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Method for preparing dopaminergic neurons reserving age characteristics through non-nerve cell transformation

A dopaminergic, neuronal technology used in the research and treatment of Parkinson's disease

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 that can produce dopaminergic neurons from normal people or PD patients at the onset stage with high efficiency and high purity

Method used

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  • Method for preparing dopaminergic neurons reserving age characteristics through non-nerve cell transformation
  • Method for preparing dopaminergic neurons reserving age characteristics through non-nerve cell transformation
  • Method for preparing dopaminergic neurons reserving age characteristics through non-nerve cell transformation

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preparation example Construction

[0059] For the above-mentioned preparation method, the following steps can be specifically included in the actual preparation application:

[0060] 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 be connected 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 be optionally further introduced to facilitate the determination of viral packaging quality and titer. Degree, observation of changes in cell morphology, determination of cell purity and subsequent analysis of various specific applications.

[0061]b. Through cell transfection, the above-mentioned gene vectors are packaged into corresponding retroviruses, lentiviruses or A...

Embodiment 1

[0088] Example 1 Rapid and efficient preparation of high-purity dopaminergic neurons from human skin fibroblasts

[0089] 1. Materials

[0090] 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.

[0091] 2) Instruments and reagents:

[0092] 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 (Eppendorf5424);

[0093] B. Intelligent high-pressure steam sterilizer (Shanghai Shenan LDZM-80KCS); American SHELLAB drying oven (CE3F-2); ...

Embodiment 2

[0107] Example 2 Rapid and efficient preparation of high-purity dopaminergic neurons from human embryonic lung fibroblasts

[0108] 1. Materials

[0109] Human embryonic lung fibroblasts MRC-5 were purchased from Heyuan Biotechnology (Shanghai) Co., Ltd. All the other materials are with embodiment 1 (omitted).

[0110] 2. Preparation method

[0111] 1) Plasmid construction: ASCL1, LMX1a, NURR1 and SOX4 genes were respectively constructed into retroviral vectors, and CMV 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 T2A sequence, so as to determine the quality and titer of virus packaging, observe changes in cell morphology, determine cell purity, and be used in various subsequent specific applications.

[0112] 2) Virus packaging: each plasmid carrying the above-mentioned genes, pGP and pVSV-G was mixed with the transfection reagent Lipofectamine2000 according to the ra...

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Abstract

The invention relates to a method for preparing dopaminergic 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 solutioncontaining small molecular compounds capable of promoting transdifferentiation and growth factors is utilized; a large number of dopaminergic neurons are obtained through transdifferentiation after the differentiation culture solution is replaced for multiple times within 10-14 days; and finally, the high-purity dopaminergic neurons are obtained through separation and purification.

Description

technical field [0001] The present invention relates to the field of research and treatment of Parkinson's disease, more specifically, the present invention relates to a related method for preparing dopaminergic neurons with age-retaining characteristics from non-nervous cells. Background technique [0002] Parkinson's disease (Parkinson disease, PD) is a kind of common nervous system degenerative disease, and it usually impairs patient's motor skills, language and other functions (Jankovic, J.Neurol.Neurosurg.Psychiatr.2008, 79 (4): 368-76). The main clinical manifestations of Parkinson's disease are resting tremor, bradykinesia, muscle rigidity, and posture and gait disturbance. [0003] Dopaminergic neurons are the main subtype of nerve cells that begin to be invaded in the early stage of Parkinson's disease and undergo continuous degeneration and death. This pathological process is closely related to various symptoms of PD patients. The use of dopaminergic neurons as a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10C12Q1/02
CPCC12N15/86C12N5/0619G01N33/5058C12N2740/15043C12N2800/107C12N2510/00C12N2506/45C12N2503/02G01N2500/10
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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