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A technique of Schwann cells and culture method, applied in the field of cell biology, can solve the problem of unsuitable naked mole rat, etc., and achieve the effect of high repeatability and simple operation method
Active Publication Date: 2017-05-31
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY +1
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[0005] However, in the prior art, no matter the methods for the separation, purification and cultivation of Schwann cells in mice or rats are not applicable to naked mole rats, there is no relevant report on the methods for separation, purification and cultivation of Schwann cells in naked mole rats at present.
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Embodiment 1
[0038] Example 1: Separation, purification and culture of naked mole rat Schwann cells
[0039] 1. Experimental materials
[0040] Naked mole rats at embryonic days 58-65 were provided by the Experimental Animal Center of the Second Military Medical University of the Chinese People's Liberation Army. DNase I was purchased from Solarbio Biotechnology Co., Ltd., trypsin, L-polylysine, PDGFa, penicillin-streptomycin mixture, etc. were purchased from Sigma Company, DMEM, low-sugar DMEM, Australian-sourced fetal bovine serum, etc. were purchased from Thermo Fisher Scientific company, petri dishes, T25 and T75 culture flasks were purchased from Corning company.
[0041] The mixed nerve cell medium is a low-sugar DMEM medium containing 15% imported fetal bovine serum by volume.
[0042] Schwann cell purification medium is Neurobasal containing 2% B27 by volume fraction and PDGFa with mass volume fraction 0.01 mg / ml added.
[0043] 2. Isolation, purification and culture of naked mo...
Embodiment 2
[0046] Example 2: Identification of Naked Mole Rat Schwann Cells
[0047] Identification of the naked mole-rat Schwann cells obtained in Example 1: the Schwann cells in the proliferation medium were fixed with 4% paraformaldehyde, and identified in combination with morphological identification, immunochemical refinement and other methods.
[0048] 1. Cell morphology identification:
[0049] The identification method was described in references (Wenjing Yang, Lin Xiao, Cui Li, Xiuyun Liu, Mingdong Liu, Qi Shao, Dan Wang, Aijun Huang and Cheng He. TIP30 inhibit soligodendrocyte precursor cell differentiation via cytoplasmic sequestration of Olig1. Glia. 2015; 63( 4): 684-98.).
[0050] The result is as figure 2 As shown, under the light microscope, the cell body of Schwann cells is round or oval, with a strong refractive index, and there is an obvious halo around the cell body, and bipolar or tripolar protrusions protrude from the cell body to the surrounding. After 24 hours...
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Abstract
The invention relates to the technical field of cell biology, and in particular to a naked mole rate Schwann cell separation & purification and culture method. According to the method provided by the invention, Schwann cells are separated & purified and cultured from marrow DRG (dorsal root ganglion) of a fetal naked mole rate with the comprehensive use of a plurality of mediums, so that a reasonable method suitable for culturing the Schwann cells of a heterothermic mammal of rodent species, namely the naked mole rate, is found out. With the application of the method provided by the invention, a great amount of naked mole rate Schwann cells, which are normal in functions and activity, are conveniently, efficiently and economically obtained; the cells, when cultured under a hypoxia condition, can still reserve biological characteristics under an in-vivo state in an in-vitro environment, so as to facilitate further research, which is directly conducted in a pure in-vitro cell culture model, on special physiological functions of the naked mole rate Schwann cells; therefore, important theoretical bases are provided for researching a biological mechanism therein and for application to related clinical fields.
Description
technical field [0001] The invention relates to the technical field of cell biology, in particular to a method for separating, purifying and culturing cells, in particular to a method for separating, purifying and cultivating naked mole-rat Schwann cells. Background technique [0002] Schwann cells (Schwann cells) are myelinating cells of the spinal cord. The myelin sheath structure formed around the axons of neurons provides nutrition, support and protection to neurons, and the existence of myelin sheath can ensure the information between neurons. delivery rate. Immature Schwann cells can be stimulated by the microenvironment to initiate differentiation again. Spinal cord transection injury, amyotrophic lateral sclerosis and other diseases are usually accompanied by myelin damage (Billiards SS, Haynes RL, Folkerth RD, Borenstein NS, Trachtenberg FL, Rowitch DH, Ligon KL, Volpe JJ, KinneyHC.2008. Myelin abnormalities without oligodendrocyte loss in perivascular leukomalaci...
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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079
CPCC12N5/0622C12N2500/02C12N2501/135C12N2509/00
Inventor 崔淑芳杨文静孙伟程继帅林丽芳丛薇蔡丽萍余琛琳赵善民徐晨李周桐
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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