Method for constructing primary ciliary dyskinesia hydrocephalus model

Abstract

The invention belongs to the technical field of animal models, and particularly discloses a method for constructing a primary ciliary dyskinesia hydrocephalus model, which comprises the following steps: screening benzimidazole as a molding agent; performing benzimidazole solution administration on the experimental animals, and setting a blank control group; carrying out experimental verification through pathological morphology and behavioral methods; the ventricle of the experimental animal is obviously enlarged, the athletic ability is obviously reduced, and if the condition is met, the primary ciliary dyskinesia hydrocephalus model is successfully constructed. According to the new method for constructing the primary ciliary dyskinesia hydrocephalus model, the constructed hydrocephalus model is good in stability and repeatability, objective, simple and high in operability, and other possible interference factors are reduced; and a necessary experimental basis is provided for research on the mechanism of hydrocephalus caused by primary ciliary dyskinesia and development of treatment means.

Description

Technical Field

[0001] The present invention relates to the technical field of animal models, and in particular to a method for constructing a hydrocephalus model of primary ciliary dyskinesia. Background Art

[0002] Hydrocephalus is a neurological disease caused by disorders in cerebrospinal fluid production or absorption. Its high disability rate and insidious course make it a major problem that the public health system urgently needs to address. Taking idiopathic normal pressure hydrocephalus (iNPH) as an example, studies have shown that the prevalence of iNPH is as high as 2.9% in people over 65 years old. Its clinical treatment faces severe challenges. On the one hand, its pathogenesis and treatment targets are not yet clear. On the other hand, the treatment methods are limited. Currently, the treatment mainly relies on surgery, which has problems such as high risk and many complications, and there is a lack of targeted therapeutic drugs.

[0003] Primary ciliary dyskinesia is a genetic disease, and its core lesion is the abnormal structure and function of cilia. Cilia are widely distributed in multiple organs and tissues of the human body, including the respiratory tract, reproductive system, and ependymal cells of the ventricular system. Under normal circumstances, these cilia rhythmically swing to promote the circulation of cerebrospinal fluid in the ventricular system, thereby maintaining the dynamic balance of intracranial pressure. When the ciliary function is impaired, the cilia of ependymal cells in the ventricle may not be able to effectively promote the circulation of cerebrospinal fluid, resulting in the accumulation of cerebrospinal fluid. The continuously accumulated cerebrospinal fluid causes an increase in intracranial pressure in the ventricle, gradually forming ventricular dilation, that is, hydrocephalus. Long-term hydrocephalus and increased intracranial pressure can further compress the brain parenchyma, affect the development of nerve function, and even cause irreversible brain damage. Currently, the pathogenesis of hydrocephalus is not yet clear, and animal models can provide new insights into the pathogenesis of the disease and lay the foundation for the development of new treatment methods.

[0004] Using the method of gene knockout of the Efcab2 gene (the expressed protein is DRC8) to construct an animal model will cause the animal to carry a lifelong gene defect, and such animals often face the technical problem of homozygous lethality. By using drug targeting to bind to the protein expressed by the Efcab2 gene and administering the drug at a certain time period, it is possible to avoid interference in the early development of the animal and achieve the effect of gene knockout at a specific time period. Therefore, developing an inhibitor targeting the DRC8 protein and constructing a PCD hydrocephalus animal model has important scientific research and clinical value.

[0005] As a model organism, zebrafish has a wide range of applications in constructing disease models due to its characteristics such as in vitro fertilization, transparent embryos, low breeding cost, and strong regenerative ability. The present invention proposes a new method for constructing a hydrocephalus animal model of primary ciliary dyskinesia using benzimidazole. Summary of the Invention

[0006] The object of the present invention is to provide a method for constructing a hydrocephalus model of primary ciliary dyskinesia, which uses a benzimidazole solution to construct a zebrafish hydrocephalus model and can simply and stably establish an animal hydrocephalus model of primary ciliary dyskinesia. By adopting a non-invasive method to construct the hydrocephalus model of primary ciliary dyskinesia, while reducing the harm to the animal model, a hydrocephalus model of primary ciliary dyskinesia is successfully established by soaking and administering benzimidazole to the animal model. It has good stability and repeatability, is objective and simple, has strong operability, reduces other possible interference factors, and provides a necessary experimental basis for studying the mechanism of hydrocephalus of primary ciliary dyskinesia and the development of treatment means.

[0007] To achieve the above object, the present invention provides a method for constructing a hydrocephalus model of primary ciliary dyskinesia, comprising the following steps:

[0008] Step 1: Screen a modeling agent compound according to the substructural protein DRC8 of the inner dynein regulatory complex (N-DRC). The screening database is the CNS central nervous system drug database. Compounds are screened according to the free binding energy, and the top 100 compounds are further screened in combination with medicine and toxicology to determine benzimidazole as the modeling agent;

[0009] Step 2: Under normal feeding conditions, administer a benzimidazole solution to the experimental animals, and set up a blank control group;

[0010] Step 3: After the experimental animals are treated with the benzimidazole solution, conduct experimental verification through pathological morphology and behavioral methods;

[0011] Step 4: Compared with the control group in terms of pathological morphology and behavioral indicators, if the ventricles of the experimental animals are significantly enlarged and the motor ability is significantly decreased, and this condition is met, the construction of the hydrocephalus model of primary ciliary dyskinesia is successful.

[0012] Preferably, in step 1, the benzimidazole is 4,5-dimethyl-1H-benzimidazole.

[0013] Preferably, in step 2, the feeding conditions are temperature: 28.5 °C, 14 hours of light / 10 hours of darkness.

[0014] Preferably, in step 2, the experimental animals are zebrafish.

[0015] Preferably, in step 2, the administration concentration of the benzimidazole solution is 200 μg / ml or 300 μg / ml.

[0016] Preferably, the administration concentration of the benzimidazole solution is 300 μg / ml.

[0017] Preferably, in step two, the benzimidazole solution is administered by immersion.

[0018] The advantages and beneficial effects of the present invention using the above method for constructing a primary ciliary dyskinesia hydrocephalus model are as follows:

[0019] 1. The present invention adopts a non-invasive method to construct a primary ciliary dyskinesia hydrocephalus model. Under the condition of reducing the harm to the animal model, by administering benzimidazole to the animal model through immersion, a primary ciliary dyskinesia hydrocephalus model is successfully established.

[0020] 2. The animal model constructed by the present invention has good stability and repeatability, is objective and simple, and has strong operability, reducing other possible interference factors, providing a necessary experimental basis for studying the mechanism of primary ciliary dyskinesia hydrocephalus and the development of treatment means.

[0021] 3. The primary ciliary dyskinesia hydrocephalus animal model prepared by the present invention can monitor the degree of hydrocephalus in real time by measuring the size of the cerebral ventricle and detecting the motor level during the research process. Therefore, the modeling process of the present invention's solution is a dynamic process, and the ventricular dilation situation at different time points can be observed, providing a good experimental platform for studying primary ciliary dyskinesia hydrocephalus. At the same time, this model also provides a good tool for developing new treatment means.

[0022] Next, through the drawings and examples, the technical solution of the present invention will be further described in detail. Description of the Drawings

[0023] Figure 1 is the predicted structure diagram of zebrafish DRC8 protein of the present invention;

[0024] Figure 2 is the computer simulation result of the interaction between DRC8 and 4,5-dimethyl-1H-benzimidazole in Example 1 of the present invention, where A is the surface and molecular structure of DRC8 protein, and B is the protein molecular interaction relationship;

[0025] Figure 3 is the experimental flow chart of zebrafish in Example 2 of the present invention;

[0026] Figure 4 is the ventricular detection result diagram of zebrafish of the present invention, where A is the ventricular diagram of zebrafish in the experimental group (300 μg / mL) (n = 20), and B is the ventricular diagram of zebrafish in the control group (n = 20). Detailed Embodiments

[0027] The technical solution of the present invention will be further described below through the drawings and examples.

[0028] Unless otherwise defined, the technical terms or scientific terms used in this invention shall have the ordinary meanings as understood by those with ordinary skills in the field to which this invention pertains.

[0029] Unless otherwise defined, the instruments, equipment, reagent materials, etc. used in this invention are all obtained from regular commercial sources.

[0030] Experimental animals: Zebrafish embryos fertilized for 20 hours.

[0031] Experimental materials: CNS central nervous system drug database (from Enamine), benzimidazole; PTU; stereomicroscope.

[0032] Example 1

[0033] A method for constructing a primary ciliary dyskinesia hydrocephalus model, comprising the following steps:

[0034] Step 1, N-DRC (intraflagellar transport-dynein regulatory complex) is an important structure in motile cilia. The research group screened compounds according to the substructural protein DRC8 of N-DRC. The screening database was the CNS central nervous system drug database (from Enamine), which contained 47,360 compounds. Compounds were screened according to the free binding energy, and the top 100 compounds were further screened in combination with medicine and toxicology. Finally, benzimidazole was determined as the modeling agent.

[0035] DRC8 protein structure: Predicted using alphafold, and at the same time, the interaction sites were predicted according to other homologous proteins.

[0036] Virtual screening: Screening was performed using Autodockvina according to the predicted binding sites.

[0037] Dataset determination: The dataset used was the Enamine CNS dataset with 47,360 small molecules, a small molecule compound database with a high CNSMPO score. This means that the molecule has a high blood-brain barrier permeability.

[0038] Score evaluation: Generally speaking, the larger the molecular weight of the molecule, the higher the upper limit of the docking score, while for CNS drugs, we usually hope for a smaller molecular weight. Therefore, the average heavy atom binding energy was selected, and the formula is as follows:

[0039] Score = docking score / number of heavy atoms

[0040] Its meaning is the docking score divided by the number of heavy atoms in the molecule. Using this score for sorting helps to find the structure with the highest binding efficiency.

[0041] Post-treatment: Sort according to the average heavy atom binding energy, and at the same time count the residue numbers of the interaction between the target protein and the molecule. Do not select the molecules that bind to residues 150-162 with low confidence. Investigate the pharmacological and toxicological properties of the remaining molecules.

[0042] The modeling agent benzimidazole determined in the final result is the parent structure of 4,5-dimethyl-1H-benzimidazole. The chemical structural formula of benzimidazole is as follows:

[0043]

[0044] The chemical structural formula of the parent structure of 4,5-dimethyl-1H-benzimidazole is as follows:

[0045]

[0046] Step 2: Under the breeding conditions of temperature: 28.5°C, 14 hours of light / 10 hours of darkness, administer benzimidazole solution to zebrafish. The administration concentration of the benzimidazole solution is 200-300 μg / ml. The administration method of the benzimidazole solution is immersion administration, and a blank control group is set up.

[0047] Step 3: After the experimental animals are treated with the benzimidazole solution, conduct experimental verification through pathological morphology and behavioral methods.

[0048] Step 4: Compared with the control group in terms of pathological morphology and behavioral indicators, the cerebral ventricles of the experimental animals are significantly enlarged and the motor ability is significantly decreased. If this condition is met, the construction of the primary ciliary dyskinesia hydrocephalus model is successful.

[0049] Example 2

[0050] (1) Preparation of zebrafish larvae

[0051] Wild-type zebrafish are all from the Chinese Zebrafish Resource Center and are maintained according to the standard protocol. The zebrafish are maintained at 28°C, 14 hours of light / 10 hours of darkness. Zebrafish embryos are obtained by natural mating and further washed and stored in a culture solution (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4) containing 2 mg / L methylene blue. The pH of the culture medium is maintained at 7.1 and changed daily.

[0052] (2) Experimental grouping and treatment

[0053] a. Model group: Zebrafish embryos 20 hours after fertilization were randomly transferred to a 24-well cell culture plate, with 10 eggs per well. The embryos were exposed to 200 μg / ml and 300 μg / ml benzimidazole solutions, which were prepared from E3 solution (5 mM NaCl, 0.17 mM KCl, 0.4 mM CaCl2, and 0.33 mM MgSO4).

[0054] b. Blank control group: zebrafish embryos fertilized 20 hours ago were randomly transferred to a 24-well cell culture plate, with 10 eggs per well, and the embryos were placed in E3 solution.

[0055] (3) Imaging the zebrafish ventricle using a stereomicroscope

[0056] Embryos were embedded in low-melting-point agarose at 54 hours of development and ready for use. After the stereomicroscope was set up, images of the zebrafish ventricles were acquired. Ventricular size was measured using ImageJ.

[0057] In summary, the animal model of primary ciliary dyskinesia-hydrocephalus prepared by the present invention allows real-time monitoring of the extent of hydrocephalus by measuring ventricular size and detecting motility levels during research. Therefore, the modeling process of the present invention is a dynamic one, allowing observation of ventricular dilation at different time points. This provides an excellent experimental platform for studying primary ciliary dyskinesia-hydrocephalus and a valuable tool for developing novel treatments.

[0058] Therefore, the present invention adopts the above-mentioned method for constructing a primary ciliary dyskinesia hydrocephalus model, adopts a non-invasive method to construct a primary ciliary dyskinesia hydrocephalus model, and successfully establishes a primary ciliary dyskinesia hydrocephalus model by immersing the animal model in benzimidazole while reducing damage to the animal model. The primary ciliary dyskinesia hydrocephalus model has good stability and repeatability, is objective and simple, has strong operability, and reduces other possible interference factors, providing a necessary experimental basis for studying the mechanism of primary ciliary dyskinesia hydrocephalus and the development of treatment methods.

[0059] 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 the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for constructing a primary ciliary dyskinesia hydrocephalus model, characterized in that, It includes the following steps: Step 1: Screen modeling agent compounds based on the substructural protein DRC8 of the innexin-dynein regulatory complex. The screening database is the CNS central nervous system drug database. Screen the compounds according to the free binding energy. Further screen the top 100 compounds in combination with medicine and toxicology to determine benzimidazole as the modeling agent; Step 2: Under normal feeding conditions, administer benzimidazole solution to the experimental animals and set up a blank control group; Step 3: After the experimental animals are treated with benzimidazole solution, conduct experimental verification through pathological morphology and behavioral methods; Step 4: Compared with the control group in terms of pathological morphology and behavioral indicators, if the ventricles of the experimental animals are significantly enlarged and the motor ability is significantly decreased, and this condition is met, the construction of the primary ciliary dyskinesia hydrocephalus model is successful.

2. The method for constructing a primary ciliary dyskinesia hydrocephalus model according to claim 1, wherein: In Step 1, the benzimidazole is 4,5-dimethyl-1H-benzimidazole.

3. A method for constructing a primary ciliary dyskinesia hydrocephalus model according to claim 1, characterized in that: In Step 2, the feeding conditions are temperature: 28.5 °C, 14 hours of light / 10 hours of darkness.

4. A method for constructing a primary ciliary dyskinesia hydrocephalus model according to claim 1, characterized in that: In Step 2, the experimental animals are zebrafish.

5. A method for constructing a primary ciliary dyskinesia hydrocephalus model according to claim 1, characterized in that: In Step 2, the administration concentration of the benzimidazole solution is 200 μg / ml or 300 μg / ml.

6. The method for constructing a primary ciliary dyskinesia hydrocephalus model according to claim 5, characterized in that: The administration concentration of the benzimidazole solution is 300 μg / ml.

7. A method for constructing a primary ciliary dyskinesia hydrocephalus model according to claim 1, characterized in that: In Step 2, the administration method of the benzimidazole solution is immersion administration.

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