A method for isolating and culturing naked mole rat skeletal muscle myoblasts

A technique for the isolation and cultivation of myoblasts, applied in cell dissociation methods, cell culture active agents, bone/connective tissue cells, etc. Problems such as low separation purity

Active Publication Date: 2021-05-07
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] It can be seen that the purity of the primary isolation of naked mole rat skeletal muscle myoblasts is not high, which is not conducive to the research on the hypoxia resistance of naked mole rats, so that no naked mole rat skeletal muscle myoblasts have been seen abroad. Reports on research on hypoxia

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  • A method for isolating and culturing naked mole rat skeletal muscle myoblasts
  • A method for isolating and culturing naked mole rat skeletal muscle myoblasts

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Example 1 Method for Isolation and Culture of Naked Mole Rat Skeletal Muscle Myoblasts

[0038] (1) After suffocating 5 newborn naked mole rats, soak them in 75% ethanol for 5 minutes, remove the skeletal muscle tissue as soon as possible in the ultra-clean bench, and cut the lung tissue into 1mm after rinsing with pre-cooled washing solution until clear 3 Small pieces, transferred into a penicillin vial.

[0039] (2) Add 5 mL of tissue digestion solution, digest at 37°C for 30 minutes, and shake once every 5 minutes; %, Dnase I0.01%.

[0040] (3) Remove the digested cell suspension, add an inhibitory solution equal in volume to the tissue digestion solution to terminate digestion; the inhibitory solution consists of: 90% low-sugar DMEM medium, 10% FBS, 1% PS double antibody, 0.01% DNase I;

[0041] (4) The cell suspension was filtered through a cell sieve, the filtrate was collected, and the cells were collected by centrifugation. 2 After culturing in the environment...

Embodiment 2

[0042] Example 2 Cell Identification, Purity and Vitality Evaluation of the Skeletal Muscle Myoblasts of Example 1

[0043] 1. Cell Identification

[0044] Identification by DESMIN immunofluorescence staining

[0045] Desmin is one of the constituents of the intermediate filaments of the cytoskeleton in muscle cells, and it is the earliest muscle-derived marker protein expressed. Detecting the expression of desmin in cells by immunocytochemical staining is the best method known to identify skeletal muscle myoblasts.

[0046] After the skeletal muscle myoblasts reach the ideal density, fix the sample with 4% (W / V) paraformaldehyde for 10 minutes; wash with PBS for 5 minutes, and treat with 0.1% (V / V) Triton X-100 for 10 minutes to increase the permeability of the cell membrane ; goat serum was blocked for 1 hour; incubated with the primary antibody (desmin was diluted with PBS 1:1000 containing 0.1% (V / V) Triton X-100), overnight at 4°C; in addition, PBS was used instead of t...

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Abstract

The invention relates to the field of in vitro culture of cells, in particular to a method for separating and culturing naked mole-rat skeletal muscle myoblasts. The naked mole-rat is obtained by using trypsin combined with collagenase and neutral protease to digest and separate, and filtering and differentially adhering to the wall Skeletal muscle myoblasts. The present invention establishes a set of methods for isolating, cultivating and identifying naked mole-rat skeletal muscle myoblasts, and evaluates the maintenance of the biological characteristics of the cells cultured by this method, confirming that this culture method can obtain naked mole rat skeletal myoblasts, which provides the possibility to further study the functional changes of naked mole rat skeletal myoblasts under hypoxic physiological conditions.

Description

technical field [0001] The invention relates to the field of in vitro culture of cells, in particular to a method for separating and culturing naked mole rat skeletal muscle myoblasts. Background technique [0002] The naked mole rat is a digging rodent distributed in parts of East Africa, taxonomically belonging to the class Mammalia, Rodentia, Porcupine suborder, Limeridae, Anisocephaly subfamily, Naked mole rat genus, Naked mole rat Rat species. Naked mole rats live in the dark underground all their lives and can adapt to low oxygen and high carbon dioxide concentrations. The low-oxygen tolerance of naked mole rats has attracted widespread attention from scientists. Skeletal muscle is an important object of hypoxia research. As a sports organ, skeletal muscle is highly sensitive to oxygen concentration. [0003] The muscle satellite cells (muscle satellite cells) isolated from skeletal muscle with the ability to self-renew and differentiate into muscle fibers are colle...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/077
CPCC12N5/0658C12N2501/998C12N2509/00
Inventor 崔淑芳丛薇李周桐杨文静余琛琳赵善民林丽芳程继帅刘攀徐晨
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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