动物造模 z-bio 2022-05-06 642

　　Objective To isolate and culture naked mole rat skin fibroblasts, identify them, and explore whether naked mole rat skin fibroblasts have the ability to secrete exosomes.

　　Methods The back skin of newborn naked mole-rats was aseptically taken and cut into pieces with a size of 1-2 mm². The tissue pieces were flattened in a cell culture dish, and then cultured with low-glucose DMEM (containing 20% fetal bovine serum) culture medium after adhering to the wall. , purify the subcultured cells by trypsin digestion; detect the expression of vimentin by cell immunofluorescence and western blotting, and identify the fibroblasts; when the cells grow to about 80% confluence, change to low-glucose DMEM (containing 20 The supernatant was collected after 48 hours of culture, and the exosomes were isolated by ultracentrifugation. The morphology was observed by transmission electron microscopy, the particle diameter was analyzed by nanofluidics, and the specificity of exosomes was detected by western blotting. Protein expression and exosome identification.

　　RESULTS: After culturing for 30 hours, a small amount of cells in the back skin tissue block of the newborn naked mole rat could be seen to dissociate from the tissue block, and after 5 days of culture, a large number of cells could be seen to grow free from the tissue block. Cell immunofluorescence and Western blotting confirmed that the isolated cells expressed vimentin. The cell supernatant was centrifuged by ultracentrifugation. Membrane cupholder-like exosomes could be observed under transmission electron microscope. The particle size of nanoflow analysis was 46.75-206.75 nm. , tumor susceptibility gene 101 (tumor susceptibilitv gene, TSG101) and heat shock protein 70 (heat shock protein 70, Hsp70) are specific.

　　Conclusion The isolated and cultured cells have the morphological characteristics of fibroblasts and express vimentin, a characteristic molecule of fibroblasts. Naked mole rat skin fibroblasts have the ability to secrete exosomes, which provides a basis for studying the biologically active molecules carried by naked mole rat exosomes and their biological functions.