A method for producing age-preserving basal forebrain cholinergic neurons from non-neuronal transformation

A technology of cholinergic neurons and nerve cells, applied in the direction of nervous system cells, biochemical equipment and methods, botany equipment and methods, etc., can solve problems such as unprepared forebrain cholinergic neurons

Active Publication Date: 2022-07-05
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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  • A method for producing age-preserving basal forebrain cholinergic neurons from non-neuronal transformation
  • A method for producing age-preserving basal forebrain cholinergic neurons from non-neuronal transformation
  • A method for producing age-preserving basal forebrain cholinergic neurons from non-neuronal 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 and application:

[0052] A. the above-mentioned genes are respectively constructed into commercially available or own retroviral vectors, lentiviral vectors or AAV viral vectors, and the promoters that regulate the expression level 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 linked by 2A sequences (for example, T2A, E2A, P2A, F2A, etc.) or IRES sequences from different sources, and a green or red fluorescent reporter gene can optionally be further introduced to facilitate the determination of viral packaging quality and titer, observe changes in cell morphology, determine cell purity and subsequent analysis for various specific applications.

[0053] b. Package the above-mentioned gene vectors into corresponding retroviruses, lentiviruses or AAV viruses through...

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 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 in 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 workbench (Suzhou purification, SW-CJ-2FD); fluorescence microscope (Thermo EVOS M5000); ultra-low temperature refrigerator (Thermo 900 series-902); Instrument TGL-20M); normal temperature high-speed centrifuge (Eppendorf 5424);

[0086] B. Intelligent high pressure steam sterilizer (Shanghai Shen'an LDZM-80KCS); American SHELLAB drying box (CE3F-2); electric heating digital dis...

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. The rest of the materials are the same as those in Example 1 (omitted).

[0104] 2. Preparation method

[0105] 1) Plasmid construction: Four genes, ASCL1, LHX8, GBX1 and SOX4, were respectively constructed into retrovirus vectors, and EF1α was used as the promoter for regulating gene expression level in the vector. At the same time, a green fluorescent reporter gene was 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 use for various subsequent specific applications.

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

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Abstract

The present invention relates to a method for preparing age-preserving basal forebrain cholinergic neurons from non-neuronal cell transformation. The preparation method of the present invention optimizes key cell direct transdifferentiation gene combinations and promoters for regulating expression levels, packages viruses that can efficiently infect donor cells of various ages, and adopts a suitable coating matrix to promote donor cells and The transformed neuron cells grow adherently to the wall, and the induced differentiation medium containing small molecular compounds and growth factors that can promote transdifferentiation is used. Brain cholinergic neurons are finally isolated and purified to obtain high-purity basal forebrain cholinergic neurons.

Description

technical field [0001] The present invention relates to the field of research and treatment of Alzheimer's disease, and more particularly, the present invention relates to a method for preparing age-preserving basal forebrain cholinergic neurons from non-neuronal cell transformation. Background technique [0002] Alzheimer's disease (AD) is a neurodegenerative disease with progressive cognitive impairment as its main clinical manifestation. Clinically, it is characterized by generalized dementia manifestations such as memory impairment, aphasia, apraxia, agnosia, impairment of visuospatial skills, executive dysfunction, and personality and behavioral 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 Alzheimer's. As the world's population ages, the incidence of AD is rising rapidly, with one person getting sick almost every 7 seconds, and it is predicted to increase to 90 million by 2050....

Claims

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Application Information

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Patent Type & Authority Patents(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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