New application of dcf1 gene

Abstract

The invention relates to new application of a dcf1 gene. A wild type mouse (serving as contrast) and a mouse with the dcf1 deleted are mainly adopted; western blotting is used for detecting reducing of myelin basic protein; immunohistochemistry and a transmission electron microscope are used for observing changing of the form of a myelin sheath; and then a behavioral experiment is used for showing that the mouse with the dcf1 deleted can simulate clinical symptoms of multiple sclerosis, then the mouse with the dcf1 deleted is subjected to rescue, and it is discovered that the dcf1 can cause myelin sheath remodeling and regeneration.

Description

technical field

[0001] The present invention relates to a new application of dcf1 gene. Background technique

[0002] Demyelinating diseases of the central nervous system refer to diseases that occur in the brain or spinal cord and are characterized by the destruction of myelin sheath and the invasion of inflammatory cells. Clinically, multiple sclerosis and neuromyelitis optica are the most common demyelinating diseases of the central nervous system. Myelin lesions in patients with multiple sclerosis (MS) cause blockage or destruction of information transmission between the brain and other areas of the body, and then manifest clinical symptoms of MS, including muscle weakness, impaired coordination and balance, paralysis, and tingling , restlessness and other sensory disturbances, cognitive and memory disturbances. The pathogenesis of MS is unclear, but it may be an autoimmune disease. There is no effective treatment for demyelinating disease, and drug therapy can only s...

Examples

Embodiment 1

[0012] Example 1: The target area, corpus callosum and hippocampus were taken out of the pre-cooled PBS. Then homogenize in a homogenizer with Western and IP cell lysate until there is no obvious tissue block and it becomes a transparent homogenate. Continue to lyse on ice for 30 min, and then collect the lysate in a 1.5ml EP tube. The supernatant was collected by centrifugation at 8000 rpm for 10 minutes at 4°C. Continue to centrifuge the supernatant at 4°C, 15,000rpm for 20min, collect the supernatant again to be the tissue protein extract, add the corresponding amount of Loading buffer, heat and denature it for later use. Afterwards, Western blotting was performed. The electrophoresis conditions are stacking gel 80V, 20 minutes; separating gel 120V, 90 minutes. The transfer condition was 25V for 60 minutes. The primary antibody dilution ratio is 1:1000, and the secondary antibody dilution ratio is 1:10000. ( figure 1 A) Schematic of Western blotting (MBP / GAPDH) of the...

Embodiment 2

[0013] Example 2: Detection of myelin sheath morphology by transmission electron microscopy

[0014] Experimental mice were first perfused (4% paraformaldehyde and 1% glutaraldehyde in fixative). Then the mouse brain was taken out, placed in a fixative solution containing 2.5% glutaraldehyde, and the area to be observed (hippocampus, corpus callosum) was cut into 1mm 2 size and then fixed in the 2.5% glutaraldehyde fixative described above. This was followed by post-fixation with osmium tetroxide for 2 hours. Alcohol gradually dehydrates. Finally, the tissue block was embedded in epoxy resin. Slice with an ultra-thin microtome. Slices with a thickness of 500nm are placed on glass slides and stained with toluidine blue, and can be observed under Confocal; Next observe. ( figure 2 A, B) Schematic diagram of ultrastructural electron microscopy of myelin in the corpus callosum and hippocampus. It can be seen that in both the corpus callosum and hippocampus, the myelin she...

Embodiment 3

[0015] Example 3: Immunohistochemical detection

[0016] Firstly, the experimental mice were perfused and fixed with 4% PFA, dehydrated with sucrose, and embedded. Then cryosection (20um) was performed. Finally, immunohistochemistry was performed. Wash 3 times with 0.01M PBS, 5 minutes each; Permeabilize with 0.1%TritonX-100-PBS for 30 minutes; Wash 3 times with 0.01M PBS, 5 minutes each; Block with 2% BSA-PBS for 1 hour at room temperature; Blot dry 2% After BSA-PBS, directly drop the primary antibody diluted in PBS (MBP, 1:500). Incubate at 37°C for 2 hours, or overnight at 4°C; blot dry the primary antibody, wash with 0.01M PBS 3 times, 5 minutes each time. Under dark conditions, add secondary antibody diluted in PBS (1:500 dilution ratio). Incubate at 37°C for 2 hours; blot dry the secondary antibody, add DAPI (1:1000) diluted in PBS, and incubate at room temperature for 10 minutes; blot dry DAPI, wash 3 times with 0.01M PBS, 5 minutes each time; seal with fluorescent ...

technical field

[0001] The present invention relates to a new application of dcf1 gene. Background technique

[0002] Demyelinating diseases of the central nervous system refer to diseases that occur in the brain or spinal cord and are characterized by the destruction of myelin sheath and the invasion of inflammatory cells. Clinically, multiple sclerosis and neuromyelitis optica are the most common demyelinating diseases of the central nervous system. Myelin lesions in patients with multiple sclerosis (MS) cause blockage or destruction of information transmission between the brain and other areas of the body, and then manifest clinical symptoms of MS, including muscle weakness, impaired coordination and balance, paralysis, and tingling , restlessness and other sensory disturbances, cognitive and memory disturbances. The pathogenesis of MS is unclear, but it may be an autoimmune disease. There is no effective treatment for demyelinating disease, and drug therapy can only s...

Examples

Embodiment 1

[0012] Example 1: The target area, corpus callosum and hippocampus were taken out of the pre-cooled PBS. Then homogenize in a homogenizer with Western and IP cell lysate until there is no obvious tissue block and it becomes a transparent homogenate. Continue to lyse on ice for 30 min, and then collect the lysate in a 1.5ml EP tube. The supernatant was collected by centrifugation at 8000 rpm for 10 minutes at 4°C. Continue to centrifuge the supernatant at 4°C, 15,000rpm for 20min, collect the supernatant again to be the tissue protein extract, add the corresponding amount of Loading buffer, heat and denature it for later use. Afterwards, Western blotting was performed. The electrophoresis conditions are stacking gel 80V, 20 minutes; separating gel 120V, 90 minutes. The transfer condition was 25V for 60 minutes. The primary antibody dilution ratio is 1:1000, and the secondary antibody dilution ratio is 1:10000. ( figure 1 A) Schematic of Western blotting (MBP / GAPDH) of the...

Embodiment 2

[0013] Example 2: Detection of myelin sheath morphology by transmission electron microscopy

[0014] Experimental mice were first perfused (4% paraformaldehyde and 1% glutaraldehyde in fixative). Then the mouse brain was taken out, placed in a fixative solution containing 2.5% glutaraldehyde, and the area to be observed (hippocampus, corpus callosum) was cut into 1mm 2 size and then fixed in the 2.5% glutaraldehyde fixative described above. This was followed by post-fixation with osmium tetroxide for 2 hours. Alcohol gradually dehydrates. Finally, the tissue block was embedded in epoxy resin. Slice with an ultra-thin microtome. Slices with a thickness of 500nm are placed on glass slides and stained with toluidine blue, and can be observed under Confocal; Next observe. ( figure 2 A, B) Schematic diagram of ultrastructural electron microscopy of myelin in the corpus callosum and hippocampus. It can be seen that in both the corpus callosum and hippocampus, the myelin she...

Embodiment 3

[0015] Example 3: Immunohistochemical detection

[0016] Firstly, the experimental mice were perfused and fixed with 4% PFA, dehydrated with sucrose, and embedded. Then cryosection (20um) was performed. Finally, immunohistochemistry was performed. Wash 3 times with 0.01M PBS, 5 minutes each; Permeabilize with 0.1%TritonX-100-PBS for 30 minutes; Wash 3 times with 0.01M PBS, 5 minutes each; Block with 2% BSA-PBS for 1 hour at room temperature; Blot dry 2% After BSA-PBS, directly drop the primary antibody diluted in PBS (MBP, 1:500). Incubate at 37°C for 2 hours, or overnight at 4°C; blot dry the primary antibody, wash with 0.01M PBS 3 times, 5 minutes each time. Under dark conditions, add secondary antibody diluted in PBS (1:500 dilution ratio). Incubate at 37°C for 2 hours; blot dry the secondary antibody, add DAPI (1:1000) diluted in PBS, and incubate at room temperature for 10 minutes; blot dry DAPI, wash 3 times with 0.01M PBS, 5 minutes each time; seal with fluorescent ...

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