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Primary microglial cell/injured neuron co-culture system and construction method and application thereof

A technology of microglia and co-culture system, applied in co-culture system, primary microglia/injured neuron co-culture system and its construction field, can solve the problem of poor nerve regeneration effect and no microglia cells. For problems such as phagocytosis of injured neurons and unfavorable simulation of spinal cord microenvironment, it can achieve the effect of considerable quantity, stable character and high activity

Inactive Publication Date: 2018-07-06
NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the methods for studying the phagocytosis of microglia in the CNS are mainly in vivo studies, or in vitro studies on the phagocytosis of ink or fluorescent microspheres by a single microglia or cell line, but no primary microglia in vitro have been reported. Phagocytosis of injured neurons by cytoplasmic cells
This is not only not conducive to simulating the microenvironment after spinal cord injury, but also often leads to the inability of the extracted primary microglia to perform phagocytosis well, and the effect of promoting nerve regeneration is poor

Method used

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  • Primary microglial cell/injured neuron co-culture system and construction method and application thereof
  • Primary microglial cell/injured neuron co-culture system and construction method and application thereof
  • Primary microglial cell/injured neuron co-culture system and construction method and application thereof

Examples

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Embodiment 1

[0045] Primary microglia / injured neuron co-culture system, the system includes primary microglial cells and injured neurons, wherein the injured neurons are phagocytized by primary microglial cells; the system is In vitro co-culture system.

[0046] The construction method of the primary microglia / injured neuron co-culture system comprises the following steps:

[0047] Step 1. Isolation and culture of primary microglia

[0048] Take newborn SD rats, disinfect them with 75% alcohol one by one for 30 seconds, decapitate and take out the brain and brainstem completely; under a stereomicroscope, peel off the vascular membrane, remove the brainstem and hippocampus, cut up the cerebral cortex, and temporarily store it in 0°C precooling DMEM high-sugar+10% FBS medium; cut the brain cortex into pieces, centrifuge to remove the supernatant, add 0.125% trypsin 5mL to resuspend, transfer to a 10cm culture dish, and store at 37°C, 5% CO2 Digest in an incubator for 15 minutes; add 1 mL of...

Embodiment 2

[0060] The primary microglia / injured neuron co-culture system described in Example 1 is applied to the evaluation of drug efficacy for promoting nerve regeneration, including the following steps:

[0061] Step 1. Experimental Group Intervention

[0062] The traditional Chinese medicine compound "Ji Suikang" (crude drug amount 20g / kg) that promotes nerve regeneration was given to healthy SD male rats orally to prepare drug-containing serum, and rats were given the same amount of normal saline to prepare blank serum as a control.

[0063] The primary microglia / damaged neuron co-culture system was divided into three drug-containing serum groups: 2.5% (low), 5% (medium), and 10% (high), and a 10% blank serum group (negative control group) was set at the same time. ), 2ug / mL LPS+10% blank serum group (positive control group), recovered primary cultured microglial cells, added drug-containing serum or blank serum according to the grouping conditions to prepare DMEM / F12+B27 condition...

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Abstract

The invention discloses a primary microglial cell / injured neuron co-culture system and a construction method and application thereof. The system comprises primary microglial cells and injured neurons,wherein the injured neurons are swallowed by the primary microglial cells intracellularly; and the system is an in vitro co-culture system. According to the primary microglial cell / injured neuron co-culture system disclosed by the invention, compared with the disadvantages of resting microglial cells cultured by a traditional method, the microenvironment of nerve injury is simulated to activate the microglial cells, and the phagocytic function of the microglial cells is maintained; the microglial cells inside the system can be activated into macrophagocytes by the injured neurons, so that thephagocytic capacity of neuron fragments is strengthened; in the system constructed by the method, the primary cells are considerable in quantity, stable in characteristics and relatively high in activity, and the reliable co-culture system can be provided for subsequent fundamental research of nerve injury or degenerative diseases.

Description

technical field [0001] The invention belongs to the field of cell biology, and relates to a co-culture system, in particular to a primary microglia / damaged neuron co-culture system and its construction method and application. Background technique [0002] Spinal cord injury is a highly disabling disease that seriously affects physical and mental health. Its treatment and rehabilitation costs are high and difficult, and it brings a heavy burden to individuals, families and society. Therefore, it has always been a difficult and hot topic in medical research. . Microglia are macrophage-derived immunoregulatory cells in the central nervous system (CNS). After neuropathological changes such as spinal cord injury, microglia are rapidly activated, regulate immune responses, and clean up cell debris. The regulation of the microenvironment is of great significance. How to study the role of microglia in the CNS microenvironment is the key to further study the comprehensive regulatio...

Claims

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Application Information

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IPC IPC(8): C12N5/079C12N5/0793
CPCC12N5/0619C12N5/0622C12N2509/00
Inventor 郭杨潘娅岚马勇周龙云苑文超郑苏阳黄桂成王磊
Owner NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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