A method for constructing a precise and controllable animal model of congenital scoliosis

Abstract

The present invention relates to the field of biomedical technology, and provides a method for constructing an animal model of congenital scoliosis by precisely controlling the location of vertebral deformity through micromanipulation. The present invention blocks the signal conduction that regulates the development of specific vertebrae during a specific developmental period by directional implantation of non-permeable materials into the developing chicken embryo, causing the vertebra to form developmental deformities and maintain normal development at non-intervention sites. The location of the vertebral deformity is precisely and controllably constructed at a set location, making the construction of the scoliosis model more targeted, and facilitating the study of the molecular regulatory mechanism of the formation of congenital scoliosis in different segments such as the cervical, thoracic, and lumbar vertebrae. The present invention solves the current problem of being unable to obtain an animal model of congenital scoliosis that is precise, controllable, and selective in the cervical, thoracic, and lumbar segments, and provides a reliable tool for studying the pathogenesis of congenital scoliosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a method for constructing a precise and controllable congenital scoliosis animal model. Background Art

[0002] Scoliosis is a three-dimensional deformity of the spine characterized by loss of its normal physiological curvature, resulting in abnormal spinal alignment and vertebral rotation. Scoliosis primarily includes idiopathic scoliosis, congenial scoliosis (CS), and neuromuscular scoliosis. It can cause deformities of the spine, ribs, and thorax, severely impacting patients' physical and mental health. CS is a major type of scoliosis, with an incidence of 0.5% to 1% of cases. It presents at an early age, is characterized by severe vertebral rotation, spinal stiffness, and rapid progression of deformity. Clinically, CS can be divided into three types: Type I: vertebral formation disorders, including hemivertebrae and butterfly vertebrae; Type II: vertebral malsegmentation, including blocky vertebrae, blocked vertebrae, or bony bridges; and Type III: a mixed type (combining symptoms of Types I and II). The etiology of CS remains unclear, including the key molecular regulatory mechanisms. Furthermore, the lack of efficient and convenient animal models for CS has hindered research into the causes and mechanisms of CS.

[0003] There are currently available methods for constructing animal models of scoliosis, such as: (1) internal and external spinal tethering methods, which involve tethering the inferior angle of the scapula of young rats to the pelvis on the same side of the body, or tethering adjacent ribs, or wearing a scoliosis vest mold to create idiopathic scoliosis; (2) surgical pinealectomy method, which involves surgically removing the pineal gland of mice or chickens to reduce the release of melatonin, thereby causing scoliosis; the idiopathic scoliosis animal model constructed by this method is far from congenital scoliosis with vertebral deformity and is not suitable for studying the pathogenesis of congenital scoliosis.

[0004] Existing methods for creating animal models of congenital scoliosis primarily include gene editing, nutrient restriction, and exposure to hypoxia. Gene editing for scoliosis (CS) models involves knocking out scoliosis-susceptibility genes (such as TBX6, DLL3, MESP2, LFNG, and HES7), thereby creating a mouse model of congenital scoliosis. This method facilitates the study of genetic influences on scoliosis, but the location of scoliosis cannot be controlled, making it impossible to accurately create animal models of scoliosis with cervical, thoracic, or lumbar vertebral deformities. This limits researchers' understanding of the molecular mechanisms regulating scoliosis formation at different spinal segments or associated symptoms (such as cardiopulmonary function). Nutrient restriction for CS models involves limiting vitamin A intake during pregnancy, leading to vitamin A deficiency, which can cause CS (vitamin A is converted to retinoic acid, which plays an important role in osteochondral development during the embryonic period). This method can mimic CS caused by malnutrition or vitamin deficiency during pregnancy, but similarly, it cannot control the location of spinal deformity. The CS model is constructed by exposing pregnant female mice to a hypoxic environment, either by creating an intrauterine hypoxic environment or by exposing them to a carbon monoxide environment (carbon monoxide competes with oxygen for hemoglobin binding sites, interfering with tissue oxygenation and leading to a hypoxic environment). This method is suitable for studying CS caused by intrauterine hypoxia in newborns. However, because mice are reptiles and cannot stand and walk, they cannot simulate the effects of gravity on the degree of scoliosis, nor can they control the location of scoliosis.

[0005] Existing methods for constructing animal models of scoliosis cannot control the location of scoliosis, which restricts research on the specific pathogenesis of congenital scoliosis at different segments. Therefore, it is extremely necessary to develop animal models of congenital scoliosis that can accurately, controllably, and selectively construct cervical, thoracic, and lumbar vertebral deformities. Summary of the Invention

[0006] The main purpose of the present invention is to provide a method for constructing a precise and controllable congenital scoliosis animal model, as well as a congenital scoliosis animal model constructed using the construction method and its application, aiming to provide a congenital scoliosis animal model that can accurately, controllably and selectively construct cervical, thoracic and lumbar vertebral deformities, to make up for the defects of the existing scoliosis animal model in which the location of scoliosis is uncontrollable, and to provide a more precise animal model tool for the study of congenital scoliosis.

[0007] The present invention, by implanting an impermeable material into developing egg embryos, blocks signaling that regulates the development of specific spinal vertebrae during specific developmental periods, maintains relative normality in other non-interventional locations and the contralateral spinal segments, and precisely and controllably positions vertebral deformities at predetermined locations. This allows for more targeted construction of congenital scoliosis models, allowing for precise construction of congenital scoliosis models of the cervical, thoracic, and lumbar vertebrae, enabling segmental research into the molecular regulatory mechanisms underlying the formation of congenital scoliosis in these cervical, thoracic, and lumbar vertebrae. The present invention addresses the current difficulty in accurately, controllably, and selectively constructing animal models of congenital scoliosis in the cervical, thoracic, and lumbar segments.

[0008] The present invention uses chickens as a model animal for several reasons: first, fertilized chicken eggs are readily available and can be incubated in vitro. Embryo manipulation can be performed by opening a window in the eggshell, and postoperative incubation allows for continuous incubation and dynamic observation of the embryo's development. This allows the selective and precise microscopic insertion of non-permeable materials, such as PET (polyethylene terephthalate) film, at different developmental stages and spinal segments of the chicken embryo's spine. This allows intervention in intertissue interactions during early embryonic development and the construction of congenital scoliosis models with cervical, thoracic, or lumbar vertebral deformities, which are difficult to achieve with other mammalian models. Second, chicken eggs are low-cost, incubation conditions are simple, and the incubation cycle is short, making it relatively convenient and efficient to obtain animal models of congenital scoliosis with vertebral deformities in the cervical, thoracic, and lumbar segments. Third, compared to quadrupeds, chickens can stand and walk, and their spines are affected by gravity, which more realistically and accurately simulates the course of scoliosis in humans when the spine is subject to the influence of gravity during upright walking.

[0009] To achieve the above objectives, the present invention provides a method for constructing an animal model of congenital scoliosis with precise and controllable vertebral deformity formation sites. Non-permeable materials are directionally implanted into the developing chicken embryo to hinder the development of vertebrae during the cervical, thoracic and / or lumbar development periods (blocking the conduction of unilateral signals that regulate vertebral development during the cervical, thoracic and / or lumbar development periods). The model is obtained after the chicken embryo's bone formation or embryonic development is completed.

[0010] The construction method is used to block the conduction of unilateral signals regulating vertebral development during cervical vertebrae development, thereby hindering the development of the cervical vertebrae. After the chicken embryo's bone formation or embryonic development is completed, a congenital cervical scoliosis animal model is obtained.

[0011] The construction method is used to block the conduction of unilateral signals regulating vertebral development during the thoracic vertebrae development period, thereby hindering the development of the thoracic vertebrae. After the chicken embryo's skeleton is formed or the embryonic development is completed, a congenital thoracic scoliosis animal model is obtained.

[0012] The construction method is used to block the conduction of unilateral signals regulating vertebral development during the lumbar vertebrae development period, thereby hindering the development of the lumbar vertebrae. After the chicken embryo's skeleton is formed or the embryonic development is completed, an animal model of congenital lumbar scoliosis is obtained.

[0013] The construction method is used to hinder the development of vertebrae during the development period of the cervical vertebrae, thoracic vertebrae and / or lumbar vertebrae, and to induce deformity of a single vertebra or multiple vertebrae.

[0014] The construction method of the present invention can construct a congenital scoliosis model that is highly similar to the clinical classification (type I, type II, type III), and can be constructed in batches and industrialized and used for scientific research on congenital scoliosis. The construction method of the present invention is not subject to ethical restrictions on animal experiments.

[0015] Preferably, the non-permeable material is any material that can hinder signal conduction during cervical, thoracic and / or lumbar vertebrae development (blocking the conduction of unilateral signals that regulate vertebrae development during cervical, thoracic and / or lumbar vertebrae development).

[0016] More preferably, the impermeable material is polyethylene terephthalate (PET) film, plastic wrap, or the like. When selecting an impermeable insert, a control experiment must be conducted. After removing the vitelline membrane, the selected material is placed on the dorsal side of the embryo and incubated until the desired developmental stage. The embryo is observed for developmental abnormalities to confirm the suitability of the insert. The impermeable material must first be non-toxic, and preliminary experiments must be conducted to demonstrate that the insert itself does not induce deformities.

[0017] Preferably, the thickness of the non-permeable material is 0.002-0.003 mm.

[0018] Preferably, the polyethylene terephthalate (PET) film is an aluminized film.

[0019] Preferably, the period of directed implantation is specifically as follows: during the cervical vertebra development period, the directional implantation is at the 18th to 20th somite stage of the chick embryo; during the thoracic vertebra development period, the directional implantation is at the 24th to 26th somite stage of the chick embryo; during the lumbar vertebra development period, the directional implantation is at the 29th to 31st somite stage of the chick embryo.

[0020] Further preferably, the period of the directed implantation is specifically as follows: during the cervical vertebra development period, the directed implantation is at the 19th somite stage of the chick embryo; during the thoracic vertebra development period, the directed implantation is at the 25th somite stage of the chick embryo; during the lumbar vertebra development period, the directed implantation is at the 30th somite stage of the chick embryo.

[0021] Preferably, in the construction method, the targeted implantation refers to implantation between the somites and the neural tube and notochord of the chicken embryo, thereby blocking signal conduction between the somites and the neural tube and notochord.

[0022] Further preferably, the targeted implantation refers to implantation between a single segment or a unilateral segment of a multi-segment body and the neural tube and the notochord.

[0023] Further preferably, in the construction method, deformities can be induced in the cervical, thoracic and lumbar segments separately or simultaneously, and the position of the directional implantation is implanted between the unilateral somites and the neural tube and notochord in the 14th to 19th somites, the 19th to 25th somites and / or the 26th to 30th somites of the chicken embryo, thereby blocking the conduction of unilateral signals that regulate vertebral development during spinal development, leading to vertebral dysplasia of the spine and the formation of congenital scoliosis.

[0024] Further preferably, in the construction method, the purpose is to induce deformities in multiple vertebrae separately or simultaneously, and the position of the directional implantation is to implant between the unilateral somite of any one or more of the 14th to 19th somite segments, the 19th to 25th somite segments and / or the 26th to 30th somite segments of the chicken embryo and the neural tube and the notochord.

[0025] Further preferably, in the construction method, the position of the directional implantation is implanted between the unilateral somite and the neural tube and notochord in the 14th to 19th somite of the chicken embryo, thereby blocking the conduction of unilateral signals regulating vertebral development during the cervical vertebrae development period, leading to cervical vertebrae dysplasia and formation of congenital scoliosis of the cervical segment.

[0026] Further preferably, in the construction method, the position of the directional implantation is implantation between a unilateral somite of any one or more somites from the 14th to 19th somites of the chicken embryo and the neural tube and notochord.

[0027] Further preferably, in the construction method, the position of the directional implantation is implanted between the unilateral somite and the neural tube and notochord in the 19th to 25th somite of the chicken embryo, thereby blocking the conduction of unilateral signals regulating vertebral development during the thoracic vertebrae development period, leading to thoracic vertebral hypoplasia and formation of congenital scoliosis of the thoracic segment.

[0028] Further preferably, in the construction method, the position of the directional implantation is implantation between a unilateral somite of any one or more somites from the 19th to 25th somites of the chicken embryo and the neural tube and notochord.

[0029] Further preferably, in the construction method, the position of the directional implantation is implanted between the unilateral somite and the neural tube and notochord in the 26th to 30th somite of the chicken embryo, thereby blocking the conduction of the unilateral signal regulating vertebral development during the lumbar development period, leading to lumbar vertebral dysplasia and forming congenital scoliosis of the lumbar segment.

[0030] Further preferably, in the construction method, the position of the directional implantation is implantation between the neural tube and the notochord of any one or more somites from the 26th to 30th somites of the chicken embryo on one side.

[0031] Preferably, the construction method comprises the following steps:

[0032] S1. Chicken embryo incubation to a specific period

[0033] incubating the eggs until the cervical vertebrae development period, the thoracic vertebrae development period, or the lumbar vertebrae development period;

[0034] S2, insert

[0035] cutting the connection between the unilateral somites of the cervical segment, thoracic segment and / or lumbar segment of the chicken embryo at the cervical vertebra development stage, thoracic vertebra development stage or lumbar vertebra development stage in step S1, and inserting a non-permeable material insert;

[0036] S3. Obtain model

[0037] Seal the micromanipulation window on the eggshell to keep the internal environment of the egg stable, and continue incubation until the chicken embryo bones are formed or the embryonic development is completed, and then the corresponding animal model is obtained.

[0038] In step S2, the connection between the unilateral somite of the chicken embryo during the cervical vertebra development period and the neural tube and notochord is cut, and a non-permeable material insert is inserted to construct a congenital cervical scoliosis model.

[0039] In step S2, the connection between the unilateral somite of the chicken embryo during the thoracic vertebra development period and the neural tube and notochord is cut, and a non-permeable material insert is inserted to construct a congenital thoracic scoliosis model.

[0040] In step S2, the connection between the unilateral somite of the chicken embryo during the lumbar vertebrae development period and the neural tube and notochord is cut, and a non-permeable material insert is inserted to construct a congenital lumbar scoliosis model.

[0041] Preferably, in step S1, the eggs are freshly laid fertilized eggs.

[0042] Preferably, in step S1, the cervical vertebra development period is the 18-20 somite stage of the chicken embryo; the thoracic vertebra development period is the 24-26 somite stage of the chicken embryo; and the lumbar vertebra development period is the 29-31 somite stage of the chicken embryo.

[0043] Further preferably, in step S1, the cervical vertebra development period is the 19-somite stage of the chicken embryo; the thoracic vertebra development period is the 25-somite stage of the chicken embryo; and the lumbar vertebra development period is the 30-somite stage of the chicken embryo.

[0044] Preferably, the non-permeable material is any material that can block signal conduction during cervical, thoracic and / or lumbar vertebrae development (blocking the conduction of unilateral signals that regulate vertebrae development during cervical, thoracic and / or lumbar vertebrae development).

[0045] More preferably, the impermeable material is polyethylene terephthalate (PET) film, plastic wrap, or the like. When selecting an impermeable insert, a control experiment must be conducted. After removing the vitelline membrane, the selected material is placed on the dorsal side of the embryo and incubated until the desired developmental stage. The embryo is observed for developmental abnormalities to confirm the suitability of the insert. The impermeable material must first be non-toxic, and preliminary experiments must be conducted to demonstrate that the insert itself does not induce deformities.

[0046] Preferably, the thickness of the non-permeable material is 0.002-0.003 mm.

[0047] Preferably, the polyethylene terephthalate (PET) film is an aluminized film.

[0048] Preferably, in step S2, the connection between the somites and the neural tube and notochord in the 14th to 19th somites of the chicken embryo during the cervical vertebrae development period is cut, and an insert is inserted; the signal conduction between the somites and the neural tube and notochord is cut off; resulting in cervical vertebrae dysplasia and the formation of congenital scoliosis of the cervical segment.

[0049] Further preferably, in step S2, the connection between the somites and the neural tube and notochord in the 14th to 19th somites of the 18-20 somite chicken embryo is cut, and an insert is inserted; the signal conduction between the somites and the neural tube and notochord is cut off; resulting in cervical vertebral hypoplasia and the formation of congenital scoliosis of the cervical segment.

[0050] Further preferably, in step S2, the connection between the somites and the neural tube and notochord in the 14th to 19th somites of the 19-somite chicken embryo is cut, and an insert is inserted; the signal conduction between the somites and the neural tube and notochord is cut off; resulting in cervical vertebral hypoplasia and the formation of congenital scoliosis of the cervical segment.

[0051] Preferably, in step S2, the connection between the somites and the neural tube and notochord in the 19th to 25th somites of the chicken embryo during the thoracic vertebrae development period is cut, and an insert is inserted; the signal conduction between the somites and the neural tube and notochord is cut off; resulting in thoracic vertebrae dysplasia and the formation of congenital scoliosis of the thoracic segment.

[0052] Further preferably, in step S2, the connection between the somites and the neural tube and notochord in the 19th to 25th somites of the chicken embryo at the 24-26 somite stage is cut, and an insert is inserted; the signal conduction between the somites and the neural tube and notochord is cut off; resulting in thoracic vertebral hypoplasia and the formation of congenital scoliosis of the thoracic segment.

[0053] Further preferably, in step S2, the connection between the somites and the neural tube and notochord in the 19th to 25th somites of the 25-somite chicken embryo is cut, and an insert is inserted; the signal conduction between the somites and the neural tube and notochord is cut off; resulting in thoracic vertebral hypoplasia and the formation of congenital scoliosis of the thoracic segment.

[0054] Preferably, in step S2, the connection between the somites and the neural tube and notochord in the 26th to 30th somites of the chicken embryo during the lumbar vertebrae development period is cut, and an insert is inserted; the signal conduction between the somites and the neural tube and notochord is cut off; resulting in lumbar vertebrae dysplasia and the formation of lumbar congenital scoliosis.

[0055] Further preferably, in step S2, the connection between the somites and the neural tube and notochord in the 26th to 30th somites of the 29th to 31st somite stage chicken embryo is cut, and an insert is inserted; the signal conduction between the somites and the neural tube and notochord is cut off; resulting in lumbar vertebral dysplasia and the formation of lumbar congenital scoliosis.

[0056] Further preferably, in step S2, the connection between the somites and the neural tube and notochord in the 26th to 30th somites of the 30-somite chicken embryo is cut, and an insert is inserted; the signal conduction between the somites and the neural tube and notochord is cut off; resulting in lumbar vertebral dysplasia and the formation of lumbar congenital scoliosis.

[0057] Preferably, in step S3, a sterile transparent film is used to seal the micromanipulation window on the eggshell to maintain a stable environment inside the egg and avoid water loss or bacterial contamination.

[0058] Preferably, in step S3, the incubation is continued until the chicken embryo is a 9-day embryo and the skeleton is formed; the incubation is continued until the shell is broken and the embryonic development is complete.

[0059] To achieve the above-mentioned purpose, the present invention provides an animal model of congenital scoliosis constructed using the construction method.

[0060] To achieve the above-mentioned purpose, the present invention provides the application of the congenital scoliosis animal model in the study of congenital scoliosis.

[0061] The present invention provides a method for constructing an animal model of congenital scoliosis that is precisely and controllable. Non-permeable materials are implanted in the developing chicken embryo to block the conduction of unilateral signals that regulate vertebral development during the development of the cervical, thoracic and / or lumbar vertebrae, while maintaining other non-intervention positions and the contralateral spinal segments relatively normal. The position of the scoliosis is precisely and controllably constructed at a set position. After the chicken embryo's bone formation or embryonic development is completed, an animal model of congenital cervical, thoracic or lumbar scoliosis is obtained. This provides a reliable research model for segmental studies on the molecular regulatory mechanisms of congenital scoliosis formation in the cervical, thoracic and lumbar vertebrae. The congenital scoliosis animal model constructed by the construction method of the present invention is of great help to research in the field of congenital scoliosis research, and accelerates and promotes research on the pathogenesis and signal pathways of congenital scoliosis in different segmental vertebrae. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 Schematic diagram of the structure of an egg according to an embodiment of the present invention.

[0063] Figure 2 Schematic diagram of opening a small hole at the tip of an egg (A) and extracting the egg white (B) according to an embodiment of the present invention.

[0064] Figure 3 A schematic diagram of an operating field of view exposed by an embodiment of the present invention.

[0065] Figure 4 This is a schematic diagram of a PET insert according to an embodiment of the present invention.

[0066] Figure 5 Schematic diagram of the insert for constructing a cervical CS model (A), a schematic diagram of the insert for constructing a thoracic CS model (B), and a schematic diagram of the insert for constructing a lumbar CS model (C) according to an embodiment of the present invention. a is an enlarged view of the portion within the frame in A; b is an enlarged view of the portion within the frame in B; and c is an enlarged view of the portion within the frame in C.

[0067] Figure 6 This is a schematic diagram of the closed eggshell operating window in an embodiment of the present invention.

[0068] Figure 7 This is the deformity of the congenital scoliosis model according to an embodiment of the present invention.

[0069] Figure 8 Schematic diagram of one vertebra deformed (A); schematic diagram of two vertebra deformed (B) according to an embodiment of the present invention.

[0070] The CS model in the accompanying drawings of the present invention is a congenital scoliosis model. DETAILED DESCRIPTION

[0071] Pelikan ink, FOUNT INDIA (originating from India).

[0072] Aluminum-plated film (for capacitors, Oji Paper Company), thickness 0.002-0.003mm. If this insert is used, the metal on the film surface must be dissolved with hydrochloric acid solution before use.

[0073] Plastic wrap: Dongguan Innovation Electronic Materials Co., Ltd., thickness 0.002mm.

[0074] The present invention provides a method for constructing an animal model of congenital scoliosis by precisely controlling the location of vertebral deformity through micromanipulation. By implanting an impermeable material into a developing egg embryo, the signal transduction that regulates the development of a specific vertebra during a specific developmental period is blocked, while other non-interventional locations and the contralateral spinal segment remain relatively normal. The location of the vertebral deformity is precisely and controllably constructed at a set location, making the construction of the animal model of congenital scoliosis more targeted. Thus, congenital scoliosis models of the cervical, thoracic, and lumbar vertebrae are precisely constructed, enabling segmental research on the molecular regulatory mechanisms of congenital scoliosis formation in the cervical, thoracic, and lumbar vertebrae. The present invention solves the current problem of being unable to accurately, controllably, and selectively construct animal models of congenital scoliosis in the cervical, thoracic, and lumbar segments; it overcomes the shortcomings of existing scoliosis models in which the location of scoliosis cannot be controlled, and provides a more precise animal model tool for the study of congenital scoliosis.

[0075] The present invention provides a method for constructing an animal model of congenital scoliosis with precise and controllable location of vertebral deformity formation. Non-permeable materials are directionally implanted into the developing chicken embryo to hinder the development of vertebrae during the cervical, thoracic and / or lumbar developmental periods (blocking the transmission of unilateral signals that regulate vertebral development during the cervical, thoracic and / or lumbar developmental periods). The model is obtained after the chicken embryo's bone formation or embryonic development is completed.

[0076] The construction method is used to block the conduction of unilateral signals regulating vertebral development during cervical vertebrae development, thereby hindering the development of the cervical vertebrae. After the chicken embryo's bone formation or embryonic development is completed, a congenital cervical scoliosis animal model is obtained.

[0077] The construction method is used to block the conduction of unilateral signals regulating vertebral development during the thoracic vertebrae development period, thereby hindering the development of the thoracic vertebrae. After the chicken embryo's skeleton is formed or the embryonic development is completed, a congenital thoracic scoliosis animal model is obtained.

[0078] The construction method is used to block the conduction of unilateral signals regulating vertebral development during the lumbar vertebrae development period, thereby hindering the development of the lumbar vertebrae. After the chicken embryo's skeleton is formed or the embryonic development is completed, an animal model of congenital lumbar scoliosis is obtained.

[0079] The construction method is used to hinder the development of vertebrae during the development period of the cervical vertebrae, thoracic vertebrae and / or lumbar vertebrae, and to induce deformity of a single vertebra or multiple vertebrae.

[0080] Optionally, the impermeable material is any material capable of hindering signal transmission during cervical, thoracic, and / or lumbar vertebral development (blocking the transmission of unilateral signals regulating vertebral development during cervical, thoracic, and / or lumbar development). Examples of such materials include polyethylene terephthalate (PET) film, plastic wrap, and the like. When selecting an impermeable material for insertion, a control experiment must be conducted. After removing the vitelline membrane, the selected material is placed on the dorsal side of the embryo and incubated until the desired developmental stage. The embryo is observed for developmental abnormalities to confirm the feasibility of the insert. The impermeable material must first be non-toxic, and preliminary experiments must be conducted to demonstrate that the insert itself does not induce deformities.

[0081] Optionally, the thickness of the non-permeable material is 0.002-0.003 mm.

[0082] Optionally, the polyethylene terephthalate (PET) film is an aluminized film.

[0083] Optionally, the cervical vertebrae are implanted at the 18th to 20th somite stage of the chick embryo during development; the thoracic vertebrae are implanted at the 24th to 26th somite stage of the chick embryo during development; and the lumbar vertebrae are implanted at the 29th to 31st somite stage of the chick embryo during development.

[0084] Optionally, during the development period of the cervical vertebrae, the implantation is at the 19th somite stage of the chick embryo; during the development period of the thoracic vertebrae, the implantation is at the 25th somite stage of the chick embryo; and during the development period of the lumbar vertebrae, the implantation is at the 30th somite stage of the chick embryo.

[0085] Alternatively, the targeted implantation refers to implantation between the somites and the neural tube and notochord of the chick embryo to block signal conduction between the somites and the neural tube and notochord. The scope of targeted implantation is not limited and can be implantation between a single somites or a unilateral somites of multiple somites and the neural tube and notochord.

[0086] Alternatively, the implantation is performed between a unilateral somite in the chick embryo's 14th to 19th somite, 19th to 25th somite, and / or 26th to 30th somite and the neural tube and notochord, thereby blocking the conduction of unilateral signals that regulate vertebral development during spinal column development, leading to vertebral dysplasia and congenital scoliosis. The implantation can be performed between a unilateral somite in any one or more of the chick embryo's 14th to 19th somite, 19th to 25th somite, and / or 26th to 30th somite and the neural tube and notochord.

[0087] Alternatively, the implant is placed between the neural tube and notochord on one side of the chick embryo's 14th to 19th somites, blocking the unilateral signaling that regulates vertebral development during cervical vertebrae development, leading to cervical spondylosis and congenital scoliosis. The implant is placed between the neural tube and notochord on one side of any one or more of the 14th to 19th somites in the chick embryo.

[0088] Alternatively, the implant is performed between the neural tube and the notochord on one side of the chick embryo's 19th to 25th somite, blocking the unilateral signaling that regulates vertebral development during thoracic vertebrae development, leading to thoracic spondylosis and congenital scoliosis of the thoracic spine. The implant is performed between the neural tube and the notochord on one side of any one or more of the 19th to 25th somite segments of the chick embryo.

[0089] Alternatively, the implant is performed between the neural tube and notochord on one side of the chick embryo's 26th to 30th somite, blocking the unilateral signaling that regulates vertebral development during lumbar development, leading to lumbar spondylosis and congenital lumbar scoliosis. The implant is performed between the neural tube and notochord on one side of any one or more somite segments 26th to 30th in the chick embryo.

[0090] Optionally, the construction method comprises the following steps:

[0091] S1. Chicken embryo incubation to a specific period

[0092] incubating the eggs until the cervical vertebrae development period, the thoracic vertebrae development period, or the lumbar vertebrae development period;

[0093] S2, insert

[0094] cutting the connection between the unilateral somites of the cervical segment, thoracic segment and / or lumbar segment of the chicken embryo at the cervical vertebra development stage, thoracic vertebra development stage or lumbar vertebra development stage in step S1, and inserting a non-permeable material insert;

[0095] S3. Obtain model

[0096] Seal the micromanipulation window on the eggshell to keep the internal environment of the egg stable, and continue incubation until the chicken embryo bones are formed or the embryonic development is completed, and then the corresponding animal model is obtained.

[0097] In step S2, the connection between the unilateral somite of the chicken embryo during the cervical vertebra development period and the neural tube and notochord is cut, and a non-permeable material insert is inserted to construct a congenital cervical scoliosis model.

[0098] In step S2, the connection between the unilateral somite of the chicken embryo during the thoracic vertebra development period and the neural tube and notochord is cut, and a non-permeable material insert is inserted to construct a congenital thoracic scoliosis model.

[0099] In step S2, the connection between the unilateral somite of the chicken embryo during the lumbar vertebrae development period and the neural tube and notochord is cut, and a non-permeable material insert is inserted to construct a congenital lumbar scoliosis model.

[0100] Optionally, in step S1, the eggs are freshly laid fertilized eggs.

[0101] Optionally, in step S1, the cervical vertebra development period is the 18-20 somite stage of the chicken embryo; the thoracic vertebra development period is the 24-26 somite stage of the chicken embryo; and the lumbar vertebra development period is the 29-31 somite stage of the chicken embryo.

[0102] Optionally, in step S1, the cervical vertebra development period is the 19-somite stage of the chicken embryo; the thoracic vertebra development period is the 25-somite stage of the chicken embryo; and the lumbar vertebra development period is the 30-somite stage of the chicken embryo.

[0103] Optionally, the impermeable material is any material capable of blocking signal transmission during cervical, thoracic, and / or lumbar vertebral development (blocking the transmission of unilateral signals that regulate vertebral development during cervical, thoracic, and / or lumbar development). Examples of such materials include polyethylene terephthalate (PET) film, plastic wrap, and the like. When selecting an impermeable material for insertion, a control experiment must be conducted. After removing the vitelline membrane, the selected material is placed dorsally on the embryo and incubated until the desired developmental stage. The embryo is observed for developmental abnormalities to confirm the feasibility of the insert. The impermeable material must first be non-toxic, and preliminary experiments must be conducted to demonstrate that the insert itself does not induce deformities.

[0104] Optionally, the thickness of the non-permeable material is 0.002-0.003 mm.

[0105] Optionally, the polyethylene terephthalate (PET) film is an aluminized film.

[0106] Optionally, in step S2, the connection between the somites of the 14th to 19th somites of the chicken embryo at the cervical vertebrae development stage and the neural tube and notochord is cut, and a plug is inserted; the signal transmission between the somites and the neural tube and notochord is cut off; resulting in cervical vertebrae dysplasia, forming cervical congenital scoliosis. In one embodiment, the connection between the somites of the 14th to 19th somites of the chicken embryo at the 18-20 somite stage and the neural tube and notochord is cut, and a plug is inserted; the signal transmission between the somites and the neural tube and notochord is cut off; resulting in cervical vertebrae dysplasia, forming cervical congenital scoliosis. In one embodiment, the connection between the somites of the 14th to 19th somites of the chicken embryo at the 19-somite stage and the neural tube and notochord is cut, and a plug is inserted; the signal transmission between the somites and the neural tube and notochord is cut off; resulting in cervical vertebrae dysplasia, forming cervical congenital scoliosis.

[0107] Optionally, in step S2, the connection between the somites of the 19th to 25th somites of the chicken embryo at the thoracic vertebrae development stage and the neural tube and notochord is cut, and a plug is inserted; the signal transmission between the somites and the neural tube and notochord is cut off; resulting in thoracic vertebrae dysplasia, forming thoracic congenital scoliosis. In one embodiment, the connection between the somites of the 19th to 25th somites of the chicken embryo at the 24th to 26th somites and the neural tube and notochord is cut, and a plug is inserted; the signal transmission between the somites and the neural tube and notochord is cut off; resulting in thoracic vertebrae dysplasia, forming thoracic congenital scoliosis. In one embodiment, the connection between the somites of the 19th to 25th somites of the chicken embryo at the 25th somites and the neural tube and notochord is cut, and a plug is inserted; the signal transmission between the somites and the neural tube and notochord is cut off; resulting in thoracic vertebrae dysplasia, forming thoracic congenital scoliosis.

[0108] Optionally, in step S2, the connection between the somites of the 26th to 30th somites of the chicken embryo at the lumbar development stage and the neural tube and notochord is cut, and a plug is inserted; the signal transmission between the somites and the neural tube and notochord is cut off; resulting in lumbar spondylosis, forming lumbar congenital scoliosis. In one embodiment, the connection between the somites of the 26th to 30th somites of the chicken embryo at the 29th to 31st somites and the neural tube and notochord is cut, and a plug is inserted; the signal transmission between the somites and the neural tube and notochord is cut off; resulting in lumbar spondylosis, forming lumbar congenital scoliosis. In one embodiment, the connection between the somites of the 26th to 30th somites of the chicken embryo at the 30th somites and the neural tube and notochord is cut, and a plug is inserted; the signal transmission between the somites and the neural tube and notochord is cut off; resulting in lumbar spondylosis, forming lumbar congenital scoliosis.

[0109] Optionally, in step S3, a sterile transparent film is used to seal the micromanipulation window on the eggshell to maintain a stable internal environment of the egg and avoid water loss or bacterial contamination.

[0110] Optionally, in step S3, after the operation is completed, the incubation is continued until the chicken embryo is a 9-day embryo and the skeleton is formed; the incubation is continued until the shell is broken and the embryonic development is completed, thereby obtaining the corresponding animal model.

[0111] The construction method of the present invention inserts non-permeable materials into different somite levels of chicken embryos at different developmental stages, cutting off the signal transmission between the somites and the neural tube and notochord, thereby achieving the precise and controllable construction of a congenital scoliosis chicken embryo model with cervical, thoracic or lumbar vertebral deformity.

[0112] The construction method of the present invention can construct a congenital scoliosis model that is highly similar to the clinical classification (type I, type II, type III), and can be constructed in batches and industrialized and used for scientific research on congenital scoliosis. The construction method of the present invention is not subject to ethical restrictions on animal experiments.

[0113] The present invention provides a congenital scoliosis animal model constructed using the construction method.

[0114] The present invention also provides application of the congenital scoliosis animal model in congenital scoliosis research.

[0115] The congenital scoliosis animal model of the present invention makes up for the defect of the existing scoliosis model in which the location of scoliosis is uncontrollable, provides a more accurate model for the study of congenital scoliosis, greatly helps the research in the field of congenital scoliosis, and accelerates and promotes the research on the pathogenesis and signal pathways of congenital scoliosis in different segmental vertebral deformities.

[0116] Example 1 Construction of an animal model of congenital scoliosis

[0117] A method for constructing a precise and controllable congenital scoliosis animal model, comprising the following steps:

[0118] 1. Egg Hatching

[0119] Select freshly laid fertilized eggs (the structure diagram of the egg is as follows Figure 1 As shown in the figure, lay the egg horizontally to ensure that the blastoderm is on top of the yolk, draw a mark just above the eggshell, and incubate it in an incubator with a constant temperature (38°C) and constant humidity (60%-70%) environment. Incubate it to the 19-somite stage (corresponding to the Hamburger Hamilton Stages 13, or HH stage 13, which is commonly used internationally to describe the period of chicken embryo development), the 25-somite stage (HH stage 15), and the 30-somite stage (HH stage 17).

[0120] 2. Aspirate egg white

[0121] After the eggs have incubated to the various somatic stages, remove the eggs and place them horizontally (with the eggshell marking facing upwards to ensure that the embryo is located in the middle and upper part of the egg to avoid damage to the embryo during operation). Use the tip of a pair of small scissors to poke a small hole at the tip of the egg (not the air chamber end) about 1 cm away from the apex of the tip. Use a syringe (with the needle tip facing the tip to avoid piercing the yolk) to aspirate 2 ml of egg white to lower the position of the chicken embryo and reserve space between the eggshell and the embryo to facilitate the next embryo operation. After the aspiration is completed, heat medical paraffin and drip it on the small hole in the eggshell to seal the egg and maintain the integrity of the eggshell. The schematic diagram of the small hole in the tip of the egg is as follows: Figure 2 As shown in A, the schematic diagram of extracting egg white is as follows Figure 2 As shown in B.

[0122] 3. Exposure of the operating field and confirmation of the developmental period

[0123] Draw a circular mark with a radius of 2 cm with the mark just above the eggshell as the center. Use the tip of a small pair of scissors to make an opening at the center of the eggshell. Use pointed tweezers to clamp the eggshell one by one from the inside out along the opening until the circular mark is reached, completely exposing the embryo to the operating field for easy subsequent operation. The schematic diagram of the exposed operating field is shown below. Figure 3 shown.

[0124] Prepare a color ink by mixing 4 drops of Pelikan with 5 ml of Tyrode's solution. Use a homemade glass pipette to draw an appropriate amount of color ink. Insert the pipette into the yolk along the outer side of the embryonic vascular network. Slowly move the pipette tip under the embryo and squeeze in a small amount of color ink as a background color. Observe the number of somites under a microscope to confirm the developmental stage. Select chick embryos of the appropriate developmental stage for the procedure.

[0125] 4. Microscopic inserts

[0126] A small amount of Tyrode's solution is dripped onto the chick embryo to moisten it. The vitelline membrane is peeled off with a tungsten needle. Under a microscope, the right side of the corresponding somites that form the cervical, thoracic or lumbar vertebrae is cut open between the neural tube and the notochord. An aluminum-coated PET insert is inserted into the incision, ensuring that the PET insert reaches the endoderm position and completely blocks the signal transmission between the tissues (somites, neural tube and notochord). The structure between the contralateral somites, neural tube and notochord is intact and serves as the control side. Figure 4 shown.

[0127] The specific PET insert and blocking positions are as follows:

[0128] (1) Construction of cervical vertebrae CS model: A chick embryo at the 19th somite stage (HH stage 13) was selected, and a PET insert of approximately 0.2 mm × 1 mm was inserted between the right somites of the 14th to 19th somites (cervical vertebrae primordium) of the chick embryo and the neural tube and notochord to block the signal transduction that regulates the development of the spinal vertebrae during development, leading to cervical vertebrae dysplasia and congenital scoliosis. The schematic diagram of the insert for constructing the cervical vertebrae CS model is shown in the figure. Figure 5 As shown in A; a is an enlarged view of the part in the frame of A;

[0129] (2) Construction of thoracic vertebrae CS model: Select a chicken embryo at the 25-somite stage (HH stage 15), and insert a PET insert of approximately 0.2 mm × 1 mm into the right somites of the 19th to 25th somites (thoracic vertebrae primordium) of the chicken embryo, between the neural tube and the notochord, to block the signal transduction that regulates the development of the spinal vertebrae during development, resulting in thoracic vertebrae dysplasia and congenital scoliosis. The schematic diagram of the insert for constructing the thoracic vertebrae CS model is shown in the figure. Figure 5 As shown in B; b is an enlarged view of the part in the frame of B;

[0130] (3) Construction of lumbar CS model: A chick embryo at the 30-somite stage (HH stage 17) was selected, and a PET insert of approximately 0.2 mm × 1 mm was inserted between the right somites of the 26th to 30th somites (lumbar vertebrae primordium) of the chick embryo and the neural tube and notochord to block the signal transduction that regulates the development of the spinal vertebrae during development, resulting in lumbar vertebrae dysplasia and congenital scoliosis. The schematic diagram of the insert for constructing the lumbar CS model is shown in the figure. Figure 5 As shown in C; c is an enlarged view of the part in the frame of C;

[0131] 5. Close the surgical wound

[0132] After the operation is completed, the opened chicken embryo structure (yolk membrane, etc.) is placed back to the position before the operation, the position and posture of the chicken embryo are adjusted, and the operation window of the eggshell is sealed with a sterile transparent film (to facilitate observation of embryonic development) to maintain a stable environment inside the egg and avoid water loss or bacterial contamination; the schematic diagram of the closed eggshell operation window is shown as follows Figure 6 shown.

[0133] 6. Obtain the model

[0134] The operated chicken embryos were incubated until day 9, when the bones were basically formed, and a chicken embryo model of congenital scoliosis was obtained.

[0135] The bones of the 9-day embryo were stained and the soft tissues were transparentized to observe the deformity. The deformity of the congenital scoliosis model is as follows: Figure 7 As shown;

[0136] from Figure 7 It can be seen that the animal models of congenital cervical, thoracic and lumbar scoliosis were successfully constructed.

[0137] The present invention can achieve scoliosis deformity in single or multiple vertebrae by controlling the position of the insert. The PET insert covers half of each of the two segments, thereby inducing a vertebral deformity, as shown in the schematic diagram. Figure 8 As shown in A; the PET insert covers three segments, half of each of the two segments at both ends, thereby inducing deformity of two vertebrae, as shown in the schematic diagram Figure 8 As shown in B.

[0138] Example 2 Construction of Congenital Scoliosis Animal Model

[0139] A method for constructing a precise and controllable congenital scoliosis animal model, comprising the following steps:

[0140] 1. Egg Hatching

[0141] The eggs were hatched in the same manner as in Example 1.

[0142] 2. Aspirate egg white

[0143] The egg white was aspirated as in Example 1.

[0144] 3. Exposure of the operating field and confirmation of the developmental period

[0145] The operation is the same as that in Example 1 for visual field exposure and developmental period confirmation;

[0146] 4. Microscopic inserts

[0147] A small amount of Tyrode's solution was dripped onto the chick embryo to moisten it. The vitelline membrane was peeled off with a tungsten needle. Under a microscope, an incision was made between the left somites of the corresponding somites that form the cervical, thoracic, or lumbar vertebrae and the neural tube and notochord. An aluminum-coated PET insert was inserted into the incision, ensuring that the PET insert reached the endoderm to completely block signal transmission between the tissues (somites, neural tube, and notochord). The structures between the somites, neural tube, and notochord on the contralateral side were intact and served as the control side.

[0148] The specific locations for PET inserts to be inserted and blocked are as follows:

[0149] (1) Construction of cervical CS model: A chick embryo at the 19th somite stage (HH stage 13) was selected, and a PET insert of approximately 0.2 mm × 1 mm was inserted between the left somites of the 14th to 19th somites (cervical vertebrae primordium) of the chick embryo and the neural tube and notochord to block the signal transduction that regulates the development of the spinal vertebrae during development, resulting in cervical vertebrae hypoplasia and the formation of congenital scoliosis (CS);

[0150] (2) Construction of a thoracic CS model: A chick embryo at the 25-somite stage (HH stage 15) was selected, and a PET insert of approximately 0.2 mm × 1 mm was inserted between the left somites of the 19th to 25th somites (thoracic vertebrae primordium) of the chick embryo and the neural tube and notochord. This blocked the signal transduction that regulates the development of the spinal vertebrae during development, leading to thoracic vertebrae hypoplasia and congenital scoliosis.

[0151] (3) Construction of a lumbar CS model: A chick embryo at the 30-somite stage (HH stage 17) was selected, and a PET insert of approximately 0.2 mm × 1 mm in size was inserted between the left somites, neural tube, and notochord of the 26th to 30th somites (lumbar vertebral primordium) of the chick embryo to block the signal transduction that regulates the development of the spinal vertebrae during development, leading to lumbar vertebral hypoplasia and congenital scoliosis. The difference between this step of the present embodiment and that of embodiment 1 is that the position of the microsurgical incision is changed to between the left somites, neural tube, and notochord.

[0152] 5. Close the surgical wound

[0153] The operation is the same as the sealing operation in Example 1.

[0154] 6. Obtain the model

[0155] The chick embryos were incubated until day 9, when the skeleton was essentially formed, creating a chick embryo model of congenital scoliosis. The bones of the 9-day embryos were stained and the soft tissues were transparentized. Based on the observed deformities, animal models of congenital scoliosis of the cervical, thoracic, and lumbar spine were successfully constructed.

[0156] Example 3 Construction of Congenital Scoliosis Animal Model

[0157] A method for constructing a precise and controllable congenital scoliosis animal model, comprising the following steps:

[0158] 1. Egg Hatching

[0159] The eggs were hatched in the same manner as in Example 1.

[0160] 2. Aspirate egg white

[0161] The operation is the same as that of aspirating egg white in Example 2.

[0162] 3. Exposure of the operating field and confirmation of the developmental period

[0163] The operation is the same as that in Example 1, with regard to exposure of the visual field and confirmation of the developmental period.

[0164] 4. Microscopic inserts

[0165] The operation is the same as that of the microscopy insert in Example 1.

[0166] 5. Close the surgical wound

[0167] The operation is the same as the sealing operation in Example 1.

[0168] 6. Obtain the model

[0169] The manipulated chicken embryos were incubated until hatching (approximately 21 days) to form chicks, completing embryonic development and creating a chicken model for congenital scoliosis. The chick bones were stained, and based on the observed deformities, animal models for congenital cervical, thoracic, and lumbar scoliosis were successfully constructed. This example differs from Example 1 in that the model obtained is a fully developed chick model.

[0170] Example 4 Construction of Congenital Scoliosis Animal Model

[0171] A method for constructing a precise and controllable congenital scoliosis animal model, comprising the following steps:

[0172] 1. Egg Hatching

[0173] Freshly laid fertilized eggs were selected and placed horizontally, ensuring that the blastoderm was located on top of the yolk. A mark was drawn directly above the eggshell, and the eggs were placed in an incubator with a constant temperature (38°C) and constant humidity (60%-70%) environment for incubation. The eggs were incubated to the 18-segment stage, the 24-segment stage, and the 29-segment stage, respectively. This step in this embodiment differs from that in embodiment 1 in that the incubation period is different.

[0174] 2. Aspirate egg white

[0175] The operation is the same as that of aspirating egg white in Example 2.

[0176] 3. Exposure of the operating field and confirmation of the developmental period

[0177] The operation is the same as that in Example 1, with regard to exposure of the visual field and confirmation of the developmental period.

[0178] 4. Microscopic inserts

[0179] The operation is the same as that of the microscopy insert in Example 1.

[0180] 5. Close the surgical wound

[0181] The operation is the same as the sealing operation in Example 1.

[0182] 6. Obtain the model

[0183] The chick embryos were incubated until day 9, when the skeleton was essentially formed, creating a chick embryo model of congenital scoliosis. The bones of the 9-day embryos were stained and the soft tissues were transparentized. Based on the observed deformities, animal models of congenital scoliosis of the cervical, thoracic, and lumbar spine were successfully constructed.

[0184] Example 5 Construction of Congenital Scoliosis Animal Model

[0185] A method for constructing a precise and controllable congenital scoliosis animal model, comprising the following steps:

[0186] 1. Egg Hatching

[0187] Freshly laid fertilized eggs were selected and placed horizontally, ensuring that the blastoderm was located on top of the yolk. A mark was drawn directly above the eggshell, and the eggs were placed in an incubator with a constant temperature (38°C) and constant humidity (60%-70%) environment for incubation. The eggs were incubated to the 20th, 26th, and 31st somites stages, respectively. This step in this embodiment differs from that in embodiment 1 in that the incubation periods are different.

[0188] 2. Aspirate egg white

[0189] The operation is the same as that of aspirating egg white in Example 2.

[0190] 3. Exposure of the operating field and confirmation of the developmental period

[0191] The operation is the same as that in Example 1, with regard to exposure of the visual field and confirmation of the developmental period.

[0192] 4. Microscopic inserts

[0193] The operation is the same as that of the microscopic insert in Example 1, except that the aluminized film PET insert is replaced with a plastic wrap insert of corresponding size.

[0194] 5. Close the surgical wound

[0195] The operation is the same as the sealing operation in Example 1.

[0196] 6. Obtain the model

[0197] The chick embryos were incubated until day 9, when the skeleton was essentially formed, creating a chick embryo model of congenital scoliosis. The bones of the 9-day embryos were stained and the soft tissues were transparentized. Based on the observed deformities, animal models of congenital scoliosis of the cervical, thoracic, and lumbar spine were successfully constructed.

[0198] The above are only preferred embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention description and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for constructing an animal model of congenital scoliosis with a precisely controlled vertebral deformity formation site, characterized in that: Non-permeable materials are implanted in the chick embryo to hinder the development of vertebrae during the cervical, thoracic and / or lumbar development periods. Once the chick embryo skeleton is formed or embryonic development is complete, an animal model of scoliosis at the corresponding position can be obtained. The non-permeable material is one of polyethylene terephthalate film and plastic wrap; The directional implantation refers to implantation between the unilateral somites in the 14th to 19th somites, the 19th to 25th somites and / or the 26th to 30th somites of the chicken embryo and the neural tube and the notochord; The specific period of the directed implantation is as follows: during the cervical vertebra development period, the directed implantation is at the 18th to 20th somite stage of the chick embryo; during the thoracic vertebra development period, the directed implantation is at the 24th to 26th somite stage of the chick embryo; and during the lumbar vertebra development period, the directed implantation is at the 29th to 31st somite stage of the chick embryo.

2. The construction method according to claim 1, wherein It can induce deformities in the cervical, thoracic and lumbar segments separately or simultaneously.

3. The construction method according to claim 1 or 2, characterized in that The steps include: The chicken eggs are incubated until the cervical vertebrae development period, the thoracic vertebrae development period, or the lumbar vertebrae development period; then, the connection between the unilateral somites of the cervical segment, thoracic segment, and / or lumbar segment of the chicken embryo during the cervical vertebrae development period, the thoracic vertebrae development period, and / or the lumbar vertebrae development period and the neural tube and the notochord is cut through micromanipulation, and a non-permeable material insert is directionally implanted; then, the micromanipulation window on the eggshell is closed to maintain a stable internal environment of the egg, and the incubation is continued until the chicken embryo skeleton is formed or the embryonic development is completed, thereby obtaining the corresponding animal model.

4. The construction method according to claim 3, wherein: The connection between the unilateral somite, neural tube and notochord in the 14th to 19th somite of the chicken embryo during the cervical vertebra development period is cut, and a non-permeable material insert is directionally implanted.

5. The construction method according to claim 3, wherein: The connection between the unilateral somite, the neural tube and the notochord in the 19th to 25th somite of the chicken embryo during the thoracic vertebra development period is cut, and a non-permeable material insert is directionally implanted.

6. The construction method according to claim 3, wherein: The connection between the unilateral somite, the neural tube and the notochord in the 26th to 30th somite of the chicken embryo during the lumbar vertebra development period is cut, and a non-permeable material insert is directionally implanted.

7. Use of a congenital scoliosis animal model constructed using the construction method according to any one of claims 1 to 6 in congenital scoliosis research.

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