Isolated culture method of alveolar type II epithelial cells of naked mole rats

A technique for the isolation and cultivation of epithelial cells, applied in cell dissociation methods, respiratory/lung cells, epidermal cells/skin cells, etc., can solve the problem of low purity of the primary isolation of naked mole rat alveolar epithelial type II cells, which is unfavorable for naked mole rats Mouse resistance to hypoxia and anti-tumor research, low affinity and other issues

Active Publication Date: 2017-08-08
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no commercial product of naked mole rat IgG. Mouse IgG is used for screening. Concentration still needs to be further explored
[0005] The purity of the primary isolation of naked mole rat alveolar epithelial type II cells is not high, which is not conducive to the study of hypoxia resistance and anti-tumor on naked mole rats. There are no reports on this research abroad.

Method used

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  • Isolated culture method of alveolar type II epithelial cells of naked mole rats
  • Isolated culture method of alveolar type II epithelial cells of naked mole rats
  • Isolated culture method of alveolar type II epithelial cells of naked mole rats

Examples

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Embodiment 1

[0039] Embodiment 1 Naked mole rat alveolar type II epithelial cell isolation and culture method

[0040] (1) After killing an adult naked mole rat by decapitation, soak it in 75% ethanol for 5 minutes, remove the whole lung tissue as soon as possible in an ultra-clean bench, rinse the lung tissue with pre-cooled washing solution until clear, and cut the lung tissue into 1mm 3 Small pieces, transferred into a penicillin vial.

[0041] (2) Add 2mL of tissue digestion solution and digest at 35°C for 5min. Remove the digested cell suspension, add an equal volume of inhibitor solution to the tissue digestion solution to terminate the digestion;

[0042] (3) Repeat step (2) 5 times until the tissue blocks basically disappear. Combine 20 mL of the cell suspension, filter through a 200-mesh cell sieve, collect the filtrate, and centrifuge at 100 g for 5 min to collect the cells;

[0043] (4) Resuspend the cells in 5ml adhesion medium preheated at 35°C, and insert them into a 10cm...

Embodiment 2 Embodiment 1

[0045] Example 2 Cell Identification, Purity and Vitality Evaluation of Alveolar Type II Epithelial Cells of Example 1

[0046] 1. Cell identification

[0047] ①Pro-SP-C immunofluorescence staining identification

[0048] SP-C is an alveolar surfactant protein specifically expressed by alveolar type II epithelial cells, while the other three surfactant proteins SP-A, SP-B, and SP-D are also expressed in other cells, so SP among the surfactant proteins was selected. -C can be used to identify alveolar type II epithelial cells.

[0049] When alveolar type II epithelial cells 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 h; incubated with primary antibody (SFTPC was diluted with PBS containing 0.1% (V / V) Triton X-100 1:1000) and diluted overnight at 4°C; in addition, PBS was u...

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Abstract

The invention relates to the field of in-vitro culture of cells and in particular relates to an isolated culture method of alveolar type II epithelial cells of naked mole rats. The isolated culture method comprises the following steps: combining pancreatin and elastase and carrying out digestion and isolation; filtering, carrying out differential adhesion and screening and purifying an IgG antibody to obtain the alveolar type II epithelial cells of the naked mole rats. The invention establishes one set of method for isolating, culturing and identifying the alveolar type II epithelial cells of the naked mole rats; a biological characteristic maintaining condition of the cells cultured by the method is evaluated, so that the culture method used for obtaining the high-purity alveolar type II epithelial cells of the naked mole rats is verified, the possibility for further deeply researching proliferation and differentiation, liquid transferring, synthesis and secretion of the alveolar type II epithelial cells, and function changes of the alveolar type II epithelial cells under pathophysiologic conditions of lung injuries and repairing and the like is provided.

Description

technical field [0001] The invention relates to the field of in vitro culture of cells, in particular to a method for isolating and culturing alveolar type II epithelial cells of naked mole rats. 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. Naked mole rats have the longest lifespan among rodents, up to nearly 30 years; they are super immune to cancer, and there is still no case of spontaneous cancer in naked mole rats. Longevity, anti-aging, hypoxia resistance, anti-tumor and other properties of naked mole rats have attracted widespread attention from scientists, and research results on these properties have been published in f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0625C12N5/0688C12N2509/00
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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