Isolation and Identification of Exosomes from Skin Fibroblasts of Naked Mole Rats

doi:10.3969/j.issn.1674-5817.2020.06.007

Abstract

Abstract: Objective To identify skin fibroblasts isolated and cultured from naked mole rats, and explore whether the skin fibroblasts have the ability to secrete exosomes. Methods The back skin samples of germ-free newborn naked mole rats were collected and were cut into pieces to a size of 1-2 mm². The samples were spread evenly over the plate, cultured in low-sugar DMEM (containing 20% fetal bovine serum) after cell adhesion, and treated by trypsin digestion for purification. The vimentin expressions were detected by immunofluorescence and Western blotting. When the cells were grown to about 80% confluency, they were moved to low-sugar DMEM (containing 20% fetal bovine serum without exosomes) and cultured for 48 h. Next the exosomes in supernatant were seperated by ultra-high-speed centrifuge, and determined vesicular structures under transmission electron microscope, particle size by nano flow cytometry, and specific proteins related to exosomes by Western blotting, respectively. Results A small amount of cells were seen free from the tissues after culturing for 30 h, and a large number of cells after 5 d. The cells were elongated spindle or irregular polygonal, growing radially. Immunofluorescence and Western blotting analysis confirmed that the skin fibroblasts expressed vimentin. Saucer-like membranous exosomes were observed under transmission electron microscopy, with particles size between 46.75-206.75 nm. Western blotting showed that the proteins specific to exosomes included the lysosmal membrane associated glycoprotein 3 (CD63), tumor susceptibility gene 101 (TSG101) and heat shock protein 70 (Hsp70). Conclusion The cells isolated from naked mole rats have typical fibroblast morphology and express actually vimentin. The skin fibroblasts are successfully isolated from the naked mole rats, and can secrete the exosomes, which provides a basis for studying the bioactive molecules carried by naked mole rat exosomes and their biological functions.

Key words: Naked mole rats, Skin fibroblasts, Exosomes

CLC Number:

Q95-33

FENG Yan, WU Wenqing, ZHANG Jingyuan, LI Yu, LI Leichen, YUAN Zheng, CUI Shufang. Isolation and Identification of Exosomes from Skin Fibroblasts of Naked Mole Rats[J]. Laboratory Animal and Comparative Medicine, 2020, 40(6): 506-512.

Figures/Tables 5

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