Nerve cells as well as culture method and application thereof

A technology of nerve cells and culture methods, applied to nervous system cells, animal cells, culture processes, etc., can solve problems such as lack of theoretical basis for artificial spinal cord

Inactive Publication Date: 2020-03-24
SHENZHEN ELDERLY MEDICAL RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The treatment of spinal cord injury is a difficult problem in the medical field. Artificial spinal cord is a potentially effective means of treating spinal cord i...

Method used

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  • Nerve cells as well as culture method and application thereof
  • Nerve cells as well as culture method and application thereof
  • Nerve cells as well as culture method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Isolation and Culture of Rat Dorsal Root Ganglion Neurons

[0056] In this embodiment, rat dorsal root ganglion neurons (dorsal root ganglion neuron, DRGn) are selected as the research object, and the acquisition method is as follows:

[0057] (1) Primary culture of DRGn

[0058] Take newborn rats within 24 hours, first soak them in 75% alcohol for disinfection, then use ophthalmic scissors to remove the back skin under aseptic conditions, cut out a section of spinal cord, place it on a sterilized ground glass slide with the back side up, and examine it under a dissecting microscope Cut off the ventral half of the vertebrae horizontally along both sides of the spinal canal to expose the spinal cord and ganglion, and separate the ganglion with dissecting forceps;

[0059] The ganglion capsule was peeled off, digested with 0.125% trypsin at 37°C for 30min, and diluted with Plating culture medium after dispersion to a density of 0.2×10 5 cells / mL of cell suspen...

Embodiment 2

[0062] Example 2 Identification of rat dorsal root ganglion neurons

[0063] (1) Observation of cell morphology

[0064] The DRGn cultured for 3 days was placed under an inverted phase-contrast microscope for morphological observation, and the 100-fold, 200-fold and 400-fold fields of view were randomly selected under the microscope to observe the morphology and growth state of DRGn;

[0065] The DRGn cultured for 3 days was taken out from the cell culture incubator, washed 3 times with D-Hank's solution; added 2.5% glutaraldehyde, fixed at 4°C for 2 hours, and then rinsed 3 times with 0.01M PBS, each time for 10 minutes ; Cells were fixed in 1% osmium tetroxide for 1.5 h at 4°C; rinsed 3 times with 0.01M PBS for 10 min each time; transferred to graded ethanol for dehydration, and then replaced with isoamyl acetate; CO 2 The critical point is dry; the surface of the conductive glass with cells is sprayed with gold, and observed under a scanning electron microscope.

[0066]...

Embodiment 3

[0070] Example 3 Effect of Electrical Stimulation on Nerve Cell Axon Growth

[0071] In this example, neurons are electrically stimulated from four aspects: biphasic / monophase, electrical stimulation time, electrical stimulation voltage, and electrical stimulation frequency. The cell activity is measured by the CCK-8 method. After immunocytochemical staining, the Image- ProPlus6.0 image analysis software measures the protrusion length.

[0072] The DRGn cells cultured for 1 day were divided into four experimental groups: normal group, electrical stimulation group, biphasic (biphasic, BI) BI group and monophasic (monophasic, MO) MO group, adjusting biphasic / monophasic, electrical stimulation time, Electrical stimulation parameters such as electrical stimulation voltage and electrical stimulation frequency, among which the normal group did not receive stimulation; the electrical stimulation group used direct current electrical stimulation, and the electric field strength was 6V / ...

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Abstract

The invention provides nerve cells as well as a culture method and application thereof, and particularly relates to dorsal root ganglion cells as well as an electrical stimulation culture method and application thereof in preparation of medicines for treating spinal cord injuries, medical materials or medical instruments. The culture method comprises the step of performing electrical stimulation culture on the nerve cells. According to the method, the micro-electrical stimulation technology is used for intervening and culturing the dorsal root ganglion neurons, the influence of micro-current on c-fos expression closely related to cellular morphology and neuron axon growth is researched, the mechanism that the micro-current stimulates neuron axon growth is disclosed, and a theoretical basisis provided for in-vitro preparation of artificial spinal cord tissues.

Description

technical field [0001] The invention belongs to the technical field of nerve electrical stimulation, and relates to a nerve cell and its culture method and application, in particular to a dorsal root ganglion cell and its electrical stimulation culture method and in the preparation of spinal cord injury treatment drugs, medical materials or medical devices Applications. Background technique [0002] Electrical nerve stimulation has been widely used in the treatment of pain, Parkinson's disease, coma awakening after traumatic brain injury, epilepsy, and post-hypoxic vegetative state, but the mechanism of action is still unclear. Commonly used nerve electrical stimulation includes spinal cord electrical stimulation, median nerve electrical stimulation, vagus nerve electrical stimulation, deep brain electrical stimulation, hypoglossal nerve electrical stimulation and sacral nerve electrical stimulation, etc. Each nerve electrical stimulation has its own characteristics, and its...

Claims

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Application Information

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IPC IPC(8): C12N5/0793A61K35/30A61P25/00
CPCA61K35/30A61P25/00C12N5/0619C12N2500/40C12N2529/00
Inventor 段丽红王春宝吴正治刘铨权李映红李利民曾嫱
Owner SHENZHEN ELDERLY MEDICAL RES INST
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