Method for preparing basal forebrain cholinergic neurons reserving age characteristics through non-nerve cell transformation

A technology for cholinergic neurons and nerve cells, which is applied in the directions of cells of the nervous system, biochemical equipment and methods, and botanical equipment and methods, and can solve the problem of not preparing forebrain cholinergic neurons and so on

Active Publication Date: 2021-02-05
宁波易赛腾生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite the great demand, there are still no related technologies and products on the market to prepare basal forebrain cholinergic neurons from normal people or AD patients

Method used

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  • Method for preparing basal forebrain cholinergic neurons reserving age characteristics through non-nerve cell transformation
  • Method for preparing basal forebrain cholinergic neurons reserving age characteristics through non-nerve cell transformation
  • Method for preparing basal forebrain cholinergic neurons reserving age characteristics through non-nerve cell transformation

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preparation example Construction

[0051] For the above-mentioned preparation method, the following steps can be specifically included in the actual preparation application:

[0052] a. Construct the above genes into commercially available or self-owned retroviral vectors, lentiviral vectors or AAV viral vectors, and the promoters regulating expression levels in each vector can be CMV, CAG, EF1α, PGK, TRE Tight, Any one or more of TRE3G. At the same time, the above-mentioned genes can also be connected by 2A sequences (for example, T2A, E2A, P2A, F2A, etc.) or IRES sequences from different sources, and green or red fluorescent reporter genes can be optionally further introduced to facilitate the determination of viral packaging quality and Titer, observation of changes in cell morphology, determination of cell purity, and subsequent analysis for various specific applications.

[0053] b. Through cell transfection, the above-mentioned gene vectors are packaged into corresponding retroviruses, lentiviruses or AA...

Embodiment 1

[0081] Example 1 Rapid and efficient preparation of high-purity basal forebrain cholinergic neurons from human skin fibroblasts

[0082] 1. Materials

[0083] 1) Cells: 293T cells used for virus packaging were purchased from Heyuan Biotechnology (Shanghai) Co., Ltd.; human skin fibroblasts AG08517 were purchased from Genetic Cell Repository (Coriell Institute for Medical Research, NJ, USA). 293T and human skin fibroblasts were cultured with high-glucose DMEM medium containing 10-20% FBS and 1×P / S double antibody.

[0084] 2) Instruments and reagents:

[0085] A. CO2 cell incubator (Thermo BB15); ultra-clean bench (Suzhou purification, SW-CJ-2FD); fluorescence microscope (Thermo EVOS M5000); ultra-low temperature refrigerator (Thermo 900 series-902); high-speed refrigerated centrifuge (Xiang instrument TGL-20M); room temperature high-speed centrifuge (Eppendorf 5424);

[0086] B. Intelligent high-pressure steam sterilizer (Shanghai Shenan LDZM-80KCS); American SHELLAB drying...

Embodiment 2

[0101] Example 2 Rapid and efficient preparation of high-purity basal forebrain cholinergic neurons from human embryonic lung fibroblasts

[0102] 1. Materials

[0103] Human embryonic lung fibroblasts MRC-5 were purchased from Heyuan Biotechnology (Shanghai) Co., Ltd. All the other materials are with embodiment 1 (omitted).

[0104] 2. Preparation method

[0105] 1) Plasmid construction: Four genes, ASCL1, LHX8, GBX1 and SOX4, were respectively constructed into retroviral vectors, and EF1α was used as the promoter regulating gene expression level in the vectors. At the same time, a green fluorescent reporter gene is introduced through the IRES sequence after the stop codon of the ASCL1 gene, so as to determine the quality and titer of virus packaging, observe changes in cell morphology, determine cell purity, and be used for various subsequent specific applications.

[0106] 2) Virus packaging: each plasmid carrying the above-mentioned genes, pGP and pVSV-G was mixed with ...

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Abstract

The invention relates to a method for preparing basal forebrain cholinergic neurons reserving age characteristics through non-nerve cell transformation. According to the preparation method disclosed by the invention, key cell direct transdifferentiation gene combinations and promoters for regulating and controlling the expression level are optimized, viruses capable of efficiently infecting donor-derived cells of various ages are packaged, and adherent growth of the donor-derived cells and transformed neuronal cells is promoted by adopting a proper coating matrix; an induced differentiation culture solution containing small molecular compounds capable of promoting transdifferentiation and growth factors is utilized; a large number of basal forebrain cholinergic neurons are obtained throughtransdifferentiation after the differentiation culture solution is replaced for multiple times within 10-14 days; and finally, the high-purity basal forebrain cholinergic neurons are obtained throughseparation and purification.

Description

technical field [0001] The present invention relates to the field of research and treatment of Alzheimer's disease, more specifically, the present invention relates to a related method for preparing basal forebrain cholinergic neurons that retain age characteristics from non-nervous cells. Background technique [0002] Alzheimer's disease (AD) is a neurodegenerative disease with progressive cognitive impairment as the main clinical manifestation. Clinically, it is characterized by generalized dementia such as memory impairment, aphasia, apraxia, agnosia, impairment of visuospatial skills, executive dysfunction, and personality and behavior changes. Generally, those with onset before the age of 65 are called Alzheimer's disease; those with onset after the age of 65 are called senile dementia. As the world's population is aging, the incidence of AD is rising rapidly, with one person suffering from the disease almost every 7 seconds, and it is predicted that it will increase t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/864C12N5/10C12Q1/02
CPCC12N15/86C12N5/0619G01N33/5008G01N33/5058C12N2740/10043C12N2740/15043C12N2750/14143C12N2510/00G01N2800/30
Inventor 不公告发明人
Owner 宁波易赛腾生物科技有限公司
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