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Blood-spinal cord barrier OGD/R injury model and construction method and application thereof

A technology of blood spinal cord barrier and injury model, which is applied in the field of biomedicine, can solve the problems of missing molecular markers, defects, and cannot fully simulate the physical morphological structure and function of the blood spinal cord barrier, and achieves easy operation, good repeatability, and excellent barrier. effect of function

Pending Publication Date: 2019-01-18
WUXI PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Among them, the co-culture model of astrocytes and endothelial cells is quite different from the physiological structure of BSCB. In this model, only astrocytes are the glial cells, and microglia are also involved in the composition of the BSCB structure. The model is clearly flawed
The co-culture model of pericytes and endothelial cells is also quite different from the physiological structure of BSCB, completely without glial cells, and cannot effectively simulate the physiological structure and function of BSCB in vivo
Although non-primary cultured cells proliferate rapidly and have high experimental reproducibility, they are not the structural constituent cells of BSCB under normal physiological conditions, and cannot fully simulate the physical structure and function of the blood-spinal cord barrier
In addition, after repeated subculture of bend.3 and N9 cell lines, some molecular markers may be lost, and even some biological characteristics may also change

Method used

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  • Blood-spinal cord barrier OGD/R injury model and construction method and application thereof
  • Blood-spinal cord barrier OGD/R injury model and construction method and application thereof
  • Blood-spinal cord barrier OGD/R injury model and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 In vitro blood-spinal cord barrier model and establishment of blood-spinal cord barrier oxygen-glucose deprivation / reoxygenation (oxygen glucosedeprivation / re-oxygenation, OGD / R) injury model

[0053] 1. Primary culture and identification of mixed glial cells derived from rat spinal cord

[0054] 1.1 Primary culture of mixed glial cells

[0055] (1) Disinfect instruments for 30 minutes;

[0056] (2) Take a newborn SD rat within 1 day, and immerse the whole body in 75% alcohol for disinfection for 2 minutes;

[0057] (3) Quickly take out the spinal cord by aseptic method in the aseptic operating table (fix the limbs of the rat with pins, pull the skin of the neck with tweezers, cut off the spine from the cervical cord with scissors, find the spinal canal, hook the tweezers into the spinal canal, pull back and upward to remove the spinal canal to the tail, expose the cervical cord, thoracic cord, and lumbar cord, take out the spinal cord with tweezers, and pla...

Embodiment 2

[0083] Example 2 Blood-spinal cord barrier OGD / R injury model detection application of its permeability, tight junction-related proteins, and inflammatory factors

[0084] In this example, a transmembrane resistance meter was used to measure the transendothelial intercellular resistance of the blood-spinal cord barrier OGD / R injury model constructed in Example 1 at different time points after the blood-spinal cord barrier oxygen-glucose deprivation / reoxygenation injury, and by detecting different time points The leakage rate of dextran (FITC-dextran, F-D) labeled with isothiocyanate fluorescent agent was used to evaluate the permeability of endothelial cells, and to determine the successful modeling of the BSCB oxygen-glucose deprivation / reoxygenation injury model; Differences in the secretion of leukocyte chemokines (MIP-1α, MIP-1β), leukocyte adhesion factors (ICAM-1, VCAM-1), pro-inflammatory factors (IL-1β, TNF-α, iNOS, COX-2) ; Immunofluorescence and Western Blot techniqu...

Embodiment 3

[0090] Example 3 In Vitro Drug Screening

[0091] 1. The effect of EGFR inhibitor PD168393 (a potential neuroprotective agent) on the permeability of the blood-spinal cord barrier OGD / R injury model

[0092] 1.1. Preparation of PD168393 (Abcam, ab145187, USA) working solution: add 135 μl DMSO to 1 mg PD168393 powder, add 405 μl HBSS solution after fully dissolving, and mix well to make 540 μl 5mM PD168393 stock solution, after aliquoting -20°C save.

[0093] 1.2 Experimental grouping: Divide the BSCB model in vitro into 5 groups: OGD / R injury group, add 3 different concentrations (10nM, 1nM and 0.1nM) of PD168393 intervention treatment to 3 treatment groups when OGD / R model reoxygenation, no The BSCB model treated with any treatment was used as the normal control group.

[0094] 1.3 Fluorescent dye leakage test: add 200 μl FITC-dextran (dextran labeled with isothiocyanate fluorescent agent) to the upper chamber after reoxygenation for 3 hours, 6 hours, 12 hours and 24 hours ...

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Abstract

The invention discloses a method for constructing a blood-spinal cord barrier (BSCB) oxygen-glucose deprivation / reoxygenation (OGD / R) injury model and an application thereof. Spinal cord derived microvascular endothelial cells were cultured in the upper chamber and mixed glial cells were cultured in the bottom chamber of the transwell culture system to construct the in vitro BSCB model. Microvascular endothelial cells in the upper chamber of transwell and mixed glial cells in the lower chamber of transwell interact with each other by secreting cytokines to form molecular membranes with good barrier function. OGD / R can effectively simulate the injury of BSCB induced by ischemia / reperfusion in vivo, and can more directly and accurately understand the changes of structure and function of BSCBafter ischemia / reperfusion injury. The results of the study have a high reference value, thus providing an effective basis for clinical study of BSCB.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a blood-spinal cord barrier oxygen-glucose deprivation / reoxygenation (OGD / R) injury model and its construction method and application. Background technique [0002] Spinal cord injury (SCI) often causes severe motor and sensory deficits in the limbs and trunk below the level of damage, and these neurological deficits are often irreversible. The neurological deficit after SCI is caused by primary injury and secondary injury. The primary injury refers to the injury caused by the spinal cord being directly subjected to mechanical violence, while the secondary injury is a series of "waterfalls" after the primary injury. "Self-destructive process activated by injury. Blood Spinal Cord Barrier (BSCB) plays an important role in the pathophysiology of SCI, and its destruction plays an important role in secondary injury. Therefore, how to protect the structural and funct...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079C12N5/071C12Q1/02
CPCC12N5/0697C12N5/069C12N2502/086C12N2502/28C12N2503/04G01N33/5058G01N33/5064G01N2500/10
Inventor 李在望蔡小军
Owner WUXI PEOPLES HOSPITAL
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