Construction method and application of zebrafish model for co-disease of Parkinson and osteoporosis

Abstract

The invention discloses a construction method of a zebra fish model for Parkinson's disease and osteoporosis, which comprises the following steps: injecting neurotoxin into a brain cavity of a zebra fish, simultaneously dosing dexamethasone, and culturing to obtain the zebra fish model. According to the method, the modeling period is remarkably shortened, the research and development cost is reduced, the phenotypic relevance of PD and OP is disclosed for the first time through a bigeminy verification system, a first vertebrate model is provided for the PD-OP co-disease, and the high-throughput drug screening requirement is met.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a method for constructing a zebrafish model of Parkinson's disease and osteoporosis and its application. Background Technology

[0002] Parkinson's disease (PD) and osteoporosis (OP) are both degenerative neurological and skeletal diseases prevalent in the elderly, and clinical studies have shown a significant correlation between the two. Epidemiological data shows that approximately 60% of PD patients have decreased bone mineral density, and their fracture risk is 2-3 times higher than that of healthy individuals. Current research suggests that motor dysfunction, abnormal vitamin D metabolism, and dopaminergic neuron degeneration in PD patients collectively lead to bone metabolic imbalance, but the specific molecular mechanisms remain unclear.

[0003] Existing disease models have the following main limitations:

[0004] 1. Single disease models cannot simulate comorbid states: Currently, PD research commonly uses 6-OHDA-induced rodent models, while OP research mostly uses ovariectomized rat models. Neither of these models can simultaneously simulate the dual pathological features of neurodegeneration and abnormal bone metabolism.

[0005] 2. Large mammalian models are expensive: Although non-human primates can better simulate human pathological processes, they have problems such as long experimental cycles (12-18 months) and a cost of more than 50,000 yuan per animal, making it difficult to meet the needs of large-scale drug screening.

[0006] 3. Limitations of existing zebrafish models:

[0007] (1) Neurotoxicity model: The 6-OHDA-induced zebrafish PD model only showed dopamine neuron damage and lacked bone metabolism phenotype;

[0008] (2) Skeletal development model: Dexamethasone-induced zebrafish OP model can lead to decreased bone density, but without neurological lesion features;

[0009] (3) Mismatch in model building cycle: PD phenotype usually requires at least 7 days to induce, while traditional OP modeling requires 21 days. The time difference makes it impossible to simulate the coordinated progression of the disease.

[0010] There is an urgent need in this field for an animal model that can meet the following requirements:

[0011] (1) Simultaneously presenting the characteristic loss of dopamine neurons in PD and the typical destruction of trabecular bone structure in OP;

[0012] (2) Maintaining high genetic homology between mammals and humans

[0013] (3) Achieve rapid modeling within 14 days, and keep the cost of a single experiment at the thousand-yuan level;

[0014] (4) Applicable to real-time observation of living organisms. Summary of the Invention

[0015] To address the aforementioned technical challenges, this application employs a dual-inducer synergistic intervention (6-OHDA intracerebral injection + dexamethasone water-soluble administration) combined with a multi-index verification system for transgenic zebrafish to achieve simultaneous induction and quantitative assessment of dual pathological phenotypes.

[0016] This application provides a method for constructing a zebrafish model of Parkinson's disease and osteoporosis, comprising the following steps:

[0017] A zebrafish model was obtained by injecting neurotoxins into the brain cavity of zebrafish and simultaneously administering dexamethasone, followed by culturing.

[0018] This application also provides the application of the zebrafish model constructed by the above-described construction method in drug preparation or screening.

[0019] This application also provides the application of a combination of neurotoxins and dexamethasone in the preparation of zebrafish models of Parkinson's disease and osteoporosis.

[0020] This application also provides the application of a combination of neurotoxins and dexamethasone in shortening the construction cycle of zebrafish models of Parkinson's disease and osteoporosis.

[0021] The beneficial effects of this application include, but are not limited to:

[0022] 1. Efficiently achieve simultaneous modeling of two diseases (core technology effect)

[0023] Shortened modeling cycle: Traditional mammalian models require 6-8 weeks to induce a single disease. This invention uses zebrafish juveniles (3dpf) combined with dual intervention for simultaneous induction (96 hours), and a stable PD-OP composite model can be established within 4 days (verified in Tables 1 to 3).

[0024] The success rate has been significantly improved:

[0025] The success rate of PD phenotype induction was significantly improved (dopamine neuron area decreased by 40-76%, p<0.001, Table 2);

[0026] The success rate of OP phenotype induction was significantly improved (skull fluorescence intensity decreased by 28-40%, p<0.001, Table 3).

[0027] 2. Significantly reduces R&D costs (economic benefits)

[0028] Animal cost savings: The cost of raising zebrafish is 1 / 50 of that of the mouse model (cost per zebrafish ≈ ¥0.5 vs. ¥25 per mouse);

[0029] Reduced reagent consumption: The micro-level drug delivery system (6-OHDA injection dose of only 500 ng / tail, dexamethasone water concentration of 3 μM) saves reagent consumption compared to mammalian systems.

[0030] 3. Precisely quantify disease interactions (technological effects)

[0031] The phenotypic association between PD and OP was revealed for the first time using a dual validation system:

[0032] Motor impairment is positively correlated with bone loss: when the area of ​​dopamine neurons decreases by 40%, the fluorescence intensity of the skull decreases by 28%. Figure 2 vs. Figure 4 This confirms that neurodegeneration accelerates abnormal bone metabolism.

[0033] 4. High safety and repeatability (technical effectiveness)

[0034] Zero mortality: The model survival rate was 100% under the optimal dose combination (500 ng / tail 6-OHDA + 3.00 μM dexamethasone) (Table 1);

[0035] Inter-batch variation <5%: Under the same parameters, the area of ​​dopamine neurons fluctuated within ±3.8% (n=10, Table 2), meeting the requirements for high-throughput drug screening.

[0036] 5. Promote research on comorbidity mechanisms and drug development (social benefits)

[0037] Providing the first vertebrate model for PD-OP comorbidity: filling a technological gap and supporting:

[0038] Research on the mechanisms of neuro-skeletal interaction (e.g., 6-OHDA inhibits osteogenic differentiation through oxidative stress);

[0039] Evaluation of dual-target drugs (e.g., screening of compounds that simultaneously increase dopamine neuron area by 47% and skull fluorescence intensity by 50%). Attached Figure Description

[0040] This application will be further described by way of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not limiting, wherein:

[0041] Figure 1 This is a typical diagram of the area of ​​zebrafish dopamine neurons after sample treatment according to some embodiments of this application.

[0042] Figure 2 To compare the area of ​​zebrafish dopamine neurons after sample treatment according to some embodiments of this application with that of the normal control group, ***p<0.001.

[0043] Figure 3 The image shows a typical fluorescence intensity diagram of zebrafish skulls after sample processing according to some embodiments of this application. Note: The yellow dashed box indicates the analysis area.

[0044] Figure 4 To compare the fluorescence intensity of zebrafish skulls after sample treatment according to some embodiments of this application with that of the normal control group, ***p<0.001. Detailed Implementation

[0045] To more clearly illustrate the technical solutions of the embodiments in this specification, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are merely some examples or embodiments of this specification. For those skilled in the art, these drawings can be applied to other similar scenarios without creative effort. Unless obvious from the context or otherwise specified, the same reference numerals in the drawings represent the same structures or operations.

[0046] As indicated in this specification and claims, unless the context clearly indicates otherwise, the words "a," "an," "an," and / or "the" do not specifically refer to the singular and may also include the plural. Generally speaking, the terms "comprising" and "including" only indicate the inclusion of expressly identified steps and elements, which do not constitute an exclusive list, and the method or apparatus may also include other steps or elements.

[0047] Flowcharts are used in this specification to illustrate the operations performed by the system according to embodiments of this specification. It should be understood that the preceding or following operations are not necessarily performed in exact order. Instead, the steps can be processed in reverse order or simultaneously. Furthermore, other operations can be added to these processes, or one or more steps can be removed from them.

[0048] This application provides a method for constructing a zebrafish model of Parkinson's disease and osteoporosis, comprising the following steps:

[0049] A zebrafish model was obtained by injecting neurotoxins into the brain cavity of zebrafish and simultaneously administering dexamethasone, followed by culturing.

[0050] In some embodiments, the zebrafish may be a zebrafish that is 3 days old after fertilization.

[0051] In some embodiments, preferably, the zebrafish may be a transgenic zebrafish. In some embodiments, preferably, the zebrafish may be a dopamine neuron green fluorescent zebrafish.

[0052] In some embodiments, the neurotoxin may be 6-hydroxydopamine. In some embodiments, preferably, the injection dose of 6-hydroxydopamine may be 31.2–500 ng. In some embodiments, more preferably, the injection dose of 6-hydroxydopamine may be 500 ng.

[0053] For example, the injection dose of the 6-hydroxydopamine can be 31.2, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 ng. It also includes any range characterized by combinations of the above values, which will not be elaborated here.

[0054] In some embodiments, the culture solution may be reverse osmosis water containing sea salt and dexamethasone. Preferably, in some embodiments, the concentration of sea salt in the reverse osmosis water may be 100–300 mg / L, the concentration of dexamethasone may be 0.188–3.00 μM, the conductivity may be 450–550 μS / cm, the pH may be 6.5–8.5, and the hardness may be 50–100 mg / L CaCO3.

[0055] For example, the concentration of sea salt in reverse osmosis water can be 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, or 300 mg / L. It also includes any range characterized by combinations of the above values, which will not be elaborated here.

[0056] For example, dexamethasone concentrations are 0.188, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.00, 1.10, 1.20, 1.30, 1.40, 1.50, 1.60, 1.70, 1.80, 1.90, 2.00, 2.10, 2.20, 2.30, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90, or 3.00 μM.

[0057] In some embodiments, more preferably, the concentration of sea salt in the reverse osmosis water may be 200 mg / L, and the concentration of dexamethasone may be 3.00 μM. Any range characterized by combinations of the above-mentioned values ​​is also included, which will not be elaborated here.

[0058] For example, the conductivity can be 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, or 550 μS / cm. It also includes any range characterized by combinations of the above values, which will not be elaborated here.

[0059] For example, the pH can be 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5. It also includes any range characterized by combinations of the above values, which will not be elaborated here.

[0060] For example, the hardness can be 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mg / L CaCO3. It also includes any range characterized by combinations of the above values, which will not be elaborated here.

[0061] In some embodiments, the culture temperature can be 22–29°C, and the culture time can be 72–120 hours. For example, the culture temperature can be 22, 23, 24, 25, 26, 27, 28, or 29°C. It also includes any range characterized by combinations of the above-mentioned values, which will not be elaborated here. For example, the culture time can be 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, or 120 hours. It also includes any range characterized by combinations of the above-mentioned values, which will not be elaborated here.

[0062] In some embodiments, preferably, the culture temperature can be 28°C and the culture time can be 96 hours.

[0063] This application also provides the application of the zebrafish model constructed by the above-described construction method in drug preparation or screening.

[0064] In some embodiments, the drug may be a treatment for Parkinson's disease or osteoporosis.

[0065] This application also provides the application of a combination of neurotoxins and dexamethasone in the preparation of zebrafish models of Parkinson's disease and osteoporosis.

[0066] This application also provides the application of a combination of neurotoxins and dexamethasone in shortening the construction cycle of zebrafish models of Parkinson's disease and osteoporosis.

[0067] In the experimental methods of the following examples, unless otherwise specified, they are all conventional methods. The test materials used in the following examples, unless otherwise specified, are all purchased from regular biochemical reagent companies. In the following examples, all quantitative tests are set with three repeated experiments, and the results are averaged.

[0068] 6-Hydroxydopamine (6-OHDA), a neurotoxin that selectively destroys dopamine neurons.

[0069] Dexamethasone (Dex), a glucocorticoid that induces bone loss.

[0070] Days post-fertilization (dpf), a standard unit for the developmental stage of zebrafish.

[0071] Maximum tolerated concentration (MTC), the highest treatment concentration that does not cause significant death.

[0072] Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), an international certification body for laboratory animal welfare

[0073] I. Experimental materials:

[0074] (1) Experimental animals

[0075] Zebrafish were all raised in fish culture water at 28 °C (water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, the conductivity was 450 - 550 μS / cm; pH was 6.5 - 8.5; hardness was 50 - 100 mg / L CaCO3). They were provided by the fish culture center of our company. The license number for the use of experimental animals was: SYXK(Zhe)2022 - 0004. The feeding management complied with the requirements of international AAALAC certification (certification number: 001458), and the IACUC ethical review number was: IACUC - 2024 - 10771 - 01).

[0076] (2) Reagents:

[0077] 6-Hydroxydopamine hydrochloride (batch number MKCO5002, Sigma, New Zealand);

[0078] Dexamethasone (batch number C2110208, Shanghai Aladdin Biochemical Technology Co., Ltd., China);

[0079] Dimethyl sulfoxide (DMSO, batch number BCCD8942, Sigma, Switzerland);

[0080] Sodium chloride (batch number 20220216, Sinopharm Chemical Reagent Co., Ltd., China);

[0081] Methylcellulose (batch number C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China);

[0082] Alizarin Red (batch number 030M1776, SIGMA, China);

[0083] Anhydrous magnesium chloride (batch number C1809044, Shanghai Aladdin Biochemical Technology Co., Ltd., China);

[0084] Potassium hydroxide (batch number T20110104, Sinopharm Chemical Reagent Co., Ltd., China);

[0085] Glycerin (batch number 6108115, Huankai Microbial Technology Co., Ltd., China);

[0086] Anhydrous ethanol (batch number 20240123, Sinopharm Chemical Reagent Co., Ltd., China);

[0087] Hydrogen peroxide solution (batch number G2023089, Shanghai Aladdin Biochemical Technology Co., Ltd., China).

[0088] 3. Instruments:

[0089] Dissecting microscope (SZX7, OLYMPUS, Japan);

[0090] CCD camera (VertA1, Shanghai Shisen Vision Technology Co., Ltd., China);

[0091] Precision electronic balance (CP214, OHAUS, USA);

[0092] 6-well plate (Zhejiang Beilanbo Biotechnology Co., Ltd., China);

[0093] 96-well plate (Nest Biotech, China);

[0094] Microinjection apparatus (IM300, Narishige, Japan);

[0095] Needle puller (PC-10, Narishige, Japan);

[0096] Zeiss fluorescence microscope (AXIOZoom.V16, ZEISS, Germany);

[0097] Motorized focusing continuous zoom fluorescence microscope (AZ100, Nikon, Japan).

[0098] II. Detection Methods:

[0099] 1. Determination of Model Maximum Detectable Dose (MTD) and Concentration (MTC)

[0100] Three-day-old (3dpf) transgenic dopamine-producing fluorescent green zebrafish (bred and raised by Hangzhou Huante Biotechnology Co., Ltd.) were randomly selected and placed in six-well plates, with 30 zebrafish per well (experimental group) and a volume of 3 mL per well. Except for the normal control group, all experimental groups underwent intracerebral injection of 6-hydroxydopamine and water-soluble dexamethasone to establish a zebrafish model of Parkinson's disease combined with osteoporosis (dosage and concentration are shown in Table 1). After treatment at 28℃ for 96 h, the MTD and MTC of the inducing agents for the Parkinson's disease combined with osteoporosis model were measured.

[0101] Intracerebral injection of 6-hydroxydopamine (injection site details are in references Gutzman JH, Sive H. Zebrafish brain ventricle injection[J]. J Vis Exp. 2009 Apr 6;(26):1218 and Meijer AH. Protection and pathology in TB: learning from the zebrafish model[J]. Semin Immunopathol. 2016 Mar;38(2):261-73).

[0102] 2. Model Exploration (Area of ​​Dopamine Neurons)

[0103] 3dpf transgenic dopamine neuron-bearing green fluorescent zebrafish were randomly selected and placed in six-well plates, with 30 zebrafish per well and a volume of 3 mL per well. Except for the normal control group, all experimental groups received intracerebral injection of 6-hydroxydopamine and water-soluble dexamethasone to establish a zebrafish model of Parkinson's disease combined with osteoporosis (dosage and concentration are shown in Table 2). After treatment at 28℃ for 96 h, 10 zebrafish were randomly selected and photographed under a Zeiss fluorescence microscope. ImageJ software was used to analyze and collect data, and the area of ​​zebrafish dopamine neurons was analyzed. Statistical analysis of this index was used to evaluate the success of the establishment of the Parkinson's disease combined with osteoporosis model. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software, and p < 0.05 indicated statistical significance.

[0104] 3. Model Exploration (Skull Fluorescence Intensity)

[0105] 3dpf transgenic dopamine-producing zebrafish with green fluorescence were randomly selected and placed in six-well plates, with 30 zebrafish per well and a volume of 3 mL per well. Except for the normal control group, all experimental groups received intracerebral injection of 6-hydroxydopamine and water-soluble dexamethasone to establish a zebrafish model of Parkinson's disease combined with osteoporosis (dosage and concentration are shown in Table 3). After treatment at 28℃ for 96 h, samples were collected, stained with succinate red, and 10 zebrafish from each experimental group were randomly selected and photographed under a fluorescence microscope. Data were analyzed and collected using NIS-Elements D3.20 advanced image processing software. The fluorescence intensity of the zebrafish skull was analyzed, and the statistical analysis results were used to evaluate the success of the establishment of the Parkinson's disease combined with osteoporosis model. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software, and p < 0.05 indicated statistical significance.

[0106] Detailed steps of alizarin red staining

[0107] ① Fixation: After the experiment, add 4% tissue cell fixative to each well and let it stand at room temperature for 2 hours. Remove the fixative, wash, and then dehydrate with 50% ethanol.

[0108] ② Staining: After dehydration, remove 50% ethanol and add alizarin red staining solution to each well and stain overnight.

[0109] ③ Bleaching: Remove the staining solution and wash twice. Bleach at room temperature after washing.

[0110] ④ Cleaning: Remove the bleach solution, clean with a mixture of 50% glycerin / 0.25% KOH, leave at room temperature overnight, and observe under a microscope.

[0111] ⑤ Storage: Store in a 50% glycerol / 0.1% KOH mixture at 4°C for long-term use. Take photographs in this storage solution using a dissecting microscope / fluorescence microscope.

[0112] III. Test Results:

[0113] 1. Models MTD and MTC

[0114] Under the experimental conditions, the MTD (maximum tolerated dose) and MTC (maximum tested concentration) of 6-hydroxydopamine + dexamethasone in the Parkinson's disease combined with osteoporosis model were 500 ng / tail + 3.00 μM. See Table 1 for details.

[0115] Table 1. Results of the dose and concentration exploration experiment in the Parkinson's disease combined with osteoporosis model (n=30)

[0116]

[0117] 2. Model Exploration (Area of ​​Dopamine Neurons)

[0118] Under the experimental conditions, 6-hydroxydopamine + dexamethasone at concentrations of 62.5 ng / tail + 0.375 μM, 125 ng / tail + 0.750 μM, 250 ng / tail + 1.50 μM, and 500 ng / tail + 3.00 μM induced damage to zebrafish dopamine neurons, while 31.2 ng / tail + 0.188 μM did not significantly induce damage to zebrafish dopamine neurons. See Table 2 for details. Figure 1 and Figure 2 .

[0119] Table 2. Experimental results of exploring a Parkinson's disease combined with osteoporosis model (dopamine neuron area) (n=10)

[0120]

[0121] Compared with the normal control group, ***p<0.001

[0122] 3. Model Exploration (Skull Fluorescence Intensity)

[0123] Under the conditions of this experiment, 6-hydroxydopamine + dexamethasone 250 ng / fish + 1.50 μM and 500 ng / fish + 3.00 μM induced osteoporosis in zebrafish, while 31.2 ng / fish + 0.188 μM, 62.5 ng / fish + 0.375 μM, and 125 ng / fish + 0.750 μM did not significantly induce osteoporosis in zebrafish. See Table 3 for details. Figure 3 and Figure 4 .

[0124] Table 3. Experimental results of exploring a Parkinson's disease combined with osteoporosis model (skull fluorescence intensity) (n=10)

[0125]

[0126] Compared with the normal control group, ***p<0.001

[0127] The basic concepts have been described above. Obviously, for those skilled in the art, the detailed disclosure above is merely illustrative and does not constitute a limitation of this specification. Although not explicitly stated herein, those skilled in the art may make various modifications, improvements, and corrections to this specification. Such modifications, improvements, and corrections are suggested in this specification and therefore remain within the spirit and scope of the exemplary embodiments described herein.

[0128] Furthermore, this specification uses specific terms to describe embodiments thereof. For example, "an embodiment," "one embodiment," and / or "some embodiments" refer to a particular feature, structure, or characteristic associated with at least one embodiment of this specification. Therefore, it should be emphasized and noted that references to "an embodiment," "one embodiment," or "an alternative embodiment" in different locations throughout this specification do not necessarily refer to the same embodiment. Moreover, certain features, structures, or characteristics in one or more embodiments of this specification can be appropriately combined.

[0129] In some embodiments, numbers describing the quantity of components and attributes are used. It should be understood that such numbers used in the description of embodiments are modified in some examples with the terms "approximately," "approximately," or "generally." Unless otherwise stated, "approximately," "approximately," or "generally" indicates that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may be changed depending on the characteristics required by individual embodiments. In some embodiments, numerical parameters should take into account specified significant digits and employ a general method of digit reservation. Although the numerical ranges and parameters used to confirm their breadth of range in some embodiments of this specification are approximate values, in specific embodiments, such values ​​are set as precisely as feasible.

[0130] Finally, it should be understood that the embodiments described in this specification are merely illustrative of the principles of the embodiments described herein. Other variations may also fall within the scope of this specification. Therefore, alternative configurations of the embodiments described herein are intended to be illustrative rather than limiting, and should be considered consistent with the teachings of this specification. Accordingly, the embodiments described herein are not limited to those explicitly introduced and described herein.

Claims

1. A method for constructing a zebrafish model of Parkinson's disease and osteoporosis, comprising the following steps: A zebrafish model was obtained by injecting neurotoxins into the brain cavity of zebrafish and simultaneously administering dexamethasone, followed by culturing.

2. The construction method as described in claim 1, characterized in that, The zebrafish in question are zebrafish that are 3 days old after fertilization. And / or, the zebrafish is a transgenic zebrafish, preferably, the zebrafish is a dopamine neuron green fluorescent zebrafish.

3. The construction method as described in claim 1, characterized in that, The neurotoxin is 6-hydroxydopamine. Preferably, the injection dose of 6-hydroxydopamine is 31.2 to 500 ng, and more preferably, the injection dose of 6-hydroxydopamine is 500 ng.

4. The construction method as described in claim 1, characterized in that, The culture solution is reverse osmosis water containing sea salt and dexamethasone.

5. The construction method as described in claim 1, characterized in that, The concentration of sea salt in the reverse osmosis water is 100–300 mg / L, the concentration of dexamethasone is 0.188–3.00 μM, the conductivity is 450–550 μS / cm, the pH is 6.5–8.5, and the hardness is 50–100 mg / L CaCO3; preferably, the concentration of sea salt in the reverse osmosis water is 200 mg / L, and the concentration of dexamethasone is 3.00 μM.

6. The construction method as described in claim 1, characterized in that, The culture temperature is 22–29°C, and the culture time is 72–120 hours; preferably, the culture temperature is 28°C, and the culture time is 96 hours.

7. The application of the zebrafish model constructed by the construction method according to any one of claims 1 to 6 in drug preparation or screening.

8. The application as described in claim 7, characterized in that, The medication in question is for the treatment of Parkinson's disease or osteoporosis.

9. Application of the combination of neurotoxin and dexamethasone in the preparation of zebrafish models of Parkinson's disease and osteoporosis.

10. Application of the combination of neurotoxins and dexamethasone in shortening the construction cycle of zebrafish models of Parkinson's disease and osteoporosis.

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