A method for constructing an animal model of pathological myopia and its uses

Abstract

The present invention relates to the field of animal disease models. A method for constructing and using a pathological myopia animal model provided by the present invention includes: inducing with a negative lens in combination with intervention with an mTOR activator to obtain a pathological myopia animal model; the present invention has found through research that when an mTOR activator based on the key regulatory pathway of myopic axial elongation is combined with negative lens induction to intervene in animals, it can efficiently induce animals to develop pathological myopia. All animals showed significant tigroid fundus (pathological myopia in stage M1), and 25% of guinea pigs showed diffuse and patchy choroidal atrophy around the optic disc, which is characteristic of pathological myopia (pathological myopia in stages M2 - M3). This kind of pathological myopia animal model is easy to induce and highly similar to the fundus changes of human pathological myopia, which can fill the gap in pathological myopia animal models and can be used as a new animal model for exploring the causes and mechanisms of the occurrence and development of pathological myopia.

Description

Technical Field

[0001] The present invention relates to the field of animal disease models, and particularly to a method for constructing a pathological myopia animal model and its uses. Background Art

[0002] High myopia seriously threatens human visual health. With the increasing prevalence rate year by year, the visual impairment caused by high myopia has become a serious public health problem. Currently, the global population with high myopia has reached 399 million. By 2050, it is estimated that half of the global population will have myopia, and 20% of them will be highly myopic (about 938 million people). The results of a number of epidemiological studies at home and abroad show that high myopia has ranked first among irreversible blinding eye diseases. A meta-analysis of 12 large population-based epidemiological studies globally shows that blindness and low vision caused by high myopia account for 2% of the total population. Preventing and controlling the low vision and blindness caused by high myopia is a major issue related to human visual health and the national economy and people's livelihood.

[0003] The cause of blindness in high myopia is not the increase in diopter, but rather the pathological changes in the fundus, that is, pathological myopia. Regarding the pathological myopia caused by high myopia, there is still little exploration of its pathogenesis mechanism. One of the important reasons is the lack of an animal model of pathological myopia for exploring the occurrence and discovery mechanisms. The traditional form deprivation or lens-induced animal myopia models can only simulate the eye axis elongation and corneal curvature steepening caused by myopia, but cannot produce the characteristic fundus changes of pathological myopia. When the time and intensity of form deprivation / lens induction are extended, pathological myopia cannot be induced either. In recent years, some researchers have tried to knockout the Lrp2 gene to simulate pathological myopia, but it can only simulate the enlargement of the eyeball and lacks the characteristic fundus changes of pathological myopia. The lack of an animal model of pathological myopia has greatly hindered the exploration of the related mechanisms of the occurrence and development of pathological myopia, which has led to the prevention and treatment of pathological myopia only staying at the observation of clinical manifestations and it is difficult to discover the targets for early diagnosis and treatment. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide a method for constructing a pathological myopia animal model.

[0005] For this purpose, the present invention provides the following technical solution:

[0006] A method for constructing a pathological myopia animal model, comprising: inducing an animal with a negative lens in combination with an mTOR activator to obtain a pathological myopia animal model.

[0007] Optionally, the negative lens induction is to fix a negative lens in front of the animal's eyes;

[0008] Optionally, the mTOR activator intervention includes administering the mTOR activator by intraperitoneal injection, intravenous injection, peribulbar injection, intravitreal injection, or eye drop administration;

[0009] Optionally, the mTOR activator intervention is intravitreal injection of the mTOR activator.

[0010] Optionally, the diopter of the negative lens is -10D.

[0011] Optionally, the material of the negative lens is polymethyl methacrylate, and the light transmittance is >90%.

[0012] Optionally, the diameter of the negative lens is 12.7 mm.

[0013] Optionally, the mTOR activator is a suspension containing 0.8 mg / ml of MHY1485;

[0014] Optionally, the suspension further contains a 10 mM phosphate buffer and 5 v / v% polysorbate 80, with a pH of 7.4; or

[0015] Optionally, the suspension further contains 0.9 wt% normal saline and 5 v / v% polysorbate 80.

[0016] Optionally, the animals are intervened with a combination of negative lens induction and mTOR activator intervention for 3 months; and / or

[0017] The animals induced with the negative lens are intervened for 24 hours per day; and / or

[0018] The animals intervened with the mTOR activator are injected with 5 μL per week.

[0019] Optionally, the animals are mammals;

[0020] Optionally, the animals are at least one of the animals in the order Rodentia;

[0021] Optionally, the animals are at least one of the animals in the family Cavidal;

[0022] Optionally, the animals are at least one of the animals in the genus Cavia;

[0023] Optionally, the animals are guinea pigs (Cavia porcellus);

[0024] Optionally, the guinea pigs (Cavia porcellus) are 3 - 4 weeks old;

[0025] Optionally, the pathological manifestations of the pathological myopia are tigroid fundus, diffuse and / or patchy choroidal atrophy around the optic disc.

[0026] A pathological myopia animal model constructed by the method for constructing a pathological myopia animal model as described above.

[0027] The pathological myopia animal model constructed by the method for constructing a pathological myopia animal model as described above has the following uses:

[0028] (1) Use in exploring the causes and mechanisms of the occurrence and development of pathological myopia;

[0029] (2) Use in screening diagnostic targets for pathological myopia;

[0030] (3) Use in screening therapeutic targets for pathological myopia;

[0031] (4) Use in screening diagnostic products for pathological myopia;

[0032] (5) Use in screening therapeutic products for pathological myopia.

[0033] The technical solution of the present invention has the following advantages:

[0034] 1. The method for constructing a pathological myopia animal model provided by the present invention includes: inducing with a negative lens in combination with intervention with an mTOR activator to obtain a pathological myopia animal model; the present invention's research finds that combining an mTOR activator based on the key regulatory pathway for myopic axial elongation with negative lens induction to intervene in animals can efficiently induce animals to develop pathological myopia. All animals showed significant tigroid fundus (pathological myopia in the M1 stage), and 25% of the guinea pigs showed diffuse and patchy choroidal atrophy characteristic of pathological myopia around the optic disc (pathological myopia in the M2 - M3 stage). This kind of pathological myopia animal model is easy to induce and highly similar to the fundus changes of human pathological myopia, can fill the gap in pathological myopia animal models, and can be used as a new animal model for exploring the causes and mechanisms of the occurrence and development of pathological myopia;

[0035] Furthermore, the method for constructing the animal model is simple, easy to operate, takes a short time, and has a high success rate.

[0036] 2. A method for constructing a pathological myopia animal model provided by the present invention, MHY1485 suspension: containing 0.8 mg / ml of MHY1485, a phosphate buffer solution with a concentration of 10 mM, 5% polysorbate 80, and a pH of 7.4; or containing 0.8 mg / ml of MHY1485, 0.9 wt% normal saline with a concentration of 0.9 wt%, 5% polysorbate 80, and a pH of 7.4; MHY1485 is a lipophilic compound that can penetrate the cell membrane and target the adenosine triphosphate (ATP) domain of mTOR, and specifically activate mTORC1. Since it is slightly soluble in water, in order to produce a long-lasting effect with a single injection, a combination of phosphate buffer (PBS) or 0.9% normal saline and 5% polysorbate 80 is used as a drug carrier to prepare a suspension containing 0.8 mg / ml of MHY1485. Among them, 5% polysorbate 80 can increase the viscosity of the solution, prevent MHY1485 from aggregating into lumps and precipitating in the solution, and at the same time can also significantly reduce the sedimentation rate of drug particles, finally forming a suspension that is stable for several weeks. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0038] Figure 1 It is the detection result of myopic eye axis in the experimental examples of the present invention; **** indicates P < 0.0001, with statistical differences;

[0039] Figure 2 It is the detection result of leopard pattern fundus in the embodiments of the present invention;

[0040] Figure 3 It is the detection result of diffuse and patchy choroidal atrophy around the optic disc in the embodiments of the present invention; among them, Figure a is a fundus color photograph, and Figure b is an optical coherence tomography centered on the optic disc. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0041] The following embodiments are provided to better further understand the present invention. They are not limited to the best embodiments, and do not limit the content and protection scope of the present invention. Any product obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior art features that is the same or similar to the present invention falls within the protection scope of the present invention.

[0042] For those embodiments where specific experimental procedures or conditions are not specified, the operations or conditions of the conventional experimental procedures described in the literature in this field can be followed. For reagents or instruments whose manufacturers are not specified, they are all conventional reagent products that can be obtained through commercial purchase.

[0043] The headgear involved in the following embodiments was self-made by conventional methods. For example, white medical tape was used to wrap the heads of guinea pigs and a negative lens was fixed in front of the eyes of the guinea pigs using a negative lens. Alternatively, the guinea pig headgear in the prior art can also be used, and there will be no obvious difference in the effect. For example, CN110123264A in Chinese patent literature, an adjustable glasses for guinea pigs based on 3D scanning and printing, etc.

[0044] The guinea pigs (Cavia porcellus) used had a body weight of 150 g and were 3 - 4 weeks old in terms of age.

[0045] MHY1485 is a commercially available product, CAS number: 326914 - 06 - 1.

[0046] MHY1485 suspension: containing 0.8 mg / ml of MHY1485, phosphate buffer (phosphate concentration 10 mM, PH = 7.4), 5 v / v% polysorbate 80.

[0047] Embodiment

[0048] This embodiment provides a method for constructing a pathological myopia animal model, including the following steps:

[0049] Use the headgear to fix a negative lens with a refractive power of -10 D (diopters, D) in front of the left and right eyes of the guinea pigs respectively. The material of the negative lens is polymethyl methacrylate (PMMA) material, with a diameter of 12.7 mm and a light transmittance of 92%. After fixing the negative lens, check whether the negative lens is clean and smooth every day. If the negative lens is contaminated or falls off, replace it with a new one.

[0050] Starting from the day when the guinea pigs start wearing the negative lens, inject 5 μL of MHY1485 suspension once a week, 5 μL each time. When injecting, remove the headgear and use the intravitreal injection method for injection. After injection, put on the headgear again. Through 3 months (12 weeks) of combined intervention of negative lens induction and intravitreal injection of MHY1485, a fundus model of typical pathological myopia can be induced.

[0051] Experimental Example

[0052] Using the pathological myopia animal model constructed from 20 guinea pigs according to the construction method in the embodiment, perform myopic eye axis detection (set a control group induced only by negative lenses, that is, do not inject the MHY1485 suspension during the induction process, and keep other conditions unchanged), leopard pattern fundus detection, peripapillary diffuse detection, and patchy choroidal atrophy detection.

[0053] Method for detecting myopic eye axis elongation: A-mode ophthalmic ultrasound (ultrasound frequency 11 MHz), see Dong L, Shi XH, Li YF, et al. Blockade of epidermal growth factor and its receptor and axial elongation in experimental myopia[J]. FASEB J, 2020, 34(10): 13654 - 13670.

[0054] Method for detecting leopard pattern fundus: fundus color photography, see Ohno-Matsui K, Kawasaki R, Jonas JB, et al. International photographic classification and grading system for myopic maculopathy[J]. Am J Ophthalmol, 2015, 159(5): 877 - 883e877.

[0055] Method for peripapillary diffuse detection: fundus color photography combined with optical coherence tomography, see Ohno-Matsui K, Kawasaki R, Jonas JB, et al. International photographic classification and grading system for myopic maculopath[J]. Am J Ophthalmol, 2015, 159(5): 877 - 883e877.

[0056] Method for patchy choroidal atrophy detection: fundus color photography combined with optical coherence tomography, see Ohno-Matsui K, Kawasaki R, Jonas JB, et al. International photographic classification and grading system for myopic maculopathy[J]. Am J Ophthalmol, 2015, 159(5): 877 - 883e877.

[0057] The detection results of myopic axial length are as follows Figure 1 shown in the figure. In the figure, NLIAE represents only negative lens induction, and NLIAE+MHY1485 represents the combined intervention of negative lens induction and intravitreal injection of MHY1485. Independent t-tests and rank sum tests were used for comparison between groups. During the 3-month induction process, this combined induction protocol could significantly accelerate the elongation of myopic axial length. Compared with simple negative lens induction, the combined induction in guinea pigs could additionally increase the axial length elongation by 0.43 mm (this result corresponds to the result after 12 weeks).

[0058] The detection results of leopard pattern fundus showed that all guinea pig models constructed by the combined induction protocol of Example 1 exhibited significant leopard pattern fundus (pathological myopia at stage M1). For example, the pathological fundus picture of leopard pattern of one guinea pig model is as follows Figure 2 shown

[0059] The detection results of peripapillary diffuse showed that among all guinea pig models constructed by the combined induction protocol of Example 1, 25% of the guinea pig models exhibited peripapillary diffuse and patchy choroidal atrophy, which are characteristic of pathological myopia (pathological myopia at stages M2-M3). For example, the pathological picture of peripapillary diffuse and patchy choroidal atrophy of one guinea pig model is as follows Figure 3 shown. In Figure a, the leopard pattern fundus of pathological myopia and the peripapillary yellowish-white diffuse atrophy lesions can be seen. In Figure b, the choroid at the corresponding position of the lesion becomes thinner and almost disappears

[0060] Obviously, the above examples are only for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention

Claims

1. A method for constructing an animal model of pathological myopia, characterized in that, Including: Using a negative lens induction combined with an mTOR activator to intervene in animals to obtain a pathological myopia animal model; The mTOR activator is a suspension containing 0.8 mg / ml of MHY1485; The suspension also contains a 10 mM phosphate buffer and 5 v / v% polysorbate 80, with a pH of 7.4; or The suspension also contains 0.9 wt% normal saline and 5 v / v% polysorbate 80; Using a negative lens induction and an mTOR activator intervention to jointly intervene in animals for 3 months; Using a negative lens to induce animals with 24-hour intervention per day; Using the mTOR activator to intervene in animals by injecting 5 μL per week.

2. The method for constructing a pathological myopia animal model according to claim 1, wherein The negative lens induction is to fix a negative lens in front of the animal's eyes.

3. The method for constructing a pathological myopia animal model according to claim 1, wherein The mTOR activator intervention includes administering the mTOR activator by intraperitoneal injection, intravenous injection, peribulbar injection, intravitreal injection, or eye drop administration.

4. The method for constructing a pathological myopia animal model according to claim 3, wherein The mTOR activator intervention is intravitreal injection of the mTOR activator.

5. The method for constructing a pathological myopia animal model according to any one of claims 1-4, characterized in that, The diopter of the negative lens is -10D.

6. The method for constructing a pathological myopia animal model according to any one of claims 1-4, characterized in that, The material of the negative lens is polymethyl methacrylate, and the light transmittance is >90%.

7. The method for constructing a pathological myopia animal model according to any one of claims 1-4, characterized in that, The diameter of the negative lens is 12.7 mm.

8. The method for constructing a pathological myopia animal model according to any one of claims 1-4, characterized in that, The animal is a guinea pig ( Cavia porcellus ).

9. The method for constructing a pathological myopia animal model according to claim 8, wherein The guinea pigs ( Cavia porcellus ) are 3-4 weeks old.

10. The method for constructing a pathological myopia animal model according to any one of claims 1-4, wherein The pathological manifestations of the pathological myopia are leopard pattern fundus, diffuse and / or patchy choroidal atrophy around the optic disc.

11. The pathological myopia animal model constructed by the method for constructing a pathological myopia animal model according to any one of claims 1-10 has the following uses: (1) For exploring the causes and mechanisms of the occurrence and development of pathological myopia; (2) For screening diagnostic targets for pathological myopia; (3) For screening therapeutic targets for pathological myopia; (4) For screening diagnostic products for pathological myopia; (5) For screening therapeutic products for pathological myopia.

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