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Preparation method of human adult neural stem cells and application thereof in prevention and treatment of cerebral apoplexy

A neural stem cell and stem cell line technology, applied in nervous system cells, biochemical equipment and methods, medical preparations containing active ingredients, etc. problems, to reduce research errors, to express efficiently, and to ensure stability.

Pending Publication Date: 2020-06-26
北京银丰鼎诚生物工程技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the number of cells that can be obtained by conventional NSCs culture methods is limited, which cannot meet the needs of clinical treatment.
On the one hand, in order to achieve the therapeutic effect on multiple people, it is usually necessary to obtain multiple embryonic tissue samples, so there is a large ethical controversy
On the other hand, there are differences in the biological characteristics of cells from different sample sources, and the resulting results also have certain variability, thus affecting the accuracy of scientific data

Method used

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  • Preparation method of human adult neural stem cells and application thereof in prevention and treatment of cerebral apoplexy
  • Preparation method of human adult neural stem cells and application thereof in prevention and treatment of cerebral apoplexy
  • Preparation method of human adult neural stem cells and application thereof in prevention and treatment of cerebral apoplexy

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Experimental program
Comparison scheme
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Embodiment 1

[0031] Example 1: Primary culture, subculture and cryopreservation of neural stem cells

[0032] Various liquids used in the present embodiment are illustrated as follows:

[0033]

[0034]

[0035] 1. Basal medium: composed of DMEM / F12, HEPES and sodium bicarbonate. Among them, DMEM / F12 is 1 ×; HEPES is 20mmol / L; sodium bicarbonate is 10mmol / L.

[0036] 2. Primary culture medium for neural stem cells: consisting of basal medium, B27, epidermal growth factor, basic fibroblast growth factor, insulin, putrescine, Notch 1, WNT3a, progesterone, sodium selenite, insulin growth factor, Laminin composition; B27 is 1×, epidermal growth factor concentration 20ng / ml, fibroblast growth factor concentration 20ng / ml, insulin 10mg / ml, putrescine 16.2mg / L, Notch 150nmol / L, WNT3a 20nmol / L, corpus luteum Ketone 20nmol / L, sodium selenite 5ug / L, insulin growth factor 25mg / ml, laminin 5μg / ml, the basal medium is composed of DMEM / F12, HEPES and sodium bicarbonate.

[0037]3. Neural stem c...

Embodiment 2

[0048] Example 2: Stemness Identification of Neural Stem Cells—Flow Cytometry

[0049] Methods and results: Neural stem cell spheroids were cultured for 7-9 days at different passages, digested into single cells, and the main stemness markers Nestin, Musashi, CD133, and Sox2 were determined by flow cytometry. Surface marker CD133 specifically: centrifuge to collect at least 10 cells per tube 6 Centrifuge at 500g for 5min, discard the supernatant, and wash the cells twice with 4°C pre-cooled PBS. Add 200 μL PBS containing the corresponding antibody. Vortex for 3s, and incubate at 4°C in the dark for 40-50min. Add 2mL PBS directly to the centrifuge tube, centrifuge at 4000g for 6min at 4°C, discard the supernatant, and repeat once. Add 350-500 μL PBS to resuspend the cells and perform flow cytometry. Other intracellular markers are specifically: membrane permeation treatment is required before adding antibodies, and others are consistent with surface markers. As shown in th...

Embodiment 3

[0050] Example 3: Stemness Identification of Neural Stem Cells——Immunofluorescence

[0051] Methods and results: Neural stem cell spheres cultured for 7-9 days were selected, 5 μm frozen sections were made of the spheres, and stem markers Nestin, Musashi, CD133 and differentiation markers Tuj-1, GFAP, O4 were immunofluorescence stained. Specifically, the frozen sections were dried at room temperature for 15 minutes, the cell balls to be tested were circled with a histochemical pen, soaked in PBS for 10 minutes to remove OCT; the sections were sealed with PBS containing 10% sheep serum for 1 hour at room temperature, the blocking solution was removed, and Monoclonal antibody (1:200), overnight at 4°C; wash 3 times with PBS, 5 min each time; add corresponding fluorescent secondary antibody (1:100), incubate at room temperature for 1 h; wash 3 times with PBS, 10 min each time; Take pictures under a microscope. The result is as image 3 As shown, the positive rate of Nestin, Mus...

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Abstract

The invention discloses a preparation method of human adult neural stem cells and application thereof in prevention and treatment of cerebral apoplexy. Specifically, a cell line established by the method highly expresses neural stem cell markers including Nestin, Musashi, Sox2, CD133 and the like, still keeps high dryness when being passed to a P20 generation, and is high in stability. A culture supernatant of the cell line has a remarkable improvement effect on nerve cell damage caused by hydrogen peroxide and oxygen-glucose deprivation, has a remarkable improvement effect on animal models inan acute stage and a sequelae stage of the cerebral apoplexy, and can be used as a drug for preventing and treating the cerebral apoplexy.

Description

technical field [0001] The invention belongs to the fields of cell biology and neuropharmacology, and specifically relates to a preparation method of human adult neural stem cells and their application in preventing and treating stroke. Background technique [0002] my country has entered an aging society, and the aging rate is faster than the total population growth rate. The number and proportion of patients with various geriatric diseases are also on the rise. Stroke, as a common disease of middle-aged and elderly people, has become the second largest killer after cardiovascular disease in my country, seriously threatening human life and health. At present, there is no specific drug for preventing and treating such diseases, so it is imperative to develop drugs that are targeted, have few side effects, and can effectively control the disease. [0003] Neural stem cells (NSCs) are cells with self-renewal ability and multilineage differentiation potential. In recent year...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0797A61K35/30A61P9/10
CPCC12N5/0623A61K35/30A61P9/10C12N2501/11C12N2501/115C12N2501/33C12N2500/46C12N2501/42C12N2501/415C12N2501/105C12N2501/998
Inventor 宋修云安文强秦娇刘鹏汤永永
Owner 北京银丰鼎诚生物工程技术有限公司
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