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A culture method and neuron technology, applied in the field of cell biology, can solve problems such as not applicable to naked mole rats
Active Publication Date: 2017-03-22
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY +1
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However, in the prior art, the methods for isolating, purifying and culturing DRG neurons of mice and rats are not applicable to naked mole rats. At present, there are no related reports on the methods of isolating, purifying and culturing DRG neurons of naked mole rats.
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Embodiment 1
[0040] Example 1: Isolation, purification and culture of naked mole rat DRG neurons
[0041] 1. Experimental materials
[0042] Clean-grade naked mole rats at 55-60 days of pregnancy were provided by the Experimental Animal Center of the Second Military Medical University of the Chinese People's Liberation Army.
[0043] DNase I was purchased from Solarbio Biotechnology Co., Ltd., NGF, 5-fluorouracil, trypsin, D-polylysine, collagen penicillin and streptomycin mixture, etc. were purchased from Sigma Company, DMEM, low-sugar DMEM, Australian source fetal Bovine serum, Neurobasal medium, and B27 serum-free additive factors were purchased from Thermo Fisher Scientific, and 24-well culture plates were purchased from Corning.
[0044] The components of the mixed nerve cell culture medium are low-sugar DMEM medium containing 10% fetal bovine serum by volume fraction, and the DRG neuron purification medium is Neurobasal+2% B27 by volume fraction+final concentration of 10 μM 5-fluoro...
Embodiment 2
[0053] Example 2: Identification of Naked Mole Rat DRG Neurons
[0054] Identification of naked mole-rat DRG neurons obtained in Example 1: DRG neurons in serum-free medium were fixed with 4% paraformaldehyde, and identified by morphological identification, immunorefinement chemistry and other methods.
[0055] 1. Cell morphology identification:
[0056] Identification methods described in references (Yang Y, Huang J, Mis MA, Estacion M, Macala L, ShahP, Schulman BR, Horton DB, Dib-Hajj SD, Waxman SG.Nav1.7-A1632G Mutation from aFamily with Inherited Erythromelalgia : Enhanced Firing of Dorsal Root Ganglia Neurons Evoked by Thermal Stimuli. J Neurosci. 2016 Jul 13; 36(28):7511-22.).
[0057] The result is as figure 1 (low magnification lens) and figure 2 (High magnification) shows that under the light microscope, the naked mole-rat DRG neurons have larger cell bodies, one or several shorter dendrites, and one elongated axon at the other end.
[0058] 2. Immunocytochemical...
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Abstract
The invention relates to the technical field of cytobiology, in particular to a naked mole rat DRG neuron culture method. DRG neurons are separated from embryonic naked mole rat medulla spinalis, purified and cultured by comprehensively adopting multiple culture media, and then the reasonable culture method suitable for the DRG neurons of poikilothermal rodent mammal naked mole rats is explored. According to the method, a large quantity of naked mole rat DRG neurons with the normal functional activity can be easily, conveniently, efficiently and economically obtained, it can be guaranteed that the cells still can keep the biological characteristics achieved under the body condition in an in-vitro environment by means of culture conducted under anoxic condition, then the special physiological functions of the naked mole rat DRG neurons can be conveniently further researched in a pure in-vitro cell culture environment, and an important theory basis is provided for exploration of the biological mechanism of the naked mole rat DRG neurons and application of the naked mole rat DRG neurons in the clinical related fields.
Description
technical field [0001] The invention relates to the technical field of cell biology, in particular to a cell separation, purification and culture method, in particular to a naked mole rat DRG neuron separation, purification and culture method. Background technique [0002] Chronic pain seriously affects the quality of life of human beings, and effective treatment measures are urgently needed. DRG neurons, as important sensory neurons in the body, play a major role in the occurrence and development of pain (Vallejo R, Tilley DM, DL, Kelley CA, DeMaegd M, Benyamin R. Genomics of the Effect of SpinalCord Stimulation on an Animal Model of Neuropathic Pain. Neuromodulation. 2016 Jul 8. doi:10.1111 / ner.12465.). Therefore, further revealing the physiological functions and characteristics of DRG neurons will help to clarify the functions of DRG neurons and their role in the occurrence and development of nervous system diseases. [0003] The naked mole rat is a temperature-changin...
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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0793
CPCC12N5/0619C12N2500/40C12N2509/00
Inventor 崔淑芳杨文静程继帅徐晨林丽芳蔡丽萍李周桐丛薇余琛琳赵善民
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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