A method for constructing a mouse model of optic nerve myelination defect

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to a method for constructing an optic nerve myelin formation defect mouse model. The optic nerve myelin formation defect mouse model is constructed by specifically knocking out the Ezh2 gene in oligodendrocytes to obtain an optic nerve myelin formation defect mouse. An optic nerve crush model is further constructed from the optic nerve myelin formation defect mouse, and the optic nerve crush model can be used to screen drugs that promote the intrinsic growth of neurons, and is used for the study of myelin regeneration after optic nerve injury, optic nerve myelin formation, and related drugs for treating optic nerve demyelinating diseases.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a method for constructing an optic nerve myelin formation defect mouse model. Background Art

[0002] Axon regeneration in the mammalian central nervous system (CNS) is a long-standing and challenging problem in biomedical research. As an important part of the CNS, the optic nerve has always been an important research object for CNS damage and repair. Glaucoma is the most common optic neuropathy caused by damage to axons of retinal ganglion cells (RGCs) and is the leading cause of irreversible blindness worldwide. The symptoms of optic nerve damage in mice are similar to those of glaucoma in humans, and it is the second leading cause of blindness after cataracts. Cataracts can usually be removed by surgery, but there is no effective treatment for glaucoma. In glaucoma, axons are damaged at the optic nerve head; in other optic neuropathies, axons are damaged along the entire visual pathway. Glaucoma is caused by excessive pressure on the optic nerve, and nearly 70 million people worldwide suffer from this disease. Trauma, retinal detachment, pituitary tumors, various brain cancers, and other causes can also cause damage to the optic nerve, leading to decreased vision. Since human and mammalian CNS axons cannot regenerate, once axons are lost, vision loss is irreversible. Traumatic optic neuropathy is relatively rare, usually occurs after head injury, and is severe and irreversible, similar to the optic nerve crush model. Neurons in the mature mammalian CNS cannot regenerate axons after injury for two main reasons: ① the harsh environment caused by the inhibition of scar tissue and myelin degeneration; and ② the weakening of the regenerative capacity of mature CNS neurons. Therefore, a combined strategy that simultaneously targets the intrinsic growth capacity and the inhibitory environment may be the best choice for successfully achieving CNS axon regeneration and functional recovery.

[0003] At present, the longest distance that can promote the regeneration of optic nerve axons through a combination of strategies to promote axon regeneration is about 5 mm, while the axons between adult RGCs and the superior colliculus can be as long as 15 to 20 cm, and the length of the optic nerve of adult mice is also greater than 1 cm. Therefore, the existing technology cannot achieve complete regeneration of damaged optic nerve axons and visual reconstruction. Due to the complex molecular and cellular pathological environment of optic nerve injury, after the optic nerve axon of existing wild-type mice is injured, the myelin sheath in the injured local and distal optic nerves collapses, a large amount of myelin fragments are generated, and a large amount of inhibitory cytokines are secreted. The local microenvironment of the injury is complex, which is not conducive to the regeneration of damaged axons. It is urgent to establish an optimized optic nerve myelination defect mouse model to facilitate subsequent research on optic nerve myelin regeneration and optic nerve axon injury regeneration. Summary of the invention

[0004] In view of the above technical problems, the present invention provides the following technical solutions:

[0005] In a first aspect, the present invention provides a method for constructing an optic nerve myelin formation defect mouse model, wherein the optic nerve myelin formation defect mouse model is obtained by specifically knocking out the Ezh2 gene in oligodendrocytes.

[0006] Preferably, the construction method specifically comprises the following steps:

[0007] 1) Obtain Ezh2 with floxp sites inserted at both ends of exons 14 and 15 of the Ezh2 gene fl / fl Mice;

[0008] 2) Obtain Olig1-Cre mice in which the endogenous Olig1 promoter controls the expression of Cre recombinase;

[0009] 3) Ezh2 fl / fl Mice were crossed with Olig1-Cre mice to obtain Ezh2 fl / + ;Olig1-Cre specific knockout of Ezh2 mice heterozygous generation;

[0010] 4) Ezh2 fl / + ; Olig1-Cre specific knockout of Ezh2 mice heterozygous generation and Ezh2 fl / fl Backcrossing of mice to obtain Ezh2 fl / fl ; Olig1-Cre homozygous specific knockout Ezh2 mice.

[0011] Preferably, the construction method further comprises: harvesting Ezh2 cells from 8 to 10 weeks of age fl / fl ; Olig1-Cre homozygous specific knockout Ezh2 mice.

[0012] In a second aspect, the present invention provides a method for constructing an optic nerve axon injury regeneration model, comprising the following steps:

[0013] Establish an optic nerve myelination defect mouse model according to any of the above methods;

[0014] A substance that increases the intrinsic growth force of neurons was injected into the vitreous of a mouse model with optic nerve myelination defects, and the mouse model was then crushed 1 mm behind the optic disc for 1 to 2 seconds to obtain an optic nerve axon injury and regeneration model.

[0015] Preferably, the substance that improves the intrinsic growth power of neurons is one or more of the AAV viruses of neurociliary factor CNTF, insulin-like growth factor IGF1 or osteopontin OPN.

[0016] In a third aspect, the present invention provides a use of the optic nerve myelination defect mouse model constructed according to the above method, wherein the use includes any one of the following:

[0017] 1) Use in the research of drugs to promote optic nerve myelination;

[0018] 2) Use in the research of drugs to promote optic nerve myelin regeneration;

[0019] 3) Use in drug research for the treatment of demyelinating diseases of the optic nerve;

[0020] 4) Use in the research of drugs that promote the intrinsic growth power of neurons.

[0021] In a fourth aspect, the present invention provides a use of the optic nerve axon injury and regeneration model constructed according to the above method, wherein the use includes any one of the following:

[0022] 1) Use in drug research to promote optic nerve myelination;

[0023] 2) Use in drug research to promote optic nerve myelin regeneration;

[0024] 3) Use in drug research for the treatment of demyelinating diseases of the optic nerve;

[0025] 4) Use in the research of drugs that promote the intrinsic growth power of neurons.

[0026] Compared with the prior art, the beneficial effects of the present invention are:

[0027] The present invention successfully constructs an optic nerve myelin formation defect mouse model by specifically knocking out the Ezh2 gene in oligodendrocytes. The optic nerve myelin formation defect mouse optic nerve crush model further constructed from the mouse model has the effect of optic nerve axon regeneration, can be used to screen drugs that promote the intrinsic growth power of neurons, and is used for the study of myelin regeneration after optic nerve injury, optic nerve myelin formation and related drugs for the treatment of optic nerve demyelinating diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 For Ezh2 fl / fl ; Gene knockout strategy of Olig1-Cre mice and wild-type, Ezh2 fl / fl ; General view of the optic nerve of Olig1-Cre mice; A, gene knockout strategy, B, wild type, Ezh2 fl / fl ;Gross view of the optic nerve of Olig1-Cre mice;

[0029] Figure 2 Wild type and Ezh2 fl / fl; Results of immunofluorescence staining of the optic nerve of Olig1-Cre mice with anti-MBP (marking myelin), GSTπ (marking oligodendrocytes) and DAPI;

[0030] Figure 3 Wild type and Ezh2 fl / fl ; 21 days after the optic nerve crush model of Olig1-Cre mice, immunofluorescence staining results of neural tracer CTB (labeling neuronal axons), anti-IBA1 (labeling microglia) and GFAP (labeling astrocytes);

[0031] Figure 4 Intravitreal injection of CNTF-AAV virus with wild-type and Ezh2 fl / fl ; Immunofluorescence staining of neural tracer CTB 21 days after optic nerve crush in Olig1-Cre mice;

[0032] Figure 5 Intravitreal injection of CNTF-AAV virus with wild-type and Ezh2 fl / fl ; Statistical results of optic nerve axon regeneration distance 21 days after optic nerve crush model in Olig1-Cre mice. DETAILED DESCRIPTION

[0033] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0034] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0035] The present invention is now further described in conjunction with specific examples. The following examples are only for explaining the present invention, but do not constitute a limitation of the present invention. The test samples and test processes used in the following examples include the following (if the specific experimental conditions are not indicated in the examples, they are usually in accordance with conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples, if not otherwise specified, can all be obtained from commercial channels).

[0036] The oligodendrocytes described in the present invention include oligodendrocytes at all stages of differentiation and development, such as precursor cells, immature or mature cells, and the like.

[0037] The gene number of the gene Ezh2 described in the present invention is ENSMUSG00000029687, and relevant information can be queried in the Ensembl database (www.ensembl.org).

[0038] Example 1

[0039] Establishment of a mouse model of optic nerve myelination defect

[0040] 1. Methods

[0041] By adding Ezh2 fl / fl The oligodendrocyte lineage-specific knockout Ezh2 heterozygous mice (Ezh2 fl / + ; Olig1-Cre), and Ezh2 fl / fl The homozygous second generation of mice (Ezh2 fl / fl ; Olig1-Cre, referred to as Ezh2cKO), and Ezh2 fl / fl (Wild type, hereinafter referred to as WT) mice were used as controls in the experiments.

[0042] The mouse optic nerve tissue was stained with anti-MBP (marking myelin) and GSTπ (marking oligodendrocytes). The expression of myelin-related protein MBP and oligodendrocytes (GSTπ positive) in the mouse optic nerve tissue was detected by immunofluorescence staining to further clarify the correlation between Ezh2 expression and optic nerve myelination.

[0043] 2. Results

[0044] like Figure 1 As shown, compared with wild-type mice, the optic nerve myelin development in Ezh2cKO mice was impaired and the optic nerve appeared translucent.

[0045] like Figure 2 As shown, compared with wild-type mice, the expression of myelin-associated protein MBP in the optic nerve of Ezh2cKO mice was significantly reduced, and the number of mature oligodendrocytes (GSTπ positive) was significantly reduced.

[0046] From the above results, it can be concluded that Ezh2 gene mutation in oligodendrocytes (OLs) may lead to the loss of mature OLs lineage cells, thereby causing optic nerve myelination defects. It also shows that specific knockout of Ezh2 gene in OLs can construct a mouse model of optic nerve myelination defects.

[0047] Example 2

[0048] Since neuroinflammation has been shown to regulate the proliferation and differentiation of oligodendrocyte precursor cells (OPCs), the myelin regeneration microenvironment is an important factor affecting myelin regeneration. Therefore, this example further constructed an optic nerve crush model through the optic nerve myelin formation defect mouse model constructed in Example 1, studied the status of microglia and astrocytes 21 days after optic nerve crush in Ezh2cKO mice, and verified the use of the optic nerve myelin formation defect mouse model constructed in Example 1 in the study of optic nerve axon regeneration microenvironment.

[0049] Take the Ezh2cKO mice constructed in Example 1, anesthetize the Ezh2cKO mice, prepare the skin and disinfect, and then surgically expose the optic nerve, and use fine forceps (Dumont #5FST) to crush the optic disc about 1 mm behind for 2 seconds. After surgery, use eye ointment to protect the cornea to obtain an optic nerve crush mouse model. Immunofluorescence staining of optic nerve tissue was performed with neural tracer CTB (labeling neuronal axons), anti-IBA1 (labeling microglia) and GFAP (labeling astrocytes), and the activation state of microglia and astrocytes in optic nerve crush mice was observed 21 days after optic nerve crush.

[0050] like Figure 3 As shown in the results, microglia and astrocytes were in a chronically activated state 21 days after optic nerve crush in both wild-type mice and Ezh2cKO mice, forming an inflammatory environment that was not conducive to myelin regeneration. Although the activation state of astrocytes was inhibited in Ezh2cKO mice after optic nerve injury compared with wild-type mice, no significant optic nerve axon regeneration was found in either of them. The above findings suggest that optic nerve axon regeneration requires further improvement of the intrinsic growth force of neurons.

[0051] Example 3

[0052] Successful axon regeneration is a prerequisite for myelin regeneration. This example verifies the use of the optic nerve myelin formation defect mouse model in studying myelin regeneration after optic nerve injury and screening drugs that promote the intrinsic growth of neurons.

[0053] The Ezh2cKO mice constructed in Example 1 were taken, and the neurociliary factor CNTF-AAV virus (purchased from Heyuan Biotechnology (Shanghai) Co., Ltd., because the effects of selecting one of the AAV viruses of neurociliary factor CNTF, insulin-like growth factor IGF1 and osteopontin OPN are basically the same, selecting two or three in combination has an additive effect, and the present embodiment only takes the neurociliary factor CNTF-AAV virus as an example for illustration) was injected into the vitreous body of the mice.

[0054] To improve the intrinsic growth of neurons, optic nerve crush (ONC) was performed on Ezh2cKO mice 14 days later. The specific operation process was as follows: the optic nerve of Ezh2cKO mice was exposed after anesthesia, skin preparation and disinfection, and fine forceps (Dumont #5FST) were used to crush the optic nerve about 1 mm behind the optic disc for 1 second. Eye ointment was used to protect the cornea after surgery. CTB fluorescent dye (i.e., CTB555 neural tracer) was used to mark the regenerated optic nerve axons 19 days after optic nerve crush. Wild-type mice were used as controls. 21 days after optic nerve crush, Ezh2cKO mice and wild-type mice were sliced ​​and sliced ​​to observe the regeneration of optic nerve axons in mice.

[0055] like Figures 4-5 As shown, the regeneration distance of optic nerve axons in Ezh2cKO mice was significantly prolonged compared with that in wild-type mice, indicating that the defect in optic nerve myelination is associated with optic nerve axon regeneration.

[0056] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the technical solution of the present invention, and are not intended to limit the specific implementation methods of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the claims of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A method for constructing an optic nerve myelin defect mouse model, characterized in that: Optic nerve myelination-deficient mice were obtained by specifically knocking out the Ezh2 gene in oligodendrocytes.

2. The construction method according to claim 1, characterized in that: The construction method specifically comprises the following steps: 1) Obtain Ezh2 with floxp sites inserted at both ends of exons 14 and 15 of the Ezh2 gene fl / fl Mice; 2) Obtain Olig1-Cre mice in which the endogenous Olig1 promoter controls the expression of Cre recombinase; 3) Ezh2 fl / fl Mice were crossed with Olig1-Cre mice to obtain Ezh2 fl / + ;Olig1-Cre specific knockout of Ezh2 mice heterozygous generation; 4) Ezh2 fl / + ; Olig1-Cre specific knockout of Ezh2 mice heterozygous generation and Ezh2 fl / fl Backcrossing of mice to obtain Ezh2 fl / fl ; Olig1-Cre homozygous specific knockout Ezh2 mice.

3. The construction method according to claim 2, characterized in that: The construction method also includes: harvesting Ezh2 cells born 8 to 10 weeks fl / fl ; Olig1-Cre homozygous specific knockout Ezh2 mice.

4. A method for constructing an optic nerve axon injury regeneration model, characterized in that: The following steps are involved: Constructing an optic nerve myelination defect mouse model according to the method described in any one of claims 1 to 3; A substance that improves the intrinsic growth power of neurons is injected into the vitreous of a mouse model with optic nerve myelination defects, and the mouse model is then crushed 1 mm behind the optic disc for 1 to 2 seconds to obtain an optic nerve axon injury and regeneration model; the substance that improves the intrinsic growth power of neurons is one or more of the AAV viruses of the neurociliary factor CNTF, insulin-like growth factor IGF1 or osteopontin OPN.

5. Use of an optic nerve myelination defect mouse model constructed according to the method of any one of claims 1 to 3, characterized in that: The uses include any of the following: 1) Use in the research of drugs to promote optic nerve myelination; 2) Use in the research of drugs to promote optic nerve myelin regeneration; 3) Use in drug research for the treatment of demyelinating diseases of the optic nerve; 4) Use in the research of drugs that promote the intrinsic growth power of neurons.

6. Use of the optic nerve axon injury regeneration model constructed according to the method of claim 4, characterized in that: The uses include any of the following: 1) Use in drug research to promote optic nerve myelination; 2) Use in drug research to promote optic nerve myelin regeneration; 3) Use in drug research for the treatment of demyelinating diseases of the optic nerve; 4) Use in the research of drugs that promote the intrinsic growth power of neurons.

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