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Method for producing dopaminergic nerve cell by utilizing nerve stem cell internal amplification and directional induction differentiation

A technology for neural stem cells and in vitro expansion, applied in the field of in vitro expansion and directional induction of neural stem cells into dopaminergic nerve cells

Inactive Publication Date: 2009-07-01
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

A few foreign research institutions are conducting experimental research on NSC directional induction, but there is no report in China

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0006] Embodiment 1, neural stem cell culture

[0007] Pregnant wistar rats (12.5 days) were killed, and the fetal mice were taken out under aseptic conditions, and the brain hemispheres were separated, cut into small tissue pieces by ophthalmologists, digested with trypsin, blown and beaten to form a single-cell suspension, and divided into 4×10 5 / ml were grown in plastic culture bottles. The culture medium contains DMEM / F12, 2% B27, EGF 20ng / ml, bFGF 20ng / ml, penicillin-streptomycin 100u / ug.ml -1 . 37°C 5% CO 2 Incubator culture. After the appearance of primary clones (7-10 days), the neurospheres were collected by centrifugation. Digested with trypsin, pipetted with a fine Pasteur pipette into a single-cell suspension, and then diluted with 1×10 3 / ml were planted in new culture medium, and the medium was changed every 7 days for subculture. The primary cultured cells proliferate vigorously in the serum-free and proliferation factor-free medium NSC, 1×10 6 The prima...

Embodiment 2

[0008] Example 2. Directed differentiation of neural stem cells in vitro

[0009] Freshly passaged P3-5 generation NSCs were mixed with 5×10 4 Inoculate in a 24-well plate with polylysine-coated coverslips per ml, 1ml / well, replace the culture medium with DMEM / F12, 2% B27 after 3 days of adherent culture, and add ascorbic acid to make the final concentration Respectively 1nmol, 10nmol, 100nmol. After 3 days, half of the medium was changed, and the induction was terminated on the 6th day. Uninduced neural stem cells seldom differentiate into dopaminergic neurons (differentiation rate: 0.7±0.3%), but induction with a certain concentration of ascorbic acid AA (10-100nM) can promote the differentiation of NSCs into dopaminergic neurons.

Embodiment 3

[0010] Embodiment 3, neural stem cell transplantation

[0011] The subcultured neural stem cells were digested into single cells by trypsin, then suspended in phosphate buffer saline (PBS), counted, and the cell concentration was adjusted to 10 5 / μl, PD model rats were anesthetized by intraperitoneal injection of 3.5% chloral hydrate, fixed in a brain stereotaxic instrument, and transplanted with bregma as coordinates. The transplantation point is the right striatum of the rat (AP+1.0mm, LR-2.5mm, OV+4.5mm; AP-1.0, LR3.5, OV5.2), and each rat is injected with two points, 2μl / point , the experimental group implanted a total of 4 × 10 5 cells; rats in the control group were injected with the same amount of PBS. 100,000 units of penicillin were injected subcutaneously after the operation. Apomorphine (0.5 mg / kg) was used to induce rotation behavior one week after operation, and to detect it once a week thereafter until the rats were killed.

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Abstract

The purpose of the present invention is to provide a method for inducing and inducing the differentiation into dopaminergic nerve cells in vitro after a large amount of neural stem cells are expanded, transplanting them in the striatum of the brain, and differentiating them into dopaminergic nerve cells in situ. In order to achieve the above object, the present invention adopts the following technical solutions: 1) Utilize the biology of stem cells to enrich neural stem cells by adopting the method of serum-free culture, utilize the self-renewal and proliferation characteristics of stem cells to amplify the neural stem cells with mitogenic proliferative factors , so as to achieve the purpose of obtaining a large number of neural stem cells. 2) The scheme of directional induction and differentiation of isolated cells in vitro, characterized in that specific culture passages (3-5 generation cells) are used for induction, and specific drug ascorbic acid is used for directional induction at a specific concentration (10-100nM); 3 ) in vivo directed differentiation of cells cultured, which is characterized in that a large number of neural stem cells obtained are differentiated into dopaminergic neurons in situ in the striatum of the brain by using a specific cell density and injection method. The invention provides a supplementary source after the nerve cells are lost, lays the foundation for the stem cell transplantation to replace clinical treatment of Parkinson's disease, and provides a new model for the development and screening of related drugs.

Description

technical field [0001] The present invention relates to a method for cultivating and amplifying neural stem cells without serum by utilizing the biological characteristics of neural stem cells, and a method for in vitro directional induction and differentiation of stem cells obtained under this condition, and in vivo directional differentiation into dopaminergic nerve cells. Background technique [0002] The discovery of neural stem cells (neural stem cells, NSCs) is one of the most important advances in the field of neurobiology. The existence of NSCs has been confirmed and successfully isolated and cultured, which not only challenges the theory that the central nervous system cannot regenerate after it matures, It provides an ideal carrier for the study of neurogenesis and the pathogenesis of genetic diseases of the nervous system, and also brings new hope for the repair of nervous system damage and the treatment of degenerative diseases. NSC has great advantages as a cell...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/00C12N5/06
Inventor 裴雪涛郑敏岳文焦文仓赵连旭
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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