Construction method of retinitis pigmentosa model and kit

Abstract

The invention provides a construction method of a retinitis pigmentosa model and a kit, and relates to the technical field of biology. The method of construction comprises administering a disulfiram-copper ion complex to a subject. According to the construction method, at least one defect that in the prior art, a retinitis pigmentosa animal model is short in model research window period, unstable, complex in construction method and high in cost is overcome.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for constructing a retinitis pigmentosa model and a kit. Background Art

[0002] The following statements merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] Retinitis pigmentosa (RP) is a common hereditary eye disease characterized by a progressive loss of photoreceptor cell and pigment epithelial function. RP is a leading cause of blindness and can be inherited in X-linked, autosomal recessive, or dominant ways, or sporadically. RP is primarily characterized by rod degeneration, followed by cone loss. The initial symptom is decreased night vision, followed by a gradual loss of concentric visual field. Fundus abnormalities typically manifest as a triad of osteocyte-like pigmentation, retinal vascular constriction, and waxy optic disc pallor. To date, multiple gene mutations have been found to cause retinitis pigmentosa.

[0004] Effective models can simulate the disease and assist in studying the pathogenesis of retinitis pigmentosa, as well as conducting drug screening and evaluation. Currently available retinitis pigmentosa models include the following: 1. RD1 and RD10 mouse models: These two models are currently the most commonly used for RP research. Mutations in the phosphodiesterase subunit B (PDE6B) gene in these mice lead to retinal photoreceptor cell death, ultimately causing RP. Disadvantages of these animal models include rapid onset of disease: RD1 mice typically develop symptoms within one week of birth and reach advanced stages by two weeks. RD10 mice develop symptoms two weeks after birth and reach advanced stages by four weeks. Consequently, researchers have limited time to conduct therapeutic studies. In particular, RD1 mice have not yet opened their eyes, and their eyeballs and retinas are not fully developed at one week of age. Subretinal injections of therapeutic drugs at this age are highly likely to cause retinal detachment, hindering effective observation of drug efficacy. Furthermore, experiments initiated at four weeks of age, when the mice are essentially mature, present advanced disease, with photoreceptor cell mortality exceeding 90%, making it impossible to investigate the effects of drugs on photoreceptor function and survival.

[0005] 2. Gene-edited mice: Using CRISPR / Cas9 technology to knock out key genes in the visual cycle, such as RHO or RPGR, induces photoreceptor cell death, ultimately leading to RP. Knocking out specific genes in the mouse genome using CRISPR / Cas9 technology has high technical barriers and a long production cycle, often exceeding six months, and is also expensive.

[0006] 3. Chemically induced animal models: Intraperitoneal injection of N-methyl-N-nitrosourea (MNU) into wild-type mice induces DNA damage in photoreceptor cells, leading to RP. However, models established with intraperitoneal injection of MNU often exhibit systemic toxicity, leading to multi-organ damage, which in turn affects research on specific organs.

[0007] 4. Physically induced animal models: Prolonged exposure of wild-type mice to intense light induces oxidative stress in photoreceptor cells, ultimately leading to RP. However, intense light exposure can lead to uncontrolled damage, and individual differences in mice and their light-avoidance behavior can easily lead to model instability.

[0008] In summary, the existing methods for constructing retinitis pigmentosa models have the following defects: short model research window, unstable model, complex construction method and high cost. How to improve the above defects is a technical problem to be solved.

[0009] In view of this, the present invention is proposed. Summary of the Invention

[0010] The purpose of the present invention is to provide a method for constructing a retinitis pigmentosa model to alleviate at least one of the defects of the retinitis pigmentosa model in the prior art, namely, a short model research window, an unstable model, a complex construction method, and a high cost.

[0011] In order to solve the above technical problems, the present invention adopts the following technical solutions: In a first aspect, a method for constructing a retinitis pigmentosa model is provided, the method comprising administering a disulfiram-copper ion complex to a subject, wherein the subject is a mouse or an organ, tissue or cell derived from a mouse.

[0012] In an optional embodiment, the retinitis pigmentosa model is an animal model, and the subject is a mouse at least 4 weeks old.

[0013] In an alternative embodiment, the construction method comprises administering a disulfiram-copper ion complex subretina to the mouse.

[0014] In an alternative embodiment, the construction method comprises administering 5 mM disulfiram-copper ion complex subretina to the mouse.

[0015] In an optional embodiment, the retinitis pigmentosa model is an in vitro model, and the subject is an organ, tissue or cell from a mouse; the construction method includes fully contacting the subject with a disulfiram-copper ion complex.

[0016] In an optional embodiment, the subject is a retinal photoreceptor cell of a mouse, and the construction method comprises administering 5 μM disulfiram-copper ion complex to the retinal photoreceptor cell of the mouse.

[0017] In an optional embodiment, the disulfiram-copper ion complex is prepared by the following method: a disulfiram solution and a salt solution providing copper ions are mixed, and the mixture is vortexed to form the disulfiram-copper ion complex.

[0018] In an optional embodiment, the solvent of the disulfiram solution is PBS; and / or the salt solution providing copper ions is a copper sulfate solution.

[0019] In a second aspect, a kit for constructing an in vitro model of retinitis pigmentosa is provided, wherein the kit comprises (i) and (ii); or the kit comprises (i) and (iii): (i) organs, tissues or cells; (ii) disulfiram-copper ion complex; (iii) Disulfiram and a salt providing copper ions.

[0020] In a third aspect, a retinitis pigmentosa model obtained by the construction method described in the first aspect, or the use of the kit described in the second aspect in any one of (X1) to (X6) is provided: (X1) Study the pathogenesis of retinitis pigmentosa; (X2) Preparation of products for studying the pathogenesis of retinitis pigmentosa; (X3) Study the molecular mechanism of retinitis pigmentosa; (X4) Preparation of products for studying the molecular mechanism of retinitis pigmentosa; (X5) Screening for drugs to treat retinitis pigmentosa; (X6) Prepare products for screening drugs for treating retinitis pigmentosa.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The method for constructing a retinitis pigmentosa model provided by the present invention involves administering a disulfiram-copper ion complex to subjects, inducing dual cell death through copper apoptosis and necroptosis, thereby mimicking the retinitis pigmentosa phenotype. Experimental studies have confirmed that the animal model produced by this method exhibits a reduction in the thickness of the photoreceptor layer, while no significant changes are observed in other retinal layers. This pathological change is consistent with the pathological characteristics of retinitis pigmentosa. Furthermore, the retinal tissue of the constructed animal model shows significant downregulation of visual transduction proteins such as PDE6B and RHO, which are the most common RP pathogenic genes, demonstrating that this model effectively simulates retinitis pigmentosa. In a cell model, the disulfiram-copper ion complex downregulated the expression of PDE6B and RHO in cells, inhibiting cell growth.

[0022] The construction method provided by the present invention selectively kills metabolically active photoreceptors by administering a disulfiram-copper ion complex to a subject, with minimal impact on cells in other retinal layers, demonstrating excellent specificity and safety. The present invention also offers cost and time-efficiency advantages: For example, the construction cost of a single mouse model is less than 100 yuan, significantly reducing the cost of animal model development. Furthermore, the time from administration of the disulfiram-copper ion complex to phenotypic stabilization is only two weeks, compared to over six months for gene-edited models, shortening the model development cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 OCT images of retinal morphology observation of each experimental group in Example 2; Figure 2 HE staining images of retinal sections of each experimental group in Example 2; Figure 3 The results of immunoblotting detection of the expression of visual conduction protein PDE6B and RHO protein in Example 2; Figure 4 The OCT images of the retinal morphology of each experimental group in Example 3 are as follows; Figure 5 HE staining images of retinal sections of each experimental group in Example 3; Figure 6 is the growth curve of cells in each experimental group in Example 4, * P <0.05,** P <0.01,*** P<0.001; Figure 7 The results of immunoblotting detection of PDE6B and RHO protein expression in cells of each experimental group in Example 4; Figure 8 is the growth curve of cells in each experimental group in Example 5, * P <0.05,** P <0.01. DETAILED DESCRIPTION

[0025] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0026] In this document, unless otherwise specified, all implementation methods and preferred implementation methods mentioned herein can be combined with each other to form a new technical solution; all technical features and preferred features mentioned herein can be combined with each other to form a new technical solution; the components involved or their preferred components can be combined with each other to form a new technical solution.

[0027] Unless otherwise specified, the numerical range "a-b" is an abbreviation for any combination of real numbers between a and b, where a and b are both real numbers. For example, the numerical range "6-22" indicates that all real numbers between "6-22" are listed herein, and "6-22" is merely an abbreviation for these numerical combinations. A "range" disclosed herein in the form of lower limits and upper limits can include one or more lower limits and one or more upper limits, respectively.

[0028] Herein, unless otherwise indicated, the various reactions or process steps may be performed sequentially or in any non-sequential manner. Preferably, the reaction methods herein are performed sequentially.

[0029] As used herein, "prepared from" is synonymous with "comprising." As used herein, the terms "comprising," "including," "having," "containing," or any other variations thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0030] As used herein, the conjunction "consisting of excludes any unspecified elements, steps, or components. If used in a claim, this phrase will render the claim closed so that it excludes materials other than those recited, except for normal impurities associated therewith.

[0031] As used herein, "and / or" is used to indicate that either or both of the stated situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0032] In this document, unless otherwise stated, arbitrary numbering is used to distinguish one entity or behavior from another entity or behavior, and does not necessarily require or imply any actual such relationship, order or importance between these entities or behaviors, such as numbers i, ii...; first, second...; X1, X2..., etc.

[0033] As used herein, unless otherwise stated, "optionally," "optional," "optional," or "optional" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0034] In a first aspect, a method for constructing a retinitis pigmentosa model is provided, wherein the method comprises administering disulfiram-copper ion (DSF-Cu) to a subject. 2+ The subject is a mouse or an organ, tissue or cell derived from a mouse.

[0035] DSF-Cu 2+ By binding to acylated proteins (such as DLAT) in the mitochondria of photoreceptors, it induces protein toxic stress and selectively kills photoreceptors with high metabolic activity (such as rods), while the effects on cells in other retinal layers (such as bipolar cells and ganglion cells) are minimal. The obtained model is consistent with the pathological changes of retinitis pigmentosa.

[0036] In an optional embodiment, the retinitis pigmentosa model is an animal model, and the subjects are mice at least 4 weeks old. Using mice at least 4 weeks old, the retina of the eyeball has matured, which improves the success rate of therapeutic drug injection and allows for better retinal function testing and drug intervention studies.

[0037] In an alternative embodiment, the construction method comprises administering a disulfiram-copper ion complex to the subretina of the mouse. Herein, the subretinal region refers to the space between the retinal pigment epithelium (RPE) and the neurosensory retina (NSR).

[0038] In an alternative embodiment, the construction method comprises administering 5 mM disulfiram-copper ion complex subretina to the mouse.

[0039] In an optional embodiment, administering the disulfiram-copper ion complex subretina to the subject comprises subretinal injection of the disulfiram-copper ion complex. Local administration via subretinal injection ensures that the drug reaches an effective concentration in the retina while maintaining systemic circulating concentrations below a safety threshold, thereby preventing liver and kidney damage in the animal model and avoiding systemic toxicity in the animal model.

[0040] In an optional embodiment, the retinitis pigmentosa model is an in vitro model, and the subject is an organ, tissue or cell from a mouse; the construction method includes fully contacting the subject with a disulfiram-copper ion complex.

[0041] In an optional embodiment, the subject is a retinal photoreceptor cell of a mouse, and the construction method comprises administering 5 μM disulfiram-copper ion complex to the retinal photoreceptor cell of the mouse.

[0042] In an optional embodiment, the construction method includes treating retinal photoreceptor cells with 5 μM disulfiram-copper ion complex for 12 to 36 hours, for example, but not limited to 12, 15, 20, 24, 30 or 36 hours, and further optionally for 24 hours.

[0043] In an optional embodiment, the retinal photoreceptor cells are mouse 661w retinal photoreceptor cells.

[0044] In an optional embodiment, in the construction method, the disulfiram-copper ion complex is prepared by the following method: a disulfiram solution and a salt solution providing copper ions are mixed, and the mixture is vortexed to form the disulfiram-copper ion complex.

[0045] In an optional embodiment, the vortexing is performed for 5 to 15 minutes, for example, but not limited to, 5, 10 or 15 minutes.

[0046] In an optional embodiment, the disulfiram solution and the salt solution providing copper ions are mixed in an equimolar ratio based on the molar concentration of disulfiram in the disulfiram solution and the molar concentration of copper ions in the salt solution providing copper ions.

[0047] In an optional embodiment, the solvent of the disulfiram solution is PBS.

[0048] In an optional embodiment, the molar concentration of disulfiram in the disulfiram solution is 10 mM.

[0049] In an optional embodiment, the molar concentration of copper ions in the salt solution providing copper ions is 10 mM.

[0050] In an optional embodiment, the salt solution providing copper ions is a copper sulfate solution.

[0051] In a second aspect, a kit for constructing an in vitro model of retinitis pigmentosa is provided, the kit comprising (i) and (ii); (i) organs, tissues or cells; (ii) Disulfiram-copper ion complex.

[0052] In an optional embodiment, the disulfiram-copper ion complex is prepared using the preparation method of the disulfiram-copper ion complex in the first aspect above.

[0053] Alternatively, the kit for constructing an in vitro model of retinitis pigmentosa comprises (i) and (iii): (i) organs, tissues or cells; (iii) Disulfiram and a salt providing copper ions.

[0054] In an optional embodiment, the kit comprises a disulfiram solution, and further optionally, the solvent of the disulfiram solution is PBS.

[0055] In an alternative embodiment, the salt providing copper ions comprises copper sulfate.

[0056] In a third aspect, the invention provides the retinitis pigmentosa model described in the first aspect, or the kit described in the second aspect, used in any one of (X1) to (X6): (X1) Study the pathogenesis of retinitis pigmentosa; (X2) Preparation of products for studying the pathogenesis of retinitis pigmentosa; (X3) Study the molecular mechanism of retinitis pigmentosa; (X4) Preparation of products for studying the molecular mechanism of retinitis pigmentosa; (X5) Screening for drugs to treat retinitis pigmentosa; (X6) Prepare products for screening drugs for treating retinitis pigmentosa.

[0057] It should be noted that the application of (X1) to (X6) is not for diagnostic and therapeutic purposes.

[0058] The present invention is further described below by way of specific examples. However, it should be understood that these examples are merely provided for more detailed description and are not to be construed as limiting the present invention in any form.

[0059] Example 1 Establishment of a mouse model of retinitis pigmentosa 1. Preparation of disulfiram-copper ion complex: 1. Dissolve disulfiram (DSF) in PBS solution (final concentration 10 mM) and store in the dark.

[0060] 2. Prepare copper sulfate (CuSO4) solution (10 mM, pH 7.4), mix with DSF at a 1:1 molar ratio, and vortex for 10 minutes to form DSF-Cu²⁺ complex (final concentration 5 mM).

[0061] 2. Mouse model preparation method: 1. Subretinal injection operation: 1) Animal Selection: Wild-type C57BL / 6J mice (4 weeks old), SPF-grade, were purchased from Beijing Weitonglihua Company.

[0062] 2) Mice were anesthetized by isoflurane inhalation anesthesia (5% for induction and 1.5% for maintenance) and local anesthesia was given by 0.5% proparacaine hydrochloride eye drops.

[0063] 3) Microinjection: Under a surgical microscope, a 30G needle was used to puncture the subretinal space through the corneal limbus. A Hamilton microsyringe (1 μL capacity, 0.1 μL precision) was used to slowly inject the DSF-Cu²⁺ solution (at an injection rate of approximately 0.2 μL / second). An equal volume of PBS was injected into the control group.

[0064] Example 2 1. Postoperative testing of the retinitis pigmentosa mouse model prepared in Example 1, and in vivo evaluation of the effect of disulfiram-copper ion complex in inducing photoreceptor cell degeneration in mice: 1. Short-term observation: Monitor vitreous hemorrhage and retinal detachment within 24 hours after surgery. Fundus photography 24 hours after surgery revealed no vitreous hemorrhage, and OCT examination showed no retinal detachment.

[0065] Long-term evaluation: On day 14 after surgery, retinal outer nuclear layer (ONL) thickness was measured by optical coherence tomography (OCT) to assess photoreceptor survival. Photoreceptor cell mortality was assessed by retinal sectioning and HE staining. The expression levels of visual cycle proteins were assessed by immunoblotting.

[0066] 1) Disulfiram-copper ion complex causes atrophy of the photoreceptor cell layer: Disulfiram-copper ion complex was injected subretinaly into 4-week-old C57 wild-type mice. OCT examination of the mice 14 days later revealed that the thickness of the photoreceptor cell layer in the experimental group was significantly thinner ( Figure 1 The mouse eyeballs were then taken for HE staining, which further confirmed that the thickness of the photoreceptor cell layer in the experimental group of mice was significantly thinner ( Figure 2 The thickness of the retinal layer shrank from 65 μM±2.3 μM to 23 μM±1.8 μM. No significant changes were observed in other retinal layers.

[0067] 2) Disulfiram-copper ion complex leads to decreased levels of visual transduction protein Retinal tissues of mice were collected 14 days after injection, and the expression levels of visual transduction proteins were detected by immunoblotting. The results showed that the levels of PDE6B and RHO proteins were significantly downregulated ( Figure 3 ), and these two proteins are specifically expressed in photoreceptor cells. At the same time, mutations in these two proteins are the key factors leading to retinitis pigmentosa.

[0068] Example 3 The model mice were intraperitoneally injected with N-acetylcysteine ​​(120 mg / kg body weight), a commonly used drug for the treatment of retinitis pigmentosa in clinical practice. 2+ The complex was injected once every other day after the first injection, for a total of 7 injections. Retinal outer nuclear layer (ONL) thickness was measured by OCT to assess photoreceptor survival. Retinal sections and HE staining were used to count photoreceptor cell death rates.

[0069] N-acetylcysteine ​​is a commonly used drug in the clinical treatment of retinitis pigmentosa. Intraperitoneal injection of N-acetylcysteine ​​was performed in this model mouse. The results showed that the atrophy of the photoreceptor layer of the treated mice was significantly alleviated ( Figure 4 The HE results also reached the same conclusion ( Figure 5 ), the thickness of the photoreceptor layer increased from 23 μM ± 1.8 μM to 45 μM ± 2.2 μM. The experimental results show that N-acetylcysteine, a commonly used drug for the treatment of retinitis pigmentosa, can alleviate the atrophy of the photoreceptor layer in model mice. The model mice prepared in Example 1 can develop typical symptoms of retinitis pigmentosa.

[0070] Example 4 1. Construction of in vitro model of disulfiram-copper ion complex: 1. Cell line 661w (mouse 661w retinal photoreceptor cells) was cultured in a medium containing 5 μM disulfiram-copper ion complex (prepared according to the method of Example 1) for 24 h, with PBS as a negative control.

[0071] 2. Detect cell viability by CCK8, and add DSF-Cu 2+ The complex was initially added (0 h), and the cell viability was measured by CCK8 at time points 12 h, 24 h, and 48 h.

[0072] 3. Detect the expression level of visual cycle proteins by immunoblotting.

[0073] 2. Experimental results: Disulfiram-copper ion complex significantly inhibits the growth of mouse photoreceptor cell line: The mouse photoreceptor cell line 661w was treated with disulfiram-copper ion complex. The results showed that disulfiram-copper ion complex can significantly inhibit the growth of 661w cells ( Figure 6 At the same time, immunoblotting results also showed that disulfiram-copper ion complex can downregulate the expression of PDE6B and RHO in cells ( Figure 7 ).

[0074] Example 5 The 661w cell line was treated with a disulfiram-copper ion complex (working concentration of 5 μM, prepared according to the method of Example 1) for 24 hours. The 661w cell line was also treated with a disulfiram-copper ion complex (working concentration of 5 μM, prepared according to the method of Example 1) and N-acetylcysteine ​​(10 μM) for 24 hours. The cell viability of each experimental group was detected by CCK8. The results showed that N-acetylcysteine ​​could significantly reverse the cell growth inhibition caused by disulfiram-copper ions and promote cell survival ( Figure 8 ). The experimental results show that the constructed cell model can produce typical symptoms of retinitis pigmentosa.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing a retinitis pigmentosa model, characterized in that: The method comprises administering a disulfiram-copper ion complex to a subject, wherein the subject is a mouse or an organ, tissue or cell derived from a mouse.

2. The construction method according to claim 1, characterized in that The retinitis pigmentosa model is an animal model, and the subjects are mice at least 4 weeks old.

3. The construction method according to claim 2, characterized in that It involves administering a disulfiram-copper ion complex subretinal to mice.

4. The construction method according to claim 3, wherein: It involves administering 5 mM disulfiram-copper ion complex subretinal to mice.

5. The construction method according to claim 1, characterized in that The retinitis pigmentosa model is an in vitro model, and the subject is an organ, tissue or cell from a mouse; the construction method comprises fully contacting the subject with a disulfiram-copper ion complex.

6. The construction method according to claim 5, wherein: The subject is a retinal photoreceptor cell of a mouse, and the construction method comprises administering 5 μM disulfiram-copper ion complex to the retinal photoreceptor cell of the mouse; Optionally, the retinal photoreceptor cells are mouse 661w retinal photoreceptor cells; Optionally, the processing time is 12 to 36 hours, further optionally 24 hours.

7. The construction method according to any one of claims 1 to 6, characterized in that The disulfiram-copper ion complex is prepared according to the following method: The disulfiram solution and the salt solution providing copper ions are mixed and vortexed to form a disulfiram-copper ion complex; Optionally, vortex for 5-15 minutes.

8. The construction method according to claim 7, characterized in that: The solvent of the disulfiram solution is PBS; and / or the salt solution providing copper ions is a copper sulfate solution; Optionally, the disulfiram solution and the salt solution providing copper ions are mixed in an equimolar ratio based on the molar concentration of disulfiram in the disulfiram solution and the molar concentration of copper ions in the salt solution providing copper ions; Optionally, the molar concentration of disulfiram in the disulfiram solution is 10 mM, and / or the molar concentration of copper ions in the salt solution providing copper ions is 10 mM.

9. A kit for constructing an in vitro model of retinitis pigmentosa, characterized in that: The kit comprises (i) and (ii); or the kit comprises (i) and (iii): (i) organs, tissues or cells; (ii) disulfiram-copper ion complex; (iii) Disulfiram and a salt providing copper ions.

10. Use of the retinitis pigmentosa model obtained by the construction method according to any one of claims 1 to 8, or the kit according to claim 9 in any one of (X1) to (X6): (X1) Study the pathogenesis of retinitis pigmentosa; (X2) Preparation of products for studying the pathogenesis of retinitis pigmentosa; (X3) Study the molecular mechanism of retinitis pigmentosa; (X4) Preparation of products for studying the molecular mechanism of retinitis pigmentosa; (X5) Screening for drugs to treat retinitis pigmentosa; (X6) Prepare products for screening drugs for treating retinitis pigmentosa.

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