Congenital scoliosis animal model construction method and application thereof

Abstract

The invention relates to the technical field of construction methods of animal disease models, in particular to a construction method of a congenital scoliosis animal model and application of the construction method. The invention provides a construction method of a congenital scoliosis animal model, which is characterized in that an animal is induced to form a congenital scoliosis disease model by simulating environmental factors exposed by organophosphorus pesticides. According to the method, the situation that people are directly or indirectly exposed to the organophosphorus pesticide in daily life can be truly simulated, and the animal model of the congenital scoliosis caused by the influence of the organophosphorus pesticide environmental factors is constructed; an efficient model tool is provided for exploring pathogenesis, treatment targets and prevention and treatment means of congenital scoliosis caused by organophosphorus pesticide exposure.

Description

Technical Field

[0001] The present application belongs to the technical field of methods for constructing animal disease models, and in particular, relates to a method for constructing a congenital scoliosis animal model and its application. Background Art

[0002] Scoliosis is a three-dimensional deformity of the spine in which the spine loses its normal physiological curvature and has abnormal curvature and rotation. Scoliosis mainly includes three categories: idiopathic scoliosis, congenital scoliosis and neuromuscular scoliosis, which can cause deformity and damage to the spine, ribs and thorax, seriously affecting the physical and mental health of patients. Congenital scoliosis is a relatively important category of scoliosis, with an incidence rate of 0.5‰ to 1‰. It has the earliest onset age, severe vertebral rotation, spinal stiffness and rapid deformity progression. Clinically, congenital scoliosis can be divided into three types: type I: vertebral formation disorder, including hemivertebrae, butterfly vertebrae, and wedge vertebrae; type II: vertebral segmentation malformation, including block vertebrae, blocked vertebrae or bone bridges; type III: mixed type, combined with symptoms of type I and type II. The cause of congenital scoliosis is still unclear. It is generally believed that it is affected by both genetic and environmental factors.

[0003] Based on the etiology of congenital scoliosis, the methods for constructing animal models of congenital scoliosis are mainly as follows: 1) Gene editing, constructing a congenital scoliosis model: knocking out the susceptibility gene of congenital scoliosis can construct a congenital scoliosis model mouse, but this type of construction method only considers the influence of genetic factors on scoliosis, ignoring the role of environmental factors in the formation of congenital scoliosis. 2) Restricting nutrient intake, constructing a congenital scoliosis model: restricting the intake of vitamin A in pregnant female mice, resulting in vitamin A deficiency, which can lead to the occurrence of congenital scoliosis (vitamin A can be converted into retinoic acid, which plays an important role in the development of bone and cartilage during the embryonic period). This model can simulate congenital scoliosis caused by insufficient nutrition or vitamin intake during pregnancy. 3) Exposure to hypoxic environment, constructing a congenital scoliosis model: giving pregnant female mice an intrauterine hypoxic environment, or exposing pregnant female mice to a carbon monoxide environment, can construct a congenital scoliosis model for offspring mice. This method is suitable for studying congenital scoliosis caused by intrauterine hypoxia in newborns. However, there is still a large type of congenital scoliosis affected by environmental factors, and there is no mature method to replicate the pathological characteristics of this type of disease - congenital scoliosis caused by exposure to organophosphorus pesticides.

[0004] my country is a large agricultural country, and people need to use a large number of organophosphorus pesticides in the process of growing crops. This also leads to people often coming into contact with various organophosphorus pesticides and their residues in daily life; the contact routes include skin contact, respiratory inhalation, and digestive ingestion. Long-term exposure to organophosphorus pesticides may increase the risk of congenital defects (such as neural tube defects, spina bifida, and congenital scoliosis) in the fetus, but the relationship between the onset of congenital scoliosis and exposure to organophosphorus pesticides is still unclear. There are few studies on the pathogenesis of congenital scoliosis and exposure to organophosphorus pesticides, and there is no method to simulate the animal model of congenital scoliosis caused by exposure to organophosphorus pesticides. This limits the research on the important pathogenic mechanism of congenital scoliosis induced by harmful environmental factors of organophosphorus pesticides, and also affects the in-depth exploration of the treatment targets and prevention and control methods of congenital scoliosis. Summary of the invention

[0005] The purpose of the present application is to provide a method for constructing an animal model of congenital scoliosis and its application, aiming to solve the problem that the method for constructing an animal model of congenital scoliosis provided in the prior art cannot simulate the impact of the environmental factors of organophosphorus pesticides on congenital scoliosis.

[0006] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0007] In a first aspect, the present application provides a method for constructing an animal model of congenital scoliosis. The construction method induces animals to form a disease model of congenital scoliosis by simulating environmental factors of organophosphorus pesticide exposure.

[0008] In some embodiments, the organophosphorus pesticide includes at least one of phoxim, chlorpyrifos, trichlorfon, dichlorvos, dimethoate, dicrotophos, and thiophanate-methyl.

[0009] In some embodiments, the animal is selected from birds.

[0010] In some embodiments, the congenital scoliosis animal model is characterized by a congenital scoliosis animal model with vertebral deformity, wherein the vertebral deformity includes at least one of vertebral malsegmentation, hemivertebra, butterfly vertebra, and wedge vertebra.

[0011] In some embodiments, the construction method comprises the following steps:

[0012] Providing fertilized eggs of animals, and incubating the fertilized eggs for a certain period; wherein the certain period includes any one of a gastrula period, a somite formation period, and a somite differentiation period;

[0013] The suspension of organophosphorus pesticides is injected into the yolk of the incubated fertilized eggs, and the eggs are incubated until the bones are formed, thereby constructing an animal model of congenital scoliosis.

[0014] In some embodiments, in the step of injecting the suspension of the organophosphorus pesticide into the yolk of the incubated fertilized eggs, the concentration of the suspension of the organophosphorus pesticide injected into each fertilized egg is 1.1 mg / kg to 11.0 mg / kg.

[0015] In some embodiments, the volume of the suspension of the organophosphorus pesticide injected into each fertilized egg is 100 μL to 400 μL.

[0016] In some embodiments, in the suspension of the organophosphorus pesticide, the mass percentage concentration of the organophosphorus pesticide is 0.2 wt % to 1 wt %.

[0017] In a second aspect, the present application provides a congenital scoliosis animal model, and the congenital scoliosis animal model is constructed by a method for constructing a congenital scoliosis animal model.

[0018] In a third aspect, the present application provides the use of the above-mentioned congenital scoliosis animal model in the study of the pathogenesis, therapeutic targets and prevention and treatment measures of congenital scoliosis induced by harmful environmental factors of organophosphorus pesticides.

[0019] In a fourth aspect, the present application provides the use of the method for constructing an animal model of congenital scoliosis as described above in constructing an animal disease model caused by environmental factors.

[0020] The first aspect of the present application provides a method for constructing an animal model of congenital scoliosis, which induces animals to form a disease model of congenital scoliosis by simulating environmental factors of organophosphorus pesticide exposure. The method can more realistically simulate the situation of people being directly or indirectly exposed to organophosphorus pesticides in their daily lives (using phosphorus-containing pesticides, fertilizers, or agricultural products with phosphorus-containing toxic substances residues), and construct an animal model of congenital scoliosis caused by the environmental factors of organophosphorus pesticides, providing an efficient model tool for studying the pathogenesis, treatment targets, and prevention and control measures of congenital scoliosis caused by organophosphorus pesticide exposure.

[0021] The second aspect of the present application provides an animal model of congenital scoliosis, which is constructed by the provided method for constructing an animal model of congenital scoliosis. The obtained animal model of congenital scoliosis is a disease model affected by the environmental factors of organophosphorus pesticides, and can be used to study the pathogenesis and therapeutic targets of congenital scoliosis induced by harmful environmental factors caused by such organophosphorus pesticides, and is helpful to explore corresponding prevention and treatment measures.

[0022] The third aspect of the present application provides an animal model of congenital scoliosis for use in the study of the pathogenesis, therapeutic targets and prevention and control measures of congenital scoliosis induced by harmful environmental factors of organophosphorus pesticides. Since the animal model of congenital scoliosis constructed in the present application is more consistent with the pathogenesis of congenital scoliosis caused by human exposure to environmental factors of organophosphorus pesticides, the use of this model for research is conducive to systematic research and analysis of the pathogenesis of environmental factors of congenital scoliosis, and targeted exploration of prevention and control measures for congenital scoliosis caused by exposure to harmful environmental factors of organophosphorus pesticides.

[0023] The fourth aspect of the present application provides the application of the method for constructing an animal model of congenital scoliosis as described above in constructing an animal disease model caused by environmental factors. The method for constructing the animal disease model of the present application is simple and easy to operate, and the obtained animal disease model has good effect, which provides a new idea for the construction of animal models of corresponding diseases caused by other environmental factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 This is a structural analysis diagram of an egg provided in an embodiment of the present application.

[0026] Figure 2 It is a schematic diagram of the construction process of the chicken embryo congenital scoliosis model provided in the examples of the present application.

[0027] Figure 3 This is a cross-sectional analysis diagram of the thoracolumbar segment of a 9-day-old chicken embryo provided in an example of the present application.

[0028] Figure 4 This is a sagittal analysis diagram of the spine of a 9-day chicken embryo provided in the examples of the present application.

[0029] Figure 5 This is a coronal analysis diagram of the spine of a 9-day chicken embryo provided in the examples of the present application.

[0030] Figure 6 This is a schematic diagram of the spine X-ray of a 15-day chicken embryo provided in an embodiment of the present application.

[0031] Figure 7 This is a schematic diagram of the spine X-ray of a 21-day chicken embryo provided in the examples of the present application. DETAILED DESCRIPTION

[0032] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0033] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0034] In this application, "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0035] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0036] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "an" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0037] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass in the embodiment description of the present application can be μg, mg, g, kg and other mass units known in the chemical industry.

[0038] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0039] A first aspect of an embodiment of the present application provides a method for constructing an animal model of congenital scoliosis. The construction method induces animals to form a disease model of congenital scoliosis by simulating environmental factors of organophosphorus pesticide exposure.

[0040] The first aspect of the embodiment of the present application provides a method for constructing an animal model of congenital scoliosis, which induces animals to form a disease model of congenital scoliosis by simulating the environmental factors of organophosphorus pesticide exposure. The method can more realistically simulate the situation of people being directly or indirectly exposed to organophosphorus pesticides in their daily lives (using phosphorus-containing pesticides, fertilizers, or agricultural products with phosphorus-containing toxic substances residues), and construct an animal model of congenital scoliosis caused by the environmental factors of organophosphorus pesticides, providing an efficient model tool for studying the pathogenesis, treatment targets, and prevention and control measures of congenital scoliosis caused by organophosphorus pesticide exposure.

[0041] In some embodiments, the organophosphorus pesticide includes at least one of phoxim, chlorpyrifos, trichlorfon, dichlorvos, dimethoate, dicrotophos, and thiophanate-methyl.

[0042] In some specific embodiments, the environmental factors of simulated organophosphorus pesticide exposure are environmental factors of simulated phoxim exposure. Phoxim is an organophosphorus pesticide with a chemical formula of C 12 H 15 N 2 O 3 PS, mainly acts as a contact and stomach poison, is very effective against Lepidoptera larvae. Phoxim has a broad insecticide spectrum and strong knockdown power. It is often used to control underground pests. It has a long residual period and is a residual pesticide that is easily exposed in daily life.

[0043] In some embodiments, a phoxim pesticide suspension is used to induce an animal model of congenital scoliosis, wherein the phoxim pesticide suspension used is a 50-500-fold dilution of an analytically pure phoxim reagent. In some specific embodiments, the phoxim pesticide suspension used is a 100-fold dilution of an analytically pure phoxim reagent.

[0044] In some embodiments, the congenital scoliosis animal model is characterized by a congenital scoliosis animal model with vertebral deformity, wherein the vertebral deformity includes at least one of vertebral malsegmentation, hemivertebra, butterfly vertebra, and wedge vertebra.

[0045] In some embodiments, the animal is selected from poultry. Among them, poultry includes any one of poultry and flying birds. In some specific embodiments, chickens are selected as congenital scoliosis model animals. The advantages of choosing chickens as congenital scoliosis model animal research include: ① The fertilized eggs of chickens are eggs, which can be hatched in an in vitro environment, and the injection of drugs is convenient, and it is easy to achieve exposure to the toxic substance environment of organophosphorus pesticides; ② Eggs have low cost, simple incubation conditions, and short incubation cycles, and it is relatively convenient to obtain congenital scoliosis animal models; ③ Chickens can stand and walk, and the spine is affected by gravity, which more realistically and accurately simulates the evolution process of the human body when the spine is affected by gravity when walking upright; ④ After selecting chickens as congenital scoliosis model animals, after induction with corresponding organophosphorus pesticides, the induced spinal deformities show typical characteristics of congenital scoliosis, such as malsegmentation of vertebrae, hemivertebrae, butterfly vertebrae, wedge-shaped vertebrae, etc., and the lordosis and scoliosis of the spine are clearly observed.

[0046] In some embodiments, the construction method comprises the following steps:

[0047] S01. Providing fertilized eggs of animals, and incubating the fertilized eggs for a certain period; wherein the certain period includes any period of the gastrula period, the somite formation period, and the somite differentiation period;

[0048] S02. Injecting a suspension of organophosphorus pesticides into the yolk of the incubated fertilized eggs, and continuing to incubate until the bones are formed, thereby constructing an animal model of congenital scoliosis.

[0049] In step S01, a fertilized egg of an animal is provided. In some embodiments, the fertilized egg of an animal is selected from any one of chicken eggs, duck eggs, and goose eggs.

[0050] Further, the fertilized eggs are incubated for a certain period; wherein the certain period includes any one of the gastrula period, the somite formation period, and the somite differentiation period.

[0051] In some embodiments, if the fertilized egg provided is a chicken egg, the incubation method is: provide a constant temperature and humidity condition of 37°C to 39°C and a humidity of 60% to 70%, place the egg horizontally with its long axis and mark the top of the eggshell (to ensure that the embryo is located in the upper middle part of the egg) for incubation.

[0052] After fertilization and incubation for 24 hours, the yolk of the egg develops into a vesicle and the embryo gradually forms. After the embryo is formed, as the incubation time increases, the chicken embryo development period includes the gastrula period, the somite formation period, the somite differentiation period, the vertebral precursor formation period, the cartilage period, etc. In the process of inducing animals to form an animal model of congenital scoliosis in the embodiment of the present application, it is necessary to expose them to environmental conditions containing organophosphorus pesticides before their vertebral precursor formation period in order to construct an animal model with congenital scoliosis symptoms. Since the spine of the chicken embryo gradually takes shape during the vertebral precursor formation period, if the environmental conditions containing organophosphorus pesticides are provided during the vertebral precursor formation period and thereafter for induction incubation, it may not be possible to obtain an animal model of congenital scoliosis.

[0053] Therefore, incubating fertilized eggs to any of the gastrula period, somitogenesis period, and somitogenesis differentiation period, and exposing them to environmental factors containing organophosphorus pesticides for induction and cultivation can ensure that an animal model with congenital scoliosis characteristics is obtained. However, due to the different susceptibility of embryos at different developmental stages, the earlier the embryonic development stage, such as the gastrula period or the somitogenesis period, the greater the impact on it, and the more obvious the spinal deformity of the obtained animal model, but the disadvantage is that the survival rate is low; if the environmental conditions containing organophosphorus pesticides are provided for cultivation at a later embryonic development stage, such as the somitogenesis differentiation period, the impact is small, and the spinal deformity effect of the obtained animal model is not too obvious, but the animal survival rate is high.

[0054] In addition, it should be noted that due to the different time periods of embryonic development in different species, this application uses the Hamburger Hamliton stages (HH Stage) method commonly used internationally to describe the embryonic development period of avian birds for labeling. The time nodes of the HH Stage corresponding to each developmental period discussed above are roughly as follows: gastrula stage (corresponding HHStage4~6); somite formation period (corresponding HH Stage15~17); somite differentiation period (corresponding HH Stage19~21). If other avian species are used to construct an animal model, the HH Stage method can be used to control the time point of exposure to environmental factors of organophosphorus pesticides during the step of incubating fertilized eggs.

[0055] In step S02, the suspension of the organophosphorus pesticide is injected into the yolk of the fertilized egg after incubation (the structure of the fertilized egg is as shown in the attached Figure 1 The incubation was continued until the bones were formed, thereby constructing an animal model of congenital scoliosis.

[0056] Furthermore, the step of injecting the suspension of the organophosphorus pesticide into the yolk of the incubated fertilized egg further includes the following steps:

[0057] G01. Disinfect the surface with 75% ethanol;

[0058] G02. Aspirate egg white: Place the egg horizontally (with the eggshell mark facing upwards to ensure that the embryo is located in the upper middle part of the egg to avoid damaging the embryo during operation). Make a small hole about 1 cm from the tip of the egg (not the air chamber end). Use a syringe (with the needle tip facing the tip to avoid piercing the yolk) to aspirate 1 ml of egg white to reserve space for the injection of the drug solution.

[0059] In some embodiments, the organophosphorus pesticide suspension used is prepared by diluting an analytically pure phoxim reagent to 50 to 500 times the concentration of a commercial phoxim pesticide. In some specific embodiments, the organophosphorus pesticide suspension used is prepared by diluting an analytically pure phoxim reagent to 100 times the concentration of a commercial phoxim pesticide.

[0060] In some embodiments, the mass percentage concentration of the organophosphorus pesticide in the suspension of the organophosphorus pesticide is 0.2wt% to 1wt%. In some specific embodiments, the mass percentage concentration of the organophosphorus pesticide in the suspension of the organophosphorus pesticide includes but is not limited to 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1.0wt%.

[0061] In some embodiments, in the step of injecting the suspension of the organophosphorus pesticide into the yolk of the incubated fertilized egg, the concentration of the suspension of the organophosphorus pesticide injected into each fertilized egg is 1.1 mg / kg to 11.0 mg / kg. If the concentration of the suspension of the organophosphorus pesticide injected into each fertilized egg is too high, the local concentration of the pesticide will be too high in the fertilized egg, which will have an adverse effect on the survival of the embryo and result in a low survival rate of the final animal model; if the concentration of the suspension of the organophosphorus pesticide injected into each fertilized egg is too low, it is impossible to provide exposure to the phoxim pesticide with sufficient efficacy, which may result in a low success rate in constructing the animal model, which is not conducive to conducting related pathological studies.

[0062] In some specific embodiments, the concentration of the suspension of organophosphorus pesticide injected into each fertilized egg includes but is not limited to 1.1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 5.5 mg / kg, 6 mg / kg, 6.5 mg / kg, 7 mg / kg, 7.5 mg / kg, 8 mg / kg, 8.5 mg / kg, 9 mg / kg, 9.5 mg / kg, 10 mg / kg, 10.5 mg / kg, and 11 mg / kg.

[0063] In some embodiments, the volume of the suspension of the organophosphorus pesticide injected into each fertilized egg is 100 μL to 400 μL. If the volume of the suspension of the organophosphorus pesticide injected into each fertilized egg is too small, the dosage of the organophosphorus pesticide cannot be accurately controlled, the operation error is large, and it is not conducive to controlling the severity of spinal deformity formed in the animal model; if the volume of the suspension of the organophosphorus pesticide injected into each fertilized egg is too large, it is easy to expand or break the yolk, and cause the egg white to overflow, affecting the survival of the embryo.

[0064] In some specific embodiments, the volume of the organophosphorus pesticide suspension injected into each fertilized egg includes but is not limited to 100 μL, 150 μL, 200 μL, 250 μL, 300 μL, 350 μL, and 400 μL.

[0065] Furthermore, the step of injecting the organophosphorus pesticide suspension into the yolk of the incubated fertilized egg includes: providing a 1 ml syringe to take 200 uL of a mixed 1% phoxim vegetable oil suspension, slowly inserting the needle tip along the small hole in the eggshell toward the center of the yolk, and slowly pushing the organophosphorus pesticide suspension.

[0066] In some embodiments, the organophosphorus pesticide suspension is a suspension obtained by mixing an organophosphorus pesticide and vegetable oil. Since the density of the phoxim vegetable oil suspension is relatively light, it will float to the bottom of the chicken embryo, creating an environment where the phoxim is exposed. When the embryo absorbs nutrients from the yolk, the phoxim is taken into the chicken embryo.

[0067] Furthermore, after injecting the suspension of organophosphorus pesticides, medical paraffin is heated and then dripped onto the small holes in the eggshell to seal the egg and maintain a stable internal environment of the egg.

[0068] Furthermore, the incubation is continued until the bones are formed, thereby constructing an animal model of congenital scoliosis.

[0069] In some embodiments, the time of embryo incubation is determined according to the specific experimental purpose. The incubation time includes but is not limited to incubation for 7 to 9 days, incubation to the embryonic cartilage formation period; or incubation for 12.5 to 15 days, incubation to the embryonic ossification formation period; or incubation to the hatching period (about 21 days) to obtain an animal model of congenital scoliosis.

[0070] A second aspect of an embodiment of the present application provides a congenital scoliosis animal model, and the congenital scoliosis animal model is constructed by a method for constructing a congenital scoliosis animal model.

[0071] The congenital scoliosis animal model provided in the second aspect of the embodiment of the present application is constructed by the provided method for constructing a congenital scoliosis animal model. The obtained congenital scoliosis animal model is a disease model affected by the environmental factors of organophosphorus pesticides, and can be used to study the pathogenesis and treatment targets of congenital scoliosis diseases induced by harmful environmental factors caused by such organophosphorus pesticides, and is helpful to explore corresponding prevention and treatment measures.

[0072] The third aspect of the embodiments of the present application provides the application of the above-mentioned congenital scoliosis animal model in the study of the pathogenesis, treatment targets and prevention and control measures of congenital scoliosis induced by harmful environmental factors of organophosphorus pesticides.

[0073] The third aspect of the embodiment of the present application provides an animal model of congenital scoliosis for use in the study of the pathogenesis, therapeutic targets and prevention and control measures of congenital scoliosis induced by harmful environmental factors of organophosphorus pesticides. Since the method of constructing the animal model of congenital scoliosis in the present application is similar to the pathogenesis of congenital scoliosis in the fetus due to exposure to environmental factors of organophosphorus pesticides during human pregnancy, the use of this model for research is more conducive to the study and analysis of the pathogenesis of environmental pathogenic factors of congenital scoliosis, and to the targeted exploration of prevention and control measures for congenital scoliosis caused by exposure to environmental factors of organophosphorus pesticides.

[0074] A fourth aspect of the embodiments of the present application provides the application of the method for constructing an animal model of congenital scoliosis as described above in constructing an animal disease model caused by environmental factors.

[0075] The fourth aspect of the embodiments of the present application provides the application of the method for constructing an animal model of congenital scoliosis as described above in constructing an animal disease model caused by environmental factors. The method for constructing the animal disease model of the present application is simple and easy to operate, and the obtained animal disease model has good effect, which provides a new idea for the construction of animal models of corresponding diseases caused by other environmental factors.

[0076] The following describes the invention in conjunction with specific embodiments.

[0077] Example 1

[0078] Chicken embryo model of congenital scoliosis and its construction method

[0079] The chick embryo model of congenital scoliosis induces animals to form an animal model of congenital scoliosis by simulating the environmental factors of phoxim exposure.

[0080] The specific construction method is as follows Figure 2 As shown below:

[0081] ①Select eggs: take the newly laid fertilized eggs;

[0082] ② Incubation temperature and humidity: In a constant temperature and humidity environment with a temperature of 38°C and a humidity of 60%-70%, place the eggs horizontally with their long axis, draw a mark just above the eggshell (to ensure that the embryo is located in the middle and upper part of the egg), and incubate in the incubator for 24 hours;

[0083] ③Disinfection: Disinfect the surface of the eggs with 75% ethanol;

[0084] ④ Aspirate egg white: After 24 hours of incubation, take out the egg and place it horizontally (with the eggshell mark facing upward to ensure that the embryo is located in the middle and upper part of the egg to avoid damaging the embryo during operation). Make a small hole about 1 cm from the tip of the egg (not the air chamber end) and use a syringe (with the needle tip facing the tip to avoid piercing the yolk) to aspirate 1 ml of egg white.

[0085] ⑤ Injection of drugs: Use a 1ml syringe to take 200ul (100-300) of the mixed 1% phoxim vegetable oil suspension, and slowly insert the needle tip toward the center of the yolk along the eggshell hole in step 3, and slowly push the phoxim vegetable oil suspension;

[0086] ⑥ Sealing wax: Heat the medical paraffin and drip it on the small holes in the eggshell to seal the egg and keep the internal environment of the egg stable;

[0087] ⑦ Incubation: 1) The embryos can be incubated until day 9, when the cartilage of the skeletal system has basically formed, and the deformity can be observed after cartilage staining and soft tissue transparency treatment. 2) Or the embryos can be incubated until day 12.5, when ossification is basically complete, and the deformity can be observed through double staining or X-rays. 3) The embryos can also be incubated until hatching (about 21 days) to obtain a chicken model of congenital scoliosis.

[0088] Property analysis and result explanation

[0089] (1) After the embryos in Example 1 were incubated to 9 days, the embryos of the model were analyzed, and the results were as follows:

[0090] The spinal deformity of the 9-day embryo is shown in the attached figure. Figure 3 As shown, Figure 3 (A) is a front view of the entire spine, showing spina bifida and hemivertebrae (circles) in the cervical spine, and scoliosis and wedge-shaped vertebrae (squares) in the thoracolumbar segment. Figure 3 (B) is a lateral view of the entire spine, showing that the cervical vertebral lamina is connected as one piece, the intervertebral segments are poorly segmented (black arrows), and the thoracolumbar segment has lordosis (white arrows). The lordosis of the 9-day embryo was analyzed by tissue section, as shown in Figure 2. Figure 3 (C) As shown in the cross-sectional tissue section of the thoracolumbar segment (cut along line C in Figure A), it can be seen that abnormal bone structure appears in the thoracic vertebral body at the lordosis of the thoracic segment of the 9-day embryo.

[0091] The sagittal plane of the spinal deformity of the 9-day embryo was analyzed, as shown in the attached Figure 4 As shown, Figure 4 (A) is a frontal view of the spine, showing malsegmentation, hemivertebrae and butterfly vertebrae (circles) in the cervical spine, and scoliosis (squares) in the thoracolumbar segment. Figure 4 (B) is a lateral view of the spine, showing that the cervical vertebral lamina are connected as one piece and the intervertebral segments are poorly segmented (arrow); Figure 4 (C) is an analysis of the sagittal section of the spine (cut along line C in Figure A). Figure 4 (D) is an enlarged view of the tissue section of the cervical vertebrae. It can be seen that the cervical vertebrae of the 9-day embryo are poorly segmented and the vertebral plates are connected, which is a typical feature of congenital scoliosis.

[0092] The coronal plane of the spinal deformity of the 9-day embryo was analyzed, as shown in the attached Figure 5 As shown, Figure 5 (A) is a front view of the spine, showing butterfly vertebrae (circles) in the cervical spine, and scoliosis and wedge-shaped vertebrae (squares) in the thoracolumbar region. Figure 5 (B) is a lateral view of the spine. Figure 5 (C) is a coronal section analysis of the superficial layer of the spine (cut along line C in Figure B). Figure 5 (D) is a tissue section analysis diagram of the deep layer of the coronal plane of the spine (cut along line D in Figure B). Figure 5 (E) is an enlarged view of the coronal tissue section of the thoracic and lumbar spine. It can be seen that the gaps between the vertebrae in the thoracic and lumbar segments have disappeared and the vertebrae have fused into pieces, which is a typical manifestation of malsegmentation of the vertebrae in congenital scoliosis.

[0093] The chicks that had completed ossification during the hatching process (15 days) were analyzed, e.g. Figure 6 As shown, Figure 6 A is an X-ray of the front of the chick. Figure 6 B is an X-ray of the chick's side. Figure 6 As indicated by the arrow ① in A, it can be seen that the cervical spine has a half vertebra and a butterfly vertebra. Figure 6 As indicated by the arrow ② in B, it can be seen that the lumbar vertebrae have hemivertebrae. Therefore, this model belongs to type I of congenital scoliosis classification, with vertebral formation disorder.

[0094] The chicks (21 days old) were analyzed. Figure 7 As shown, Figure 7 A is an X-ray of the front of the chick. Figure 7 B is an X-ray of the side of the chicken. According to the indication of the arrow, it can be seen that the cervical vertebrae and spinous processes are fused, which belongs to type II of congenital scoliosis classification and has poor vertebral segmentation.

[0095] In summary, the present application provides a method for constructing an animal model of congenital scoliosis, and the method of constructing an animal model of congenital scoliosis is to induce animals to form an animal model of congenital scoliosis by simulating the environmental factors of organophosphorus pesticide exposure. This method can more realistically simulate the situation of people being directly or indirectly exposed to phosphorus-containing organic pesticides in their daily lives (using phosphorus-containing pesticides, fertilizers, or ingesting agricultural products with phosphorus-containing toxic substance residues), and construct the evolution process of congenital scoliosis caused by the environmental factors of phosphorus-containing organic pesticides, solve the problem that there is currently no animal model of congenital scoliosis caused by organophosphorus pesticide exposure, and provide a theoretical basis for clinical research on the pathogenesis, treatment targets, and prevention and control measures of congenital scoliosis caused by organophosphorus pesticide exposure.

[0096] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for constructing an animal model of congenital scoliosis. It is characterized in that The construction method induces animals to form a disease model of congenital scoliosis by simulating environmental factors of organophosphorus pesticide exposure.

2. The construction method according to claim 1, It is characterized in that The organophosphorus pesticide includes at least one of phoxim, chlorpyrifos, trichlorfon, dichlorvos, dimethoate, dicrotophos and thiophanate-methyl.

3. The construction method according to claim 1, It is characterized in that The animal is selected from birds.

4. The construction method according to claim 1, It is characterized in that The congenital scoliosis animal model is characterized by a congenital scoliosis animal model with vertebral deformity, wherein the vertebral deformity includes at least one of vertebral malsegmentation, hemivertebra, butterfly vertebra or wedge vertebra.

5. The construction method according to any one of claims 1 to 4, It is characterized in that The construction method comprises the following steps: Providing a fertilized egg of an animal, and incubating the fertilized egg to a certain period; wherein the certain period includes any one of a gastrula period, a somite formation period, and a somite differentiation period; The suspension of organophosphorus pesticides is injected into the yolk of the incubated fertilized eggs, and the eggs are incubated until the bones are formed, thereby constructing an animal model of congenital scoliosis.

6. The construction method according to claim 5, It is characterized in that In the step of injecting the suspension of the organophosphorus pesticide into the yolk of the incubated fertilized eggs, the concentration of the suspension of the organophosphorus pesticide injected into each fertilized egg is 1.1 mg / kg to 11.0 mg / kg; and the volume of the suspension of the organophosphorus pesticide injected into each fertilized egg is 100 μL to 400 μL.

7. The construction method according to claim 5, It is characterized in that In the suspension of the organophosphorus pesticide, the mass percentage concentration of the organophosphorus pesticide is 0.2wt% to 1wt%.

8. An animal model of congenital scoliosis, It is characterized in that The congenital scoliosis animal model is constructed by the method for constructing a congenital scoliosis animal model according to any one of claims 1 to 7.

9. Use of the congenital scoliosis animal model as claimed in claim 8 in the study of the pathogenesis, therapeutic targets and prevention and treatment measures of congenital scoliosis induced by harmful environmental factors of organophosphorus pesticides.

10. Use of the method for constructing an animal model of congenital scoliosis according to any one of claims 1 to 7 in constructing an animal disease model caused by harmful environmental factors.

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