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The application of mir containing the specific seed base sequence at the 5' end, the improved Schwann cell and its application

A technology of Schwann cells and cells, applied in the field of improved Schwann cells, can solve problems such as inability to stagger fusion

Active Publication Date: 2020-10-16
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to overcome the defect that the existing Schwann cells and astrocytes cannot be staggered and fused, the present invention provides a mature body miR containing a specific seed base sequence at the 5' end in promoting Schwann cells and astrocytes. Application in tolerance integration, through the expression level of miR-124-3P containing a specific seed base sequence at the 5' end in Schwann cells to change the cytological characteristics of Schwann cells, thereby improving their interleaving with astrocytes Fusion to promote the regeneration and repair of Schwann cells in the central nervous system including the spinal cord

Method used

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  • The application of mir containing the specific seed base sequence at the 5' end, the improved Schwann cell and its application
  • The application of mir containing the specific seed base sequence at the 5' end, the improved Schwann cell and its application
  • The application of mir containing the specific seed base sequence at the 5' end, the improved Schwann cell and its application

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Experimental program
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Embodiment 1

[0050] Isolation, culture and identification of rat Schwann cells:

[0051] The sciatic nerve of adult female Wistar rats (200-250 g) was taken, and the muscle tissue, fat, blood vessels and epineurium were peeled off with micro forceps and ophthalmic scissors under a microscope in a sterile operating table. After obtaining the white nerve fibers, soak them in sterile PBS (phosphate buffer solution), and cut them into tissue pieces with a length of about 2 mm by using an ophthalmologist. Discard the liquid, add 0.1% type I collagenase and digest in a cell incubator at a constant temperature of 37°C for 25 minutes. Discard the type I collagenase and wash the residual enzyme solution with sterile PBS to terminate the digestion reaction. Use micro tweezers to remove the tissue block and place it in a clean cell culture dish to adhere to the bottom of the dish, add a small amount of SCM (Schwann cell culture medium: DMEM / F12+10% fetal bovine serum+fibroblast growth factor bFGF+ne...

Embodiment 2

[0055] Example 2 Comparison of miR-124-3P content in Schwann cells, astrocytes and normal bone marrow cells:

[0056] Isolation and culture of rat astrocytes:

[0057] The brains of suckling rats were taken out from 1-3 days old rats, and the cerebral cortex was peeled off under a microscope under a sterile operating table. Soak in sterile DMEM. Break the cerebral cortex tissue into a uniform particle-free suspension by pipetting with a 5ml pipette tip. Collect the tissue fluid in a sterile centrifuge tube and discard the supernatant to collect the precipitate. Tissues were digested with 0.05% trypsin and DNase (Deoxyribonuclease I, Worthington) at 37°C for 25 minutes. Use a sterile filter 40μm-sterile EASYstrainer after terminating digestion TM (Greiner bio-one) to obtain a single cell suspension. After centrifugation, the precipitate was collected and washed three times with PBS. ASM for cells (astrocyte culture medium: DMEM / F12 + 10% fetal bovine serum + fibroblast gr...

Embodiment 3

[0064] 2Taking lentivirus as an example: lentivirus transfection of Schwann cells to obtain improved Schwann cells

[0065] 2.1 Construction of improved Schwann cells (Schwann cells with high expression of miR-124-3P)

[0066] 2.1.1 Entrust Shanghai Jikai Gene Chemical Technology Co., Ltd. to construct lentivirus GV309 with titer and stable expression of miR-124-3P, and obtain miR-124-3P lentiviral vector. The viral titer of the obtained miR-124-3P lentiviral vector was 6E+8, and the expression level of miR-124-3P was about 4 times that before transfection.

[0067] 2.1.2 Determine the optimal MOI value (multiplicity of infection).

[0068] The miR-124-3P lentiviral vector was used to infect Schwann cells with gradient MOI values ​​of 1, 10, 25, 50, and 75, respectively. After 48 hours, the MOI value (MOI=50) with the infection efficiency of more than 80% and the least cytotoxicity was the best. The optimal MOI value is used for subsequent experiments.

[0069] 2.1.3 Determ...

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Abstract

The invention provides an application of a miR containing a 5'- terminal specific seed base sequence, an improved Schwann cell and applications thereof, and relates to the technical field of central nervous system damage repairing. The miR is used to promote the compatibility and integration between Schwann cells and astrocyte; and the 5'- terminal specific seed base sequence is UAAGGCAC or AAGGCAC. The results of experiments show that after the expression level of matured miR containing a 5'- terminal specific seed base sequence is artificially improved, the expression of astrocyte related gene in improved Schwann cells is obviously reduced, the expression of neurotrophic factor genes (NT-3 and BDNF) is obviously increased, and improved Schwann cells are compatible with and fused with astrocyte. The improved Schwann cells can inhibit astrocyte, the integration of Schwann cells and astrocyte is promoted, and the formation of glial scars caused by astrocyte is inhibited.

Description

technical field [0001] The invention relates to the technical field of spinal cord injury repair, in particular to the application of miR containing a specific seed base sequence at the 5' end, improved Schwann cells and applications thereof. Background technique [0002] Spinal cord injury (SCI) refers to a disease in which the spinal cord is damaged by external direct or indirect factors, resulting in sensory and motor dysfunction below the damaged segment of the body. According to the World Health Organization (WHO) statistics, there are about 3 million SCI patients in the world and about 180,000 new patients every year, most of which are caused by road traffic accidents. As a disease with high incidence and high disability rate, SCI has not yet been completely cured by medical means. Relying on surgery, drug treatment and rehabilitation nursing can not achieve the ideal medical effect, and patients will also face severe neurological dysfunction. After SCI, neurons and g...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/113C12N5/10A61K35/30A61P25/00
CPCA61K35/30A61P25/00C12N5/0622C12N15/113C12N2310/141C12N2510/00
Inventor 池光范李玉林李祉君许侃郑洋洋徐金影
Owner JILIN UNIV
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