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Method for preparing human-derived dorsal root node neurons with age characteristics through non-nerve cell transformation

A technology of nerve cells and neurons, applied in the field of human dorsal root ganglion neurons

Pending Publication Date: 2022-04-01
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 dorsal root ganglion neurons from normal people or patients with neuralgia at the onset stage with high efficiency and high purity

Method used

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  • Method for preparing human-derived dorsal root node neurons with age characteristics through non-nerve cell transformation
  • Method for preparing human-derived dorsal root node neurons with age characteristics through non-nerve cell transformation
  • Method for preparing human-derived dorsal root node neurons with age characteristics through non-nerve cell transformation

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Experimental program
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preparation example Construction

[0063] For the above-mentioned preparation method, the following steps can be specifically included in the actual preparation application:

[0064] a. Construct the above genes into commercially available or self-owned retroviral vectors, lentiviral vectors or AAV viral vectors, and the promoters regulating 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 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.

[0065] b. Through cell transfection, the above-mentioned gene vectors are packaged into corresponding retroviruses, lentiviruses ...

Embodiment 1

[0092] Example 1 Rapid and efficient preparation of high-purity dorsal root ganglion neurons from infant and adult skin fibroblasts

[0093] 1. Materials

[0094] 1) Cells: 293T cells used for virus packaging were purchased from ATCC (CRL-3216) in the United States; infant and adult skin fibroblasts were purchased from Genetic Cell Repository (Coriell Institute for Medical Research, NJ, USA, Cat. No. AG07095) and Sciencell ( Cat. No. #2320) Inc. 293T and human skin fibroblasts were cultured with high-glucose DMEM medium containing 10-20% FBS and 1×P / S double antibody.

[0095] 2) Instruments and reagents:

[0096] A. CO2 cell incubator (ESCO CLM-170B-8-CN); ultra-clean bench (ESCO AC2-5S1); fluorescence microscope (Thermo EVOS M5000); ultra-low temperature refrigerator (Haier DW-86L388J); liquid nitrogen tank (Haier YDS-175-216-F); high-speed refrigerated centrifuge (Baiyang BY-R20 type); normal temperature high-speed centrifuge (Xiangyi H1650-W);

[0097] B. Intelligent h...

Embodiment 2

[0110] Example 2 Rapid and efficient preparation of high-purity dorsal root ganglion neurons from human embryonic lung fibroblasts

[0111] 1. Materials

[0112] Human embryonic lung fibroblasts MRC-5 were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. (ZQ0006). All the other materials are with embodiment 1 (omitted).

[0113] 2. Preparation method

[0114] 1) Plasmid construction: NGN2, ISL2, BRN3b and SOX4 genes were respectively constructed into retroviral vectors, and the promoters regulating gene expression levels in the vectors were CMV. 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.

[0115] 2) Virus packaging: each plasmid carrying the above-mentioned genes, pGP and pVSV-G was mixed with the transfection reagent Lipofectamine2000...

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Abstract

The invention relates to a method for preparing dorsal root node neurons with age characteristics from non-nerve cell transformation. According to the preparation method disclosed by the invention, a key cell direct transdifferentiation gene combination and a promoter for regulating and controlling the expression level are optimized, viruses capable of efficiently infecting donor cells of various ages can be packaged, and an appropriate coating matrix is adopted to promote the donor cells and transformed nerve cells to adhere to the wall to grow; an induced differentiation culture solution containing a small molecule compound capable of promoting transdifferentiation and growth factors is utilized, a large number of dorsal root node neurons are obtained through transdifferentiation after the differentiation culture solution is replaced several times within 10-14 days, and finally the dorsal root node neurons are separated and purified to obtain the high-purity dorsal root node neurons.

Description

technical field [0001] The present invention relates to the field of research and treatment of neuralgia, more specifically, the present invention relates to a related method for preparing human-derived dorsal root ganglion neurons with age-reserving transformation from non-nervous cells. Background technique [0002] Neuralgia is a common clinical complex disease, which can be divided into many types according to the etiology or site of onset, such as postherpetic neuralgia, cancerous neuralgia, diabetic neuralgia, post-stroke neuralgia, trigeminal neuralgia, Glossopharyngeal neuralgia etc. All kinds of long-term chronic neuralgia will seriously affect the quality of life of patients, and some severe pains may even cause patients to commit suicide due to unbearable pain. All kinds of neuralgia are age-related (the induced diseases are also closely related to age). For example, the occurrence of postherpetic neuralgia mainly occurs after adulthood, and the older the age, th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N5/0793C12Q1/02
Inventor 刘猛六柳明杰沈雁飞
Owner 宁波易赛腾生物科技有限公司
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