A method for constructing a choroidal melanin in situ tumor animal model

Abstract

The application discloses a construction method of a choroid melanin in-situ tumor animal model, and belongs to the technical field of biological medicine. The construction method comprises the following steps: providing tumor cells, wherein the tumor cells are mouse melanoma B16 cells; mixing luciferase-labeled tumor cells and biological gold medal matrix glue at 0-4 DEG C, and then injecting the mixture into the choroid layer through the suprachoroidal space of a mouse, so that the choroid melanin in-situ tumor animal model is obtained. The biological gold medal matrix glue is used as a carrier, the adhesion of the matrix glue and the cells is strong, and the cells can be uniformly dispersed in the matrix glue, so that the cells can be uniformly dispersed in the matrix glue. The matrix glue can be gelled in 1-3 minutes at room temperature, and can be rapidly solidified at 37 DEG C, so that the cells can be fixed in the choroid layer, and the real anatomical site of human choroid melanoma occurrence can be simulated. In addition, the matrix glue can provide a good nutrient environment for tumor growth, so that a single solid tumor can be formed by a tumor cell line in a short time.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for constructing an animal model of choroidal melanoma in situ. Background Technology

[0002] Choroidal melanoma is the most common malignant tumor of the eye in adults, and it is prone to metastasis. After metastasis, the survival rate of patients is less than 9 months in 50% of cases. Choroidal melanoma occurring in the eye can not only cause local exudation in the eyeball or retinal detachment due to tumor growth pushing against the anterior retina, but it can also directly invade important intraocular tissues such as the retina and optic nerve, causing vision loss. More importantly, it can metastasize to critical organs throughout the body, such as the liver and lungs, through the bloodstream, leading to death and posing a great threat to patients. Although the clinical diagnostic level of uveal melanoma has greatly improved, a significant number of patients have already developed systemic metastases before melanoma is diagnosed. Treatment for uveal melanoma currently includes various local treatment methods such as transpupil hyperthermia, charged particle radiotherapy, 106Ru / 106Rh extrascleral radiotherapy, and various novel chemotherapy and immunotherapies. However, multicenter clinical case-control studies have found that these treatment methods do not show an advantage in reducing patient mortality compared to traditional enucleation. The underlying mechanisms of uveal melanoma development and metastasis remain incompletely understood. The key to resolving this issue lies in establishing an ideal animal model that accurately simulates the growth, invasion, and metastasis of human uveal melanoma. This is a necessary foundation and prerequisite for conducting uveal melanoma research.

[0003] Some researchers have injected cultured B16 tumor cells into the ophthalmic artery, allowing them to reach the eyes of C57BL / 6J mice via the bloodstream. Tumor formation in the choroid and ciliary body was observed, but the tumors appeared scattered in multiple locations within the eye, failing to fully and realistically simulate the development of choroidal melanoma. Furthermore, this method is technically challenging and time-consuming to establish. To explore an animal model more closely resembling human choroidal melanoma, a choroidal melanoma in situ animal model can be constructed by injecting mouse melanoma B16 cell lines into the eyes of mice. Currently, the main methods for injecting cell lines into the eye include anterior chamber injection, injection into the vitreous cavity via the ciliary body, and injection into the choroidal layer via the suprachoroidal space (e.g., injection into the choroidal layer). Figure 1 (As shown).

[0004] Primary choroidal melanoma originates in the choroid, and establishing an in situ tumor model requires mimicking the human disease as closely as possible, and its anatomy must closely resemble that of the human eye. In models constructed by injecting tumor cells into the anterior chamber, the choroidal melanoma is located in the anterior chamber; in models constructed by injecting cells into the vitreous cavity, the choroidal melanoma is located within the vitreous cavity or on the retinal surface. Anatomically, neither of these models can completely and realistically simulate human choroidal melanoma. Because the anterior chamber and vitreous cavity lack blood vessels, drug metabolism and efficacy depend on hemodynamics; therefore, neither of these models is suitable for drug treatment research on choroidal melanoma. Therefore, theoretically, injecting tumor cell lines into the suprachoroidal space holds more promise for constructing a model that closely resembles the human choroidal melanoma disease.

[0005] The choroid lies between the retina and sclera, where the two are tightly adhered. Choroidal melanoma originates in the choroid. When tumor cell lines are injected subretinally via the suprachoroidal space, the pressure of the intraocular contents can cause the injected tumor cells to easily spread into the vitreous cavity, forming tumors of varying sizes (e.g., ...). Figure 2 (As shown in the image) Even when tumor cell lines are injected into the choroidal layer, intraocular pressure still causes the formation of scattered, unevenly sized tumor masses within the choroid. These tumor models do not meet the anatomical requirements of in situ tumor models, and the scattered, multiple tumors are not conducive to efficacy evaluation.

[0006] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the purpose of this invention is to provide a method for constructing an animal model of choroidal melanoma in situ, which aims to solve the problem that the existing models cannot completely and realistically simulate human choroidal melanoma and are not suitable for drug treatment research of this disease.

[0008] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for constructing an animal model of choroidal melanoma in situ, comprising the steps of: Provide tumor cells, wherein the tumor cells are mouse melanoma B16 cells; After mixing luciferase-labeled tumor cells with bio-gold matrix gel at 0-4°C, the mixture was injected into the choroidal layer of mice via the suprachoroidal space to obtain the choroidal melanoma in situ animal model.

[0009] Optionally, luciferase-labeled tumor cells are mixed with a suspension of bio-gold matrix gel, the density of which is 5 × 10⁻⁶. 4 -15×10 4 pcs / μL; The luciferase-labeled tumor cell suspension was mixed with BioGold Matrix Gel at a volume ratio of 1:1 to 1:1.5.

[0010] Alternatively, the luciferase-labeled tumor cell suspension can be mixed with BioGold matrix gel at a volume ratio of 1:1.

[0011] Optionally, the injection volume is 4-7 μL.

[0012] Optionally, the injection is performed using a 33G ophthalmic needle.

[0013] Optionally, the injection is performed by inserting the needle perpendicular to the limbus to enter the suprachoroidal space.

[0014] Optionally, the mice are C57BL / 6, 6-8 weeks old, female, and weigh 16-22g.

[0015] In a second aspect, the present invention provides an application of an animal model of choroidal melanoma obtained by the construction method described herein in the study of the pathogenesis of choroidal melanoma.

[0016] A third aspect of the present invention provides the application of an animal model of choroidal melanoma obtained by the construction method described in the present invention in screening therapeutic drugs for choroidal melanoma.

[0017] Beneficial Effects: This invention provides a method for constructing an animal model of choroidal melanoma in situ. The method includes the following steps: providing tumor cells, specifically mouse melanoma B16 cells; mixing luciferase-labeled tumor cells with a bio-based matrix gel at 0-4°C; and injecting the mixture into the choroidal layer via the suprachoroidal space of a mouse to obtain the choroidal melanoma in situ animal model. This invention uses a bio-based matrix gel as a carrier, which exhibits strong adhesion to cells, helping to ensure uniform cell dispersion within the matrix gel. The matrix gel gels within 1-3 minutes at room temperature and rapidly solidifies at 37°C, fixing cells to the choroidal layer, thereby simulating the actual anatomical site of human choroidal melanoma. Furthermore, the matrix gel provides a favorable nutritional environment for tumor growth, enabling tumor cell lines to form single solid tumors in a short time. The successful construction of this choroidal melanoma in situ animal model contributes to a deeper understanding of the disease's pathogenesis and provides strong support for exploring effective treatment options. Attached Figure Description

[0018] Figure 1 The three most commonly used injection methods for constructing an animal model of choroidal melanoma in situ are: orange represents anterior chamber injection, blue represents intravitreal injection, and green represents suprachoroidal injection.

[0019] Figure 2 The in situ tumors formed by injecting mouse melanoma B16 cells into the eye via the suprachoroidal space were described. Fundus photography results were obtained 2 weeks after injection: the black ovals represent tumors of varying sizes that are scattered; among them, A represents tumors in the peripapillary region, and B represents tumors of varying sizes on the peripheral retinal surface.

[0020] Figure 3 A schematic diagram showing the luciferase-labeled tumor cells mixed with bio-gold matrix gel and injected into the choroidal layer via the suprachoroidal space.

[0021] Figure 4 The results show the observation of tumor growth using a slit lamp and mouse retinal imaging system. A represents fundus photography taken 1 week before injection using a mouse retinal imaging system; B represents fundus photography taken 3 days after injection using a mouse retinal imaging system; C represents fundus photography observed in the pupillary area under a slit lamp 2 weeks after injection; D represents fundus photography taken 2 weeks after injection using a mouse retinal imaging system; E represents optical coherence tomography (OCT) 2 weeks after injection; F represents in vivo imaging system (IVIS) 2 weeks after injection; and G represents the appearance of the eye 2 months after injection.

[0022] Figure 5 The results are shown in the fundus photography 4 weeks after the injection.

[0023] Figure 6 The results of optical coherence tomography (OCT) of the Enface layer are shown 4 weeks after injection.

[0024] Figure 7 This is the result of optical coherence tomography (OCT) scan 4 weeks after injection, with blue arrows indicating tumors.

[0025] Figure 8 Results of small animal in vivo imaging system (IVIS) 4 weeks after injection.

[0026] Figure 9 HE staining results of enucleated eyes 2 months after injection; A is the control eye and B is choroidal melanoma. Detailed Implementation

[0027] This invention provides a method for constructing an animal model of choroidal melanoma in situ. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0028] This invention provides a method for constructing an animal model of choroidal melanoma in situ: tumor cells are injected into the suprachoroidal space, so that the tumor cells are localized only in the choroidal layer and form a single solid tumor therein, while avoiding the spread of tumor cells to the vitreous body.

[0029] To ensure that tumor cells injected via the suprachoroidal space are entirely localized to the choroid and form a single solid tumor, while preventing tumor cell dissemination to the vitreous body, anterior chamber, and other sites, a novel material is selected as the carrier. This carrier must possess the following characteristics: the matrix gel can gel within 1-3 minutes at room temperature, rapidly solidify at 37°C, remain liquid at 0-4°C (facilitating mixing with tumor cells and ensuring uniform distribution), and rapidly solidify below 0°C. Based on these characteristics, this invention preferentially uses BioGold brand matrix gel as the carrier.

[0030] BioGold Medal Matrix is ​​a natural basement membrane matrix extracted from mouse tumors rich in extracellular matrix proteins. Its main components include laminin, type IV collagen (Col-IV), Entactin, heparan sulfate proteoglycans, and various cytokines such as insulin-like growth factor-1 (IGF-1), transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and fibroblast growth factor (bFGF). BioGold Medal Matrix gels within 1-3 minutes at room temperature and solidifies rapidly at 37°C, serving not only as an excellent carrier for tumor cells but also providing good nutritional support for tumor growth.

[0031] Based on this, embodiments of the present invention provide a method for constructing an animal model of choroidal melanoma in situ, comprising the following steps: Provide tumor cells, wherein the tumor cells are mouse melanoma B16 cells; After mixing luciferase-labeled tumor cells with bio-gold matrix gel at 0-4°C, the mixture was injected into the choroidal layer of mice via the suprachoroidal space to obtain the choroidal melanoma in situ animal model.

[0032] When using bio-based matrix gel (hereinafter referred to as matrix gel) as a carrier, it exhibits strong adhesion to tumor cells (hereinafter referred to as cells), helping to ensure uniform cell distribution within the matrix gel. Matrix gel gels within 1-3 minutes at room temperature and rapidly solidifies at 37°C, fixing cells to the choroidal layer, thus simulating the actual anatomical location of human choroidal melanoma. Furthermore, matrix gel provides a favorable nutritional environment for tumor cell growth, enabling tumor cell lines to form single solid tumors in a relatively short time. In contrast, if phosphate-buffered saline (PBS) is used as a cell carrier and injected intraocularly via the suprachoroidal space, tumor cells easily disseminate into the vitreous cavity, forming scattered tumors located on the retinal surface, failing to fully simulate the tumor development and metastasis process. The establishment of tumor models is crucial for scientific research. Model construction should closely resemble anatomical structures to simulate the disease's development and metastasis, facilitating deeper research into the pathogenesis of diseases and the exploration of effective treatment options.

[0033] With the development of science and technology, optical coherence tomography (OCT) navigation technology under a microscope can better and more accurately locate the anatomical parts of the eyeball and implant tumor cells into the choroidal layer.

[0034] In one embodiment, luciferase-labeled tumor cells are mixed with a BioGold matrix gel in suspension (suspended in PBS), and the density of the luciferase-labeled tumor cell suspension is 5 × 10⁻⁶. 4 -15×10 4 pcs / μL; Mix the luciferase-labeled tumor cell suspension with BioGold Matrix Gel at a volume ratio of 1:1 to 1:1.5 (e.g., 1:1).

[0035] In one embodiment, the injection volume is 4-7 μL (e.g., 7 μL).

[0036] In one embodiment, the mice are C57BL / 6, 6-8 weeks old, female, and weigh 16-22g.

[0037] In one embodiment, the injection is performed using a 33G ophthalmic needle.

[0038] In one embodiment, the injection is made by inserting the needle perpendicular to the limbus to enter the suprachoroidal space.

[0039] This invention provides an application of the choroidal melanoma in situ animal model obtained by the construction method described above in the study of the pathogenesis of choroidal melanoma.

[0040] This invention provides an application of an animal model of choroidal melanoma obtained by the construction method described above in screening therapeutic drugs for choroidal melanoma, providing strong evidence for drug treatment of human choroidal melanoma.

[0041] The present invention will be further described below through specific embodiments.

[0042] Example 1 I. Construction of luciferase-labeled tumor cells (where the tumor cells are mouse melanoma B16 cells), the specific steps are as follows: 1. Required materials 1) B16F10 cells; B16F10 cells are a subclone of mouse melanoma B16 cells and are widely used in tumor immunology, metastasis models, and anti-tumor drug research.

[0043] 2) GV826 lentiviral vector Using the Shanghai Jikai Gene GV826 lentiviral vector, the firefly luciferase gene was integrated into the B16F10 cell genome to construct a cell line that stably expresses luciferase for in vivo imaging (IVIS) to track tumor growth and metastasis.

[0044] The basic parameters of the GV826 lentivirus vector are shown in Table 1 below: Table 1. Basic parameters of the GV826 lentivirus vector

[0045] GV826 is a dual-promoter expression vector containing two independent expression cassettes: Expression Box 1 (EF1 Promoter Driver): P EF1 → Luciferase → T2A → Puro

[0046] Firefly luciferase expression is driven by the EF1 promoter, and the T2A self-cleaving peptide opens the purine resistance gene. Co-expression of luciferase enables stable expression of luciferase and resistance screening. Expression Box 2 (CMV Starter Driver): P CMV → MCS (XhoI / BamHI) → 3×FLAG The expression of the target gene at the multiple cloning site (MCS) is driven by the CMV promoter, with a 3×FLAG tag at the C-terminus. In this experiment, this site can be left empty or a different target gene can be inserted.

[0047] The carrier elements are shown in Table 2 below: Table 2. Carrier Components

[0048] The identification primers are shown in Table 3 below: Table 3. Identification Primers

[0049] 2. Required reagents and equipment The required reagents and equipment are shown in Table 4 below: Table 4. Required Reagents and Equipment

[0050] 3. Construction Steps Phase 1: Preliminary Experiments (MOI Optimization and Kill Curves) Objective: To determine the optimal multiple of infection (MOI) and the minimum lethal concentration of puromycin.

[0051] MOI optimization experiment: B16F10 cells were seeded in 24-well plates and infected with different MOI gradients (MOI = 1, 5, 10, 20, 50). Cell status was observed and luciferase activity was measured after 48-72 h. The MOI value with infection efficiency ≥80% and low cytotoxicity was selected. The recommended MOI range for B16F10 is 10-30.

[0052] Ganoderma-2 killing curve: B16F10 cells were seeded in 24-well plates, and different concentrations of puromycin (0, 0.5, 1.0, 1.5, 2.0, 3.0, 5.0 μg / mL) were added. The selection medium was changed every 24 h, and the lowest concentration at which all cells died within 3-5 days was observed. Experiments confirmed that the optimal selection concentration of puromycin for B16F10 cells was 2 μg / mL.

[0053] Phase 2: GV826-Luc lentivirus infection of B16F10 cells Objective: To infect B16F10 cells with GV826-Luc lentivirus and integrate the Luciferase gene into the cell genome. Day 1: Cell Plating Digest logarithmic phase B16F10 cells at 1×10⁻⁶ 5 Cells were seeded at a density of 500 μL of complete culture medium per well in 24-well plates. A control group (uninfected cells) was set up. Cells were cultured overnight to allow confluence to reach 30%-50%.

[0054] 2. Day 2: Viral Infection ① Remove the GV826-Luc lentivirus from -80°C, thaw it quickly on ice, and gently mix it. Avoid repeated freeze-thaw cycles.

[0055] ② Calculate the required viral load based on the MOI value determined in the preliminary experiment: Viral volume (μL) = MOI × Cell number ÷ Viral titer (TU / mL) × 10 6 .

[0056] ③ Remove the original culture medium and add 300 μL of fresh culture medium containing the optimal MOI viral load.

[0057] ④ Add HitransGP infection enhancement solution (according to the recommended dosage in the instructions) and gently shake the plate. Optional: Add Polybrene to a final concentration of 6-8 μg / mL.

[0058] ⑤ Incubate overnight (12-24 h) at 37°C and 5% CO2.

[0059] 3. Day 3: Change the culture medium Aspirate the culture medium containing lentivirus, gently wash once with PBS, and add 500 μL of fresh complete culture medium. Continue culturing for 24–48 h.

[0060] Phase 3: Screening for Gastromycin Resistance Objective: To screen B16F10 cell clones stably integrating GV826-Luc using gaccharin. The Luciferase and Puro enzyme in the GV826 vector... Since T2A peptide is co-expressed in equal amounts, puromycin resistance can be used as an indirect indicator of Luc expression.

[0061] 1). Day 4: Start filtering Forty-eight hours post-infection, the culture medium was replaced with complete medium containing puromycin (2 μg / mL). Simultaneously, the control group cells were observed to determine if cell death had begun.

[0062] 2). Days 4-7: Continuous screening Replace the culture medium with fresh puromycin (2 μg / mL) every 24-48 hours. Observe cell status: all uninfected control cells should die within 3-5 days, while resistant clones should gradually appear in the infected group.

[0063] 3). Days 8-12: Expanded culture Once the resistant cell clones have reached confluence, they are progressively transferred from 24-well plates to 6-well plates, then to 6 cm plates, and finally to 10 cm plates. The screening concentration can be halved to 1 μg / mL (maintenance concentration) for subsequent culture.

[0064] Phase 4: Cell line identification Objective: To verify the luciferase expression activity and stability of the B16F10-Luc cell line. ① Luciferase activity assay (in vitro) B16F10 cells and B16F10-Luc cells were digested and centrifuged, and the supernatant was discarded. B16F10 cells served as the control group, and B16F10-Luc cells served as the experimental group. Cell counts showed that the cell densities in both groups were 8.23 ​​× 10⁻⁶. 9 8.19 × 10⁻⁶ cells / μL and 8.19 × 10� 9 Cells / μL. Then, 200 μL of each of the two cell lines was taken, and 100 μL of luciferase substrate was added to detect their luminescence. The results are shown in Table 5 below: Table 5. Luminescence Results

[0065] The results showed that the experimental group cells produced a significant luminescence signal after the addition of the luciferase substrate, while no significant luminescence was detected in the control group, and the luminescence value of the experimental group was significantly higher than that of the control group. This result confirms that the B16F10-Luc cell line can stably express luciferase.

[0066] ② In vivo imaging verification B16F10-Luc cells were subcutaneously seeded into C57BL / 6 mice (e.g., 1×10⁻⁶ cells). 5 -5×10 5 One week later, D-Luciferin (150 mg / kg) was injected intraperitoneally, and the tumor luminescence signal was observed using an IVIS in vivo imaging system 10-15 minutes later.

[0067] Imaging results showed that the inoculation site emitted a significant fluorescent signal, while the control mice without cell inoculation did not show significant fluorescence, indicating that B16F10-Luc cells can still be detected with fluorescent signals in vivo. This result further validates the suitability of this cell line for in vivo tracking and dynamic monitoring of tumor growth.

[0068] ③ Stability verification After 10 or more generations of continuous passage, luciferase activity was measured every 5-10 generations to confirm stable expression of the Luciferase gene.

[0069] The test results showed that the luminescence intensity of cells in each generation did not decrease significantly, and the luminescence value remained within a stable range, indicating that the Luciferase gene was stably expressed.

[0070] Phase 5: Cell cryopreservation and preparation ① Take B16F10-Luc cells in the logarithmic growth phase, digest and count them.

[0071] ② Collect cells by centrifugation, resuspend in cryopreservation buffer (90% FBS + 10% DMSO), and adjust the cell density to 1-5 × 10⁻⁵.6 cells / mL.

[0072] ③ Dispense 1 mL / tube into cryovials, place them in a programmed cooling freezer, transition to -80°C, and then transfer to liquid nitrogen for long-term storage. ④ Freeze multiple vials from the same batch to avoid repeated thawing that could lead to fluorescence signal attenuation.

[0073] II. A method for constructing an animal model of choroidal melanoma in situ. 1. Description of animal and cell origins Animals: 8 female C57BL / 6 mice, aged 6-8 weeks, weighing approximately 16-22g, purchased from Zhuhai Baishitong Animal Co., Ltd. Quality attributes: The purchased batch tested negative for mycoplasma contamination and sterility.

[0074] Cells: Luciferase-labeled mouse melanoma B16 cells (i.e., B16F10-Luc cells) constructed using the above method.

[0075] Instruments, reagents and consumables: 1.5mL EP tubes, DMEM culture medium, Gibco fetal bovine serum, optical inverted microscope and photography system, surgical microscope, 37℃ 5%CO2 cell culture incubator, electronic balance, biosafety cabinet, sodium pentobarbital, promecaine hydrochloride eye drops, compound tropicamide eye drops, ofloxacin eye drops, ofloxacin eye ointment, 33G ophthalmic needles, insulin injections, mouse warming pads, fluorescence microscope, slit lamp, mouse retinal imaging system (IISCIENCE Eyemerafundus and IISCIENCE EyemeraOCT from South Korea) and small animal in vivo imaging system.

[0076] 2. Construction Steps (1) Sodium pentobarbital was used to anesthetize mice. 1-2 drops of mydriatic drug, promecaine hydrochloride eye drops were used for local surface anesthesia, and ofloxacin eye drops were used to prevent infection.

[0077] (2) Hold the eyelids open with your hand and gently press them to fully expose the cornea and sclera of the mouse's eye for easy injection, and operate under a surgical microscope. Maintain the protruding eye position until the injection is complete. Place your fingers on the outer side of the orbital rim to stabilize it and prevent needle displacement during injection. Expose the upper nasal region of the eye.

[0078] (3) The luciferase-labeled tumor cells (i.e., B16F10-Luc cells) were resuspended in PBS to obtain a density of approximately 8 × 10⁻⁶. 4A suspension of tumor cells / μL was prepared and mixed with BioGold Methyl Gel (purchased from Xiamen Moji Biotechnology Co., Ltd., catalog number 082745) at a 1:1 volume ratio on ice. Approximately 10 μL of the mixture was aspirated using a 33G ophthalmic needle. An insulin needle was used to puncture the limbus in the superior nasal region of the eyeball. A small amount of aqueous humor was observed to flow out after puncture, taking care to avoid vitreous herniation and contact with ocular muscles and blood vessels, while also avoiding damage to the lens and retina. Then, the 33G ophthalmic needle was vertically inserted into the limbus and injected into the choroidal layer, with an injection volume of approximately 7 μL (see [link to article]). Figure 3 Avoid contact with the lens and intraocular tissues. Gently press the injection site with a cotton swab for 3 minutes.

[0079] (4) After the suprachoroidal injection is completed, ofloxacin eye ointment is applied to the eyes, and the mice are kept warm on a warming pad during this period. After the operation is completed, the mice are placed in an incubator until they are fully awake. After confirming that the mice are fully awake, they are returned to their cages.

[0080] (5) Observe the mice daily after injection, including: eye appearance, fur, physical signs, behavior, glandular secretion, diet, heart rate, respiration, fecal characteristics, and mortality. Weigh the mice twice a week. Weigh them once before modeling; after modeling, perform slit-lamp examination, small animal in vivo systemic examination, fundus examination and optical coherence tomography (OCT) using a mouse retinal imaging system approximately once a week. Perform gross necropsy and enucleate the eyeballs after 2 months, and observe the tumor after hematoxylin-eosin (HE) staining.

[0081] Two months later, after removing the mouse's eyeballs, tissue sections were prepared. The sections were then embedded in paraffin and stained with hematoxylin and eosin (HE). The specific steps are as follows: 1. Dewaxing of paraffin sections: Place the sections in the following order: xylene I for 10 minutes → xylene II for 10 minutes → xylene III for 10 minutes → anhydrous ethanol I for 5 minutes → anhydrous ethanol II for 5 minutes → 95% alcohol for 5 minutes → 90% alcohol for 5 minutes → 80% alcohol for 5 minutes → 70% alcohol for 5 minutes → tap water for 1 minute; 2. Staining steps: Stain with hematoxylin for 1-5 minutes and then wash with water; differentiate with 1% hydrochloric acid alcohol for 3 seconds and then wash with water; react with 0.6% ammonia water for 1 minute and then wash with water; soak in 95% ethanol for 30 seconds; stain with eosin for 1-5 minutes and then wash with water. 3. Dehydration and clearing: Immerse the slices in the following order: 85% alcohol for 5 seconds → 95% alcohol for 5 seconds → anhydrous ethanol I for 5 seconds → anhydrous ethanol II for 30 seconds → xylene I for 30 seconds → xylene II for 1 minute → xylene III for 3 minutes; 4. Mounting: After removing the slides from the xylene and allowing them to dry slightly, mount them using a super-clean adhesive. See the table below for details of the reagents used.

[0082] In this context, I, II, and III represent the order of use of xylene, corresponding to the first, second, and third cylinders, respectively; I and II represent the order of use of anhydrous ethanol, corresponding to the first and second cylinders, respectively.

[0083] The reagents used in the pathological procedures are shown in Table 6 below: Table 6. Reagents Used

[0084] The results of slit-lamp and mouse retinal imaging observation of tumor growth are as follows: Tumors in all 8 mice grew normally, with a tumor formation rate of 100%; ocular observation results are shown below. Figure 4 ,in, A shows the results of fundus photography taken by a mouse retinal imaging device one week before injection. The eyeball is clear and transparent, the retinal blood vessels are uniform and clear, and no tumor formation is observed. B shows the results of fundus photography taken by a mouse retinal imaging device 3 days after injection. It can be seen that the course and distribution of retinal blood vessels in the fundus are basically normal, and no tumor formation has been observed. C shows the fundus findings observed under a slit lamp in the pupillary area 2 weeks after injection: a fundus tumor is faintly visible in the lower nasal region of the pupillary area, located in the choroidal layer, occupying about 1 / 4 of the lower nasal quadrant; D shows the results of fundus photography taken by a mouse retinal imaging device 2 weeks after injection, which shows a well-defined tumor located in the choroid layer in about 1 / 4 of the lower nasal quadrant of the retina. E shows the optical coherence tomography (OCT) results 2 weeks after injection: the white circles in the Enface layer represent the size of the tumor. F represents the results of the small animal in vivo imaging system (IVIS) 2 weeks after injection, with red indicating the formation of a single, complete tumor; G represents the result two months after injection, where the tumor protrudes outside the orbit, and the normal anatomical structure of the eyeball is no longer discernible.

[0085] Figure 5 The results of fundus photography 4 weeks after injection show that the tumor occupies about 1 / 2 of the lower nasal quadrant of the retina and has increased significantly in size.

[0086] Figure 6 Optical coherence tomography (OCT) results 4 weeks after injection: Enface layer shows tumor enlargement.

[0087] Figure 7 This is the result of optical coherence tomography (OCT) scan 4 weeks after injection, with blue arrows indicating the location of the tumor and the OCT image showing an upward arch.

[0088] Figure 8 The results of the small animal in vivo imaging system (IVIS) 4 weeks after injection show enhanced fluorescence and increased size of the tumor.

[0089] Fluorescence imaging was used to monitor changes in tumor flux, and the results are shown in Table 7 below: Table 7. Flux Changes

[0090] Quantitative assessment of tumor growth using fluorescence imaging showed that after B16F10-Luc cell transplantation, the tumor gradually increased in size and fluorescence intensity over time, which was consistent with the tumor growth observed by slit lamp and mouse retinal imaging.

[0091] Figure 9 HE staining results of enucleated eyes 2 months after injection; A is the control eye, and B is choroidal melanoma, with pathological observation indicating choroidal melanoma.

[0092] This demonstrates that the mouse choroidal melanoma model has been successfully established. The tumor is located in the choroidal layer and is a solid, single tumor, which is beneficial for conducting scientific research.

[0093] In summary, this invention provides a method for constructing an animal model of choroidal melanoma in situ. The method includes the following steps: providing tumor cells, specifically mouse melanoma B16 cells; mixing luciferase-labeled tumor cells with a bio-based matrix gel at 0-4°C; and injecting the mixture into the choroidal layer via the suprachoroidal space of a mouse to obtain the choroidal melanoma in situ animal model. This invention uses a bio-based matrix gel as a carrier, which exhibits strong adhesion to cells, helping to ensure uniform cell dispersion within the gel. The matrix gel gels within 1-3 minutes at room temperature and rapidly solidifies at 37°C, fixing cells to the choroidal layer, thus simulating the actual anatomical location of human choroidal melanoma. Furthermore, the matrix gel provides a favorable nutritional environment for tumor growth, enabling tumor cell lines to form single solid tumors in a short time. The successful construction of this choroidal melanoma in situ animal model contributes to a deeper understanding of the disease's pathogenesis and provides strong support for exploring effective treatment options.

[0094] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A method for constructing an animal model of choroidal melanoma in situ, characterized in that, Including the following steps: Provide tumor cells, wherein the tumor cells are mouse melanoma B16 cells; After mixing luciferase-labeled tumor cells with bio-gold matrix gel at 0-4°C, the mixture was injected into the choroidal layer of mice via the suprachoroidal space to obtain the choroidal melanoma in situ animal model.

2. The method for constructing an animal model of choroidal melanoma in situ according to claim 1, characterized in that, Luciferase-labeled tumor cells were mixed with BioGold Matrix gel in suspension form. The density of the luciferase-labeled tumor cell suspension was 5 × 10⁻⁶. 4 -15×10 4 pcs / μL; The luciferase-labeled tumor cell suspension was mixed with BioGold Matrix Gel at a volume ratio of 1:1 to 1:1.

5.

3. The method for constructing an animal model of choroidal melanoma in situ according to claim 2, characterized in that, The luciferase-labeled tumor cell suspension was mixed with BioGold Matrix Gel at a volume ratio of 1:

1.

4. The method for constructing an animal model of choroidal melanoma in situ according to claim 1, characterized in that, The injection volume is 4-7 μL.

5. The method for constructing an animal model of choroidal melanoma in situ according to claim 1, characterized in that, The injection was performed using a 33G ophthalmic needle.

6. The method for constructing an animal model of choroidal melanoma in situ according to claim 1, characterized in that, The injection is performed by inserting the needle perpendicular to the limbus to enter the suprachoroidal space.

7. The method for constructing an animal model of choroidal melanoma in situ according to claim 1, characterized in that, The mice were C57BL / 6, 6-8 weeks old, female, and weighed 16-22g.

8. The application of an animal model of choroidal melanoma obtained by the construction method according to any one of claims 1-7 in the study of the pathogenesis of choroidal melanoma.

9. The use of an animal model of choroidal melanoma obtained by the construction method according to any one of claims 1-7 in screening therapeutic drugs for choroidal melanoma.

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