically engineered models. Among them, drug-induced dry eye is widely used due to its simple operation, short cycle, and low cost. Currently, commonly used dry eye modeling methods include: (1) Using the drug scopolamine, by blocking the stimulation of the lacrimal gland by the parasympathetic nerve, significantly inhibiting tear secretion, a "tear secretion deficiency type" dry eye model can be quickly established; however, when used alone, the ocular surface inflammation reaction is mild, which is different from the clinically common mixed dry eye phenotype. (2) Using the drug benzalkonium chloride (BAC), long-term high-frequency eye drops can destroy the tear film lipid layer, damage the corneal epithelial tight junction, induce ocular surface inflammation, goblet cell loss and squamous metaplasia, forming an "evaporative dry eye type" dry eye model; its pathological changes are close to those of severe dry eye patients, but it is often difficult to simulate secretion deficiency type dry eye due to insufficient tear secretion. (3) Simple environmental drying method: Placing animals in a low humidity (<30%–40% RH) environment can accelerate tear evaporation, but the modeling period is long, individual differences are large, and the effect on tear secretion is limited. It often needs to be used in combination with other stimuli.

[0004] Further research is needed to develop a more effective dry eye model. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing a mouse dry eye model.

[0006] This invention provides a method for constructing a mouse dry eye model, wherein the method involves instilling benzalkonium chloride solution into the ocular surface of a mouse, simultaneously injecting scopolamine solution subcutaneously into the mouse, and placing the mouse in a dry environment to create a mouse dry eye model.

[0007] Furthermore, The concentration of the benzalkonium chloride solution is 0.1~0.2%; And / or, the concentration of the scopolamine solution is 0.2-0.3%.

[0008] Furthermore, The concentration of the benzalkonium chloride solution is 0.1%; And / or, the concentration of the scopolamine solution is 0.25%.

[0009] Furthermore, The benzalkonium chloride solution is prepared by dissolving benzalkonium chloride in 0.9% sodium chloride injection. And / or, the scopolamine solution is prepared by dissolving scopolamine hydrobromide in 0.9% sodium chloride injection.

[0010] Furthermore, The amount of benzalkonium chloride solution used was 5 μL per mouse; And / or, the amount of scopolamine solution used is 0.2 mL per mouse; And / or, the frequency of instilling benzalkonium chloride solution and subcutaneous injection of scopolamine solution is 3 times / day; Preferably, the subcutaneous injection is a subcutaneous injection in the back.

[0011] Furthermore, the method of placing mice in a dry environment was to increase airflow by blowing air through them.

[0012] Furthermore, the air blowing is done during the day, and not at night.

[0013] Preferably, the blowing time is 8 to 9 hours.

[0014] Furthermore, the molding time is 7 to 10 days; preferably, the molding time is 7 days.

[0015] Furthermore, the mouse is a C57BL / 6 mouse.

[0016] The present invention also provides the use of a mouse dry eye model obtained by the aforementioned construction method in screening drugs for the prevention and / or treatment of dry eye syndrome.

[0017] The present invention has achieved the following beneficial effects: This invention provides a method for constructing a mouse dry eye model, which induces the formation of a mouse dry eye model through a dry environment and a combination of benzalkonium chloride and scopolamine. This mouse dry eye model exhibits significantly reduced tear secretion and ocular surface inflammation, effectively mimicking dry eye syndrome. This model has promising applications in screening drugs for treating dry eye syndrome and in conducting other research on dry eye.

[0018] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0019] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0020] Figure 1 Histological images of mice in each group. Detailed Implementation

[0021] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0022] 1. The main reagents and preparation methods are as follows: Benzalkonium chloride (BAC): Manufacturer: Sigma Aldrich; Batch number: BCCD13587; Appearance: Pale yellow crystals; Specification: 100g / bottle; Storage conditions and shelf life after preparation: Store at 2~8℃, shelf life: 7 days. Preparation method: Weigh an appropriate amount of sample, add an appropriate amount of 0.9% sodium chloride injection, stir to dissolve, and make up to a final concentration of 1mg / mL (0.1%).

[0023] Scopolamine hydrobromide: Manufacturer: Chengdu Yirui Biotechnology Co., Ltd.; Batch number: MUST-21092802; Appearance: White powder; Specification: 1g / bottle; Storage conditions and shelf life after preparation: Store at 2~8℃, shelf life: 7 days. Preparation method: Weigh an appropriate amount of sample, add an appropriate amount of 0.9% sodium chloride injection, stir to dissolve, and make up to a final concentration of 2.5mg / mL (0.25%).

[0024] Cyclosporine A eye drops: Manufacturer: Shenyang Xingqi Eye Drops Co., Ltd.; Appearance: colorless emulsion; Specification: 0.4ml / 0.2mg*30 vials / box; Storage conditions and precautions: Protect from moisture, store in a sealed container at 4℃; No preparation required, use directly.

[0025] 0.9% Sodium Chloride Injection: Manufacturer: Sichuan Kelun Pharmaceutical Co., Ltd.; Batch No.: M21060704B; Appearance: Colorless and clear liquid; Specification: 500 mL / bottle; Storage conditions: 15~25℃.

[0026] 2. Experimental animals 2.1 Basic information Species: C57BL / 6 mice; Grade: ordinary grade; Female; Age: 6 - 8 weeks; Body weight: 18 - 22 g, and the individual body weight should be within the range of the average body weight ± 20%.

[0027] Animal welfare: The experimental institution is Chengdu Huaxi Haiqi Pharmaceutical Technology Co., Ltd., which is certified by AAALAC (International Association for Assessment and Accreditation of Laboratory Animal Care). This experiment has been approved by the institution's IACUC (Institutional Animal Care and Use Committee), approval number: IACUC - S2021020 - P001 - 01.

[0028] 2.2 Environmental adaptation All animals need to adapt to the environment for at least 7 days before the experiment. Healthy animals are selected as the test animals. During the adaptation period, the general condition of the mice is mainly checked to see if it is good, whether the body weight meets the requirements, the anterior segment of the eye (conjunctiva, cornea, anterior chamber, iris, lens, etc.) is examined once with a slit lamp, and the phenol red cotton tear flow is detected once to confirm the eye health of the mice. Animals that do not pass the above inspections are not included in the experiment.

[0029] 2.3 Animal feeding conditions Feeding location: In the ordinary animal house area (Building 1) of Chengdu Huaxi Haiqi Pharmaceutical Technology Co., Ltd. (Experimental Animal Use License Number: SYXK (Chuan) 2013 - 123); The type of feeding cage is an ordinary mouse feeding cage; The feeding density is 5 mice per cage.

[0030] Environmental conditions: The standard of the feeding environment conditions is the national standard of the People's Republic of China GB14925 - 2010; The feeding environment control system: The EBI400 automatic control fresh air central air - conditioning system of Honeywell Company; Temperature: Room temperature 16 - 26 °C (daily temperature difference ≤ 4 °C); Humidity: Relative humidity 40 - 70%; Lighting: Artificial lighting, 12 / 12 - hour day - night light - dark alternation.

[0031] Feed and drinking water Type: Mouse growth and reproduction feed; Manufacturer: Beijing Keao Xieli Feed Co., Ltd.; Feed production license: Jing Si Zheng (2014) 06054; Feeding method: Free intake.

[0032] Type: Experimental animal drinking water; Water supply method: Contained in a drinking water bottle, free intake.

[0033] Example 1. Construction of a dry eye model in mice As mentioned above, female C57BL / 6 mice were selected; age: 6-8 weeks; weight: 18-22g. Before modeling, the mice were allowed to acclimatize to the environment for 7 days and were examined. Only qualified mice were used for modeling. The modeling method is as follows: Starting from day 1 (D1), 5 μL of 0.1% BAC solution was instilled into the ocular surface of mice three times a day (every time at 4.0 ± 1.0 hours interval). Simultaneously, 0.2 mL of a 0.25% scopolamine solution containing 0.5 mg was injected subcutaneously into the back of the mice, also three times a day. Furthermore, air circulation was increased daily to create a dry environment. Specifically, a small mini fan was placed in the mouse cage during the day (approximately 8 hours) to increase airflow, and the mouse refuge box was removed to avoid obstructing airflow. No air circulation was provided at night.

[0034] The mouse dry eye model was created from day 1 (D1) to day 7 (D7).

[0035] The following experimental examples demonstrate the beneficial effects of the present invention.

[0036] Experimental Example 1: Validation of the Effect of the Mouse Dry Eye Model of the Present Invention 1. Experimental Design 1.1 Group Design Group design: The experiment was divided into three groups: negative control group, model control group, and cyclosporine group; Animal number and sex: 10 animals / group, female.

[0037] 1.2 Modeling (1) BCA eye drops: 5 μL of 0.1% BAC solution was instilled into the ocular surface of all mice in the model control group and cyclosporine group, and 5 μL of 0.9% sodium chloride injection was instilled into the ocular surface of mice in the negative control group. The frequency of eye drops for all mice was 3 times / day (with an interval of 4.0±1.0 hours each time) for 7 days (D1~D7).

[0038] (2) Subcutaneous injection of scopolamine: 0.2 mL of scopolamine solution containing 0.5 mg (concentration of 0.25%) was injected subcutaneously on the back of all mice in the model control group and cyclosporine group, and 0.2 mL of 0.9% sodium chloride solution was injected subcutaneously on the back of mice in the negative control group. All mice were injected 3 times a day. Scopolamine was administered at the same time as eye drops. The injection time was 7 days (D1~D7).

[0039] (3) Blowing air increases airflow and creates a dry environment. Small mini fans were placed in the cages of all mice in the model control group and cyclosporine group to increase airflow, and the mouse refuge boxes in the cages were removed to avoid obstructing airflow. The exposure lasted for 7 days (D1-D7), with fans running during the day (approximately 8 hours) but not at night. Mice in the negative control group were not exposed to fans.

[0040] 1.3 Administration The drug administration information for each group of mice is shown in Table 1.

[0041] Table 1. Drug administration information for mice in each group 1.4 Observation Indicators (1) General state observation Near-death and death observation: at least once a day; Routine observation: Observe at least once a day. If toxic symptoms appear, the observation frequency can be increased. The observation content includes, but is not limited to, local eye reactions (whether there is congestion, edema, increased secretions, etc.), animal appearance, coat, general behavior, mental state, gland secretion, skin and mucous membrane color, respiratory status, fecal characteristics, genital condition, and other toxic symptoms. (2) Tear flow rate test Testing times: before modeling, D7, D10, D13, D17; Animals examined: all surviving mice in each group; Examination method: After anesthetizing the mice with sodium pentobarbital (approximately 50 mg / kg, intraperitoneal injection), the tear fluid was tested using phenol red cotton. The phenol red cotton thread was inserted into the outer corner of the mouse's eye for 60 seconds.

[0042] (3) Slit-lamp examination and corneal fluorescein staining examination Testing times: before modeling, D7, D10, D13, D17; Animals examined: all surviving mice in each group; Examination Method: Mice were anesthetized with sodium pentobarbital (approximately 50 mg / kg, intraperitoneal injection), and the structures of the anterior segment of the mouse were observed using a slit lamp. Subsequently, 2 μL of 1% sodium fluorescein solution was instilled into the ocular surface of the mouse, and the eyelids were gently closed for approximately 2 seconds. Before observation, residual sodium fluorescein solution was rinsed off with physiological saline. Under a slit lamp microscope, the staining of the ocular surface was examined under cobalt blue diffuse light, and scores were assigned according to Table 2 below. Photographs were taken and recorded. When determining the tear film breakup time, the animal's eyelids were artificially closed, and the time from the last opening of the eye to the appearance of the first tear film breakup spot was taken as the tear film breakup time.

[0043] Table 2. Corneal fluorescein sodium staining scoring table (4) Gross anatomical and histopathological examination Dissection time: D18; Animals to be dissected: All surviving animals in each group; Anesthesia and Dissection: Anesthetize the animal with sodium pentobarbital according to its body weight (approximately 60 mg / kg intraperitoneally, dosage can be adjusted according to the animal's health condition), and euthanize it by exsanguination from the abdominal aorta. Remove both eyeballs. The left eyeball is fixed in modified Davison's fixative. The cornea and conjunctiva of the right eyeball are separated, labeled with the animal number and tissue name, and placed in the same tissue tube for storage below -66°C until examination. Slide preparation and staining: Paraffin sections of the left eyeball were prepared according to the standard procedures for histopathology. The sections were stained with hematoxylin and eosin and examined under a microscope.

[0044] Estimated number of slides: 5 slides per eye (sagittal plane, evenly distributed from the nasal side to the temporal limbus) * 30 mice = 150 slides.

[0045] All slides are examined under a microscope by a pathologist, and the lesions are classified into five grades based on their distribution, severity, and morphological characteristics using appropriate diagnostic terminology: minor (Grade 1), mild (Grade 2), moderate (Grade 3), severe (Grade 4), and serious (Grade 5). For lesions that are difficult to grade, "presence" can be used for marking.

[0046] (5) Cytokine PCR detection Test samples: right eye tissue from all surviving animals in each group; Detection indicators: TNF-α (tumor necrotizing factor alpha), IFN-γ (interferone gamma); Detection method: Tissue homogenate was obtained from the right eye of the animal, RNA samples were extracted, genomic DNA was removed, and reverse transcription was performed to qualitatively detect the levels of TNF-α and IFN-γ, with β-actin expression level used as an internal control. Results are expressed as amplified count (CT value).

[0047] 1.5 Statistical Analysis and Result Determination Tear volume length values ​​are expressed as mean ± standard deviation ( ). The results of fluorescein staining, slit-lamp examination McDonald scores, and qRT-PCR detection are expressed as mean (interquartile range). Pathological data are analyzed descriptively.

[0048] Statistical analysis was performed, and comparisons were made between the negative control group, the model control group, and the cyclosporine group.

[0049] First, the LEVENE test is used to test for homogeneity of variance. If the variances are homogeneous (P>0.05), then one-way ANOVA is used for statistical testing; if the variances are unequal (P>0.05), then... P If the variance is ≤0.05, then the Kruskal-Wallis H rank-sum test (KW test) is used for statistical analysis. When the one-way ANOVA shows a statistically significant difference ( P If the variance is ≤0.05, then Dunnett's t-test (Dunnett's method) is used for statistical analysis; when the one-way ANOVA shows no statistically significant difference ( P If the result is greater than 0.05, the statistical analysis ends. When the Kruskal-Wallis H rank-sum test shows a statistically significant difference (…), the statistical analysis concludes. P If the value is ≤0.05, the Mann-Whitney U test (MW test) is used to compare differences between groups; when the Kruskal-Wallis H rank-sum test shows no statistically significant difference ( P If the result is greater than 0.05, the statistical analysis ends.

[0050] 2. Results 2.1 General Situation Both the model control group and the cyclosporine group showed significant corneal epithelial damage, corneal opacity, and conjunctival hyperemia after the modeling period, which is considered to be related to local damage and inflammatory response caused by ocular surface modeling. These signs gradually subsided during the drug administration period, which is considered to be related to the self-limiting nature of the model and drug treatment.

[0051] 2.2 Tear flow rate Table 3 shows the results of phenol red cotton assay for tear flow in each group of animals at each time point in the experiment. After the modeling period, both the model control group and the cyclosporine group showed a significant decrease in tear secretion compared to the negative control group, indicating that the dry eye model was successfully established. After 3 and 6 days of cyclosporine treatment, the tear secretion in the cyclosporine group returned to the pre-modeling level and showed no significant difference compared to the negative control group, while the tear secretion in the model control group remained significantly lower than the pre-modeling level and the negative control group level. At D17, the tear secretion in the model control group recovered and showed no significant difference compared to the negative control group and the cyclosporine group, indicating that the dry eye model is self-limiting.

[0052] Table 3. Results of phenol red cotton detection on tear flow in each group of animals at different time points. Note: In the table P The value represents the result of a three-group analysis of variance test. a This indicates a significant difference compared to the negative control group at the same time point; b This indicates that there was a significant difference between the cyclosporine group and the model control group at the same time point.

[0053] 2.3 Slit-lamp inspection The McDonald scores obtained by slit-lamp examination of animals in each group at each time point are shown in Tables 4-6 below. After the modeling period, both the model control group and the cyclosporine group showed significant corneal opacity, conjunctival hyperemia, conjunctival edema, increased secretions, and corneal neovascularization, indicating diffuse ocular surface inflammation. These symptoms persisted in the model control group until the end of the treatment period, showing slight relief compared to the end of the modeling period, indicating the persistence of ocular surface inflammation. Compared with the model control group, the cyclosporine group showed slightly milder corneal opacity, corneal neovascularization, and conjunctival inflammation after 10 days of treatment, with a significantly lower conjunctival score than the model control group.

[0054] Table 4. Corneal opacity scores of animals in each group at each time point. Note: The corneal opacity score in the table is the product of the corneal opacity degree score and the opacity area score. P The values ​​are the results of three Kruskal-Wallis tests. a This indicates a significant difference compared to the negative control group at the same time point; b This indicates that there was a significant difference between the cyclosporine group and the model control group at the same time point.

[0055] Table 5. Corneal neovascularization scores of animals in each group at each time point. Note: P The values ​​are the results of three Kruskal-Wallis tests. a This indicates a significant difference compared to the negative control group at the same time point; b This indicates that there was a significant difference between the cyclosporine group and the model control group at the same time point.

[0056] Table 6. Conjunctival scores of animals in each group at each time point Note: The conjunctival score in the table is the sum of the scores for conjunctival congestion, edema, and secretions. P The values ​​are the results of three Kruskal-Wallis tests. a This indicates a significant difference compared to the negative control group at the same time point; b This indicates that there was a significant difference between the cyclosporine group and the model control group at the same time point.

[0057] 2.4 Corneal fluorescein staining Table 7 shows the results of corneal fluorescein sodium staining scores for each group at each time point in the experiment. Before modeling, corneal fluorescein staining was mild in all groups. After the modeling period, significant patchy corneal fluorescein staining appeared in both the model control group and the cyclosporine group. During the treatment period (D10), the corneal fluorescein staining score in the cyclosporine group was lower than that in the model control group, but still significantly higher than that in the negative control group. During the treatment periods (D13 and D17), the corneal fluorescein staining score in the cyclosporine group gradually decreased further, showing no significant difference from the negative control group, but no significant difference compared with the model control group.

[0058] Table 7. Results of corneal fluorescein sodium staining scores for each group of animals at each time point. Note: In the table P The values ​​are the results of three Kruskal-Wallis tests. a This indicates a significant difference compared to the negative control group at the same time point; b This indicates that there was a significant difference between the cyclosporine group and the model control group at the same time point.

[0059] 2.5 Pathological examination Gross anatomical observation On day 18 of the experiment, no gross abnormalities were found in the mice in each group after gross anatomical observation.

[0060] Microscopic observation Histopathological examination revealed corneal epithelial atrophy and mixed inflammatory cell infiltration in the model control group, while corneal epithelial atrophy was observed in the cyclosporine group (see Table 8 for details). Corneal epithelial atrophy and mixed inflammatory cell infiltration were correlated with model establishment.

[0061] Table 8. Incidence and classification of ocular lesions In the model control group, mild (4 / 10) to mild (3 / 10) corneal epithelial atrophy and mild (3 / 10) mixed inflammatory cell infiltration of the cornea were observed. In the cyclosporine group, mild (3 / 10) to mild (2 / 10) corneal epithelial atrophy was observed. Compared with the model control group, the ocular lesions in the cyclosporine group were milder. Histological images are shown below. Figure 1 .

[0062] 2.6 qRT-PCR detection The results of qRT-PCR detection of corneal and conjunctival tissues in each group at the experimental endpoint are shown in Table 9. Compared with the negative control group, the expression levels of TNF-α in the corneal and conjunctival tissues of the model control group and the cyclosporine group were significantly decreased, while there was no significant difference between the model control group and the cyclosporine group. There was no significant difference in the expression levels of IF-γ in the corneal and conjunctival tissues of the three groups of animals.

[0063] Table 9. Results of qRT-PCR detection of corneal and conjunctival tissues from different groups at different time points. Note: The value in the table is 2. -△△CT value, P The values ​​are the results of three Kruskal-Wallis tests. a This indicates a significant difference compared to the negative control group at the same time point; b This indicates that there was a significant difference between the cyclosporine group and the model control group at the same time point.

[0064] 3. Conclusion This invention successfully established a dry eye model in C57BL / 6 mice under dry environment and combined conditions of benzalkonium chloride and scopolamine. This model exhibits significant characteristics of dry eye, including reduced tear secretion and ocular surface inflammation. The model is self-limiting, with a stable time window of approximately 6 days. Treatment with 0.05% cyclosporine A eye drops effectively stimulated tear secretion and reduced ocular surface inflammation compared to the control group.

[0065] In summary, this invention provides a method for constructing a mouse dry eye model, which induces the formation of a mouse dry eye model through a dry environment and a combination of benzalkonium chloride and scopolamine. This mouse dry eye model exhibits significantly reduced tear secretion and ocular surface inflammation, effectively mimicking dry eye syndrome. This model has promising applications in screening drugs for treating dry eye syndrome and in conducting other research on dry eye.

Claims

1. A method for constructing a mouse dry eye model, characterized in that: The method of constructing the model involves instilling benzalkonium chloride solution into the ocular surface of mice, simultaneously injecting scopolamine solution subcutaneously into the mice, and placing the mice in a dry environment to create a mouse dry eye model.

2. The construction method according to claim 1, characterized in that: The concentration of the benzalkonium chloride solution is 0.1~0.2%; And / or, the concentration of the scopolamine solution is 0.2-0.3%.

3. The construction method according to claim 2, characterized in that: The concentration of the benzalkonium chloride solution is 0.1%; And / or, the concentration of the scopolamine solution is 0.25%.

4. The construction method according to claim 3, characterized in that: The benzalkonium chloride solution is prepared by dissolving benzalkonium chloride in 0.9% sodium chloride injection. And / or, the scopolamine solution is prepared by dissolving scopolamine hydrobromide in 0.9% sodium chloride injection.

5. The construction method according to claim 1, characterized in that: The amount of benzalkonium chloride solution used was 5 μL per mouse; And / or, the amount of scopolamine solution used is 0.2 mL per mouse; And / or, the frequency of instilling benzalkonium chloride solution and subcutaneous injection of scopolamine solution is 3 times / day; Preferably, the subcutaneous injection is a subcutaneous injection in the back.

6. The construction method according to claim 1, characterized in that: The method of placing mice in a dry environment is to increase airflow by blowing air through them.

7. The construction method according to claim 6, characterized in that: The ventilation is to be done during the day, and not at night.

8. The construction method according to claim 1, characterized in that: The molding process takes 7 to 10 days; preferably, it takes 7 days.

9. The construction method according to any one of claims 1 to 8, characterized in that: The mice in question were C57BL / 6 mice.

10. Use of a mouse dry eye model obtained by the construction method according to any one of claims 1 to 9 in screening drugs for the prevention and / or treatment of dry eye syndrome.

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