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Kit for preparing human body dopaminergic neurons and application thereof

A kit and dopamine technology, applied in the field of biopharmaceuticals, can solve the problem that the efficiency of dopamine neurons needs to be further improved, and achieve the effects of reducing the risk of personal injury, avoiding gene integration, and reducing experimental costs.

Pending Publication Date: 2021-10-19
山东大学深圳研究院 +3
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Professor Connor used transcription factors to synthesize chemically modified RNA, and its defects in the efficiency of dopamine neurons produced by the induced culture system need to be further improved

Method used

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  • Kit for preparing human body dopaminergic neurons and application thereof
  • Kit for preparing human body dopaminergic neurons and application thereof
  • Kit for preparing human body dopaminergic neurons and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Skin fibroblast transfection experimental steps:

[0050] 1. Collect skin tissue and obtain fibroblasts:

[0051] 1) Sign the skin donor’s informed consent, and collect skin tissue (2-3mm thick, 6mm in diameter) under local anesthesia;

[0052] 2) The collected tissue was first washed 3 times with 10 mL of sterile PBS (containing 1% double antibody P / S) under aseptic conditions;

[0053] 3) Cut the tissue block into tissue fragments with sterile ophthalmic scissors, and the time should not exceed 10 minutes;

[0054] 4) Aspirate 5ml of cell culture solution A to resuspend the tissue fragments;

[0055] 5) Add 1ml of cell suspension to each 35mm cell culture dish, and then add 1ml of cell culture medium A. Try not to change the medium for the first 3 days, and perform a full medium change after the cell tissue block adheres to the wall;

[0056] 6) After the confluence of the cells reaches 85%, subculture and cryopreserve.

[0057] Diagram of the isolation and cultu...

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Abstract

The invention discloses a kit for preparing human body dopaminergic neurons and application thereof, and belongs to the field of biopharmacys. The kit comprises a fibroblast efficient separation culture solution, chemical modification mRNA, an induction culture solution, a small molecule compound induction combination 1 and an induction combination 2 for promoting maturation and survival. The invention also provides an application of the kit in reprogramming the skin fibroblasts of the Parkinson's patient to generate the dopaminergic neurons. According to the kit, the combination of cmRNA-ASLC1, cmRNA-LMX1A and cmRNA-NURR1 is generated by using the chemically modified mRNA (cmRNA) so as to directly reprogram the human fibroblasts into the dopamine nerve cells, the inherent risk of genome integration and insertion mutagenesis in the DNA method is eliminated by using the chemically modified mRNA (cmRNA), the transgenic tracer agent is not left, the small molecule combination is used to optimize the induction culture system. The generation efficiency and the maturity of the dopaminergic neurons are improved.

Description

technical field [0001] The invention relates to a kit and its application, in particular to a kit for preparing human dopamine neurons and its application, and belongs to the field of biopharmaceuticals. Background technique [0002] Parkinson's disease (Parkinson's disease, PD) is the second largest neurodegenerative disease after senile dementia. The main manifestations of patients are resting tremor, muscle rigidity, decreased movement, and abnormal posture and gait, which are mainly caused by degeneration of dopaminergic neurons in the substantia nigra pars compacta of the midbrain. [0003] The current clinical treatment methods for Parkinson's disease are mostly limited to drug therapy and neurosurgery, which can only delay the development of the disease. Mature neurons are non-renewable, making it difficult to effectively replenish damaged nerve cells, so transplantation of exogenous nerve cell replacement therapy has become one of the potential therapeutic approache...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N5/077
CPCC12N5/0619C12N5/0656C12N2506/1307C12N2501/60C12N2510/00C12N2501/115C12N2501/727C12N2501/01
Inventor 韩发彬芦现杰刘延明王伟宋娜王学翔
Owner 山东大学深圳研究院
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