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Method for amplifying NSCs and inhibiting it to neuroglial cell differentiation and its use

A technology of glial cells and nerve cells, applied in the field of biomedicine, can solve the problems of insufficient differentiation and insufficient number, and achieve the effect of promoting proliferation

Inactive Publication Date: 2007-01-10
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, research on neural stem cells at home and abroad has only confirmed its existence, achieved its in vitro expansion and a certain degree of directional induced differentiation, but it will take time to apply it in clinical practice, especially to overcome the shortage of its quantity and insufficient differentiation towards functional neuronal cells

Method used

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  • Method for amplifying NSCs and inhibiting it to neuroglial cell differentiation and its use
  • Method for amplifying NSCs and inhibiting it to neuroglial cell differentiation and its use
  • Method for amplifying NSCs and inhibiting it to neuroglial cell differentiation and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1. Isolation and cultivation of rat neural stem cells

[0055] Pregnant Wistar rats were taken, and when the pups reached 14±1 days of pregnancy, the pregnant rats were anesthetized by intramuscular injection of pentobarbital, laparotomy was performed under aseptic conditions and the uterus was cut open, the fetal rats were taken out, the membranes were peeled off, and D-Hanks solution was used to anesthetize the pregnant rats. Rinse gently; after the whole body of the suckling mouse is disinfected, the scalp and skull are cut in layers to expose the cerebral hemispheres on both sides, the telencephalon and midbrain are taken out, the brain tissue blocks are cleaned with D-Hanks solution, blood vessels and other connective tissues are removed, and then cut into paste After centrifuging and discarding the supernatant, transfer to 8ml 0.125% trypsin, pipette gently several times with a thin-mouthed pipette, and then digest at 37°C for 30min, shaking once every 5...

Embodiment 2

[0056] Embodiment 2. Isolation and cultivation of human brain neural stem cells

[0057] Take 3-4 month-old embryos, take out the telencephalon and midbrain, carefully peel off the meninges, wash the brain tissue block with D-Hanks and remove connective tissue such as blood vessels, cut it into paste, centrifuge, discard the supernatant, and transfer to 8ml In 0.125% trypsin, pipette lightly for a few times, then digest at 37°C for 30 minutes, shake once every 5 minutes. Add 100 μl serum and 10 μl DNase I and mix well, pass the cell suspension through a 200-mesh cell sieve, centrifuge at 200 g for 10 min, discard the supernatant, resuspend the pellet with 1 ml serum-free medium, and culture.

Embodiment 3

[0058] Example 3, Affinity Chromatography Purification of Lectin FRIL

[0059] Grind the eyebrow beans into powder, mix with 5 times the volume of Tris buffer (50mmol / L Tris-HCl, 1mmol / LMgCl 2 , 1mmol / L CaCl 2 , pH8.0) homogenate, equilibrate at 4°C for more than 4 hours; centrifuge at 20,000g at 4°C for 20min, and take the supernatant; 8.0, 20000g high-speed centrifugation at 4°C for 20min, and the supernatant was taken to obtain the crude lectin extract. The crude extract was combined with the affinity chromatography medium (mannose-Sepharose matrix, Pharmacia Company) equilibrated with Tris buffer, reacted at 4°C for more than 4 hours, then washed with Tris buffer until no protein flowed out, and then washed with 200mmol / Lα- Methyl α-D-mannoside was eluted to give a pure solution of the lectin FRIL. 15% SDS-PAGE results such as figure 2 As shown, it can be seen from the figure that there are 5 obvious bands in the range of 10kD-25kD, which are the 5 subunits of the iso...

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Abstract

The invention relates to a method to induce nerve stem cell expanding and restrict differentiation toward gial cell. It adopts a serum-free culture system containing a new type grain legumes agglutinin to induce nerve stem cell expanding and restrict differentiation toward gial cell. It provides evidence to nerve cell renewable theory and supplies daughter cell to nerve cell transplant and micro-encapsulation preparation. It could be also used as extraneous pharmacokinetics model to selecting cell medicament. The invention would have great application prospect, and great social and economic benefits.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for expanding neural stem cells (neuralstem cells, NSCs) and inhibiting their differentiation into glial cells and its application. Background technique [0002] Nervous system diseases such as Parkinson's disease, epilepsy, Alzheimer's disease, Huntington's disease, Lou Gehrig's disease, spinal cord injury, multiple lateral sclerosis, metabolic disorders and major retinal diseases, including The damage of nerve axons and the damage and loss of nerve cells are both caused by the continuous loss of neurons and there is no way to replace them. For diseases of the nervous system, due to the existence of the blood-brain barrier, conventional treatment methods are often difficult to be effective. At the same time, because the genetic, metabolic and infectious diseases of the nervous system are often widespread or even whole-brain lesions, brain cell or tissue transplantation in th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08C12N5/06C12N5/0797
Inventor 裴雪涛谢小燕李艳华陈琳师伟姚海雷
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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