关键词: 脂肪干细胞, 外泌体, 巨噬细胞, 细胞迁移, 细胞黏附

Abstract:

Objective To discuss the effect of adipose-derived stem cell-derived exosomes （ADSC-Exos） on the migration ability of the macrophages RAW264.7， and to clarify its role in promoting function of the macrophages. Methods The adipose tissue adjacent to epididymis of the SD rats was isolated to perform primary culture of the adipose-derived stem cells （ADSCs）. The adipogenic and osteogenic differentiation induction was conducted， and the multidirectional differentiation potential of the ADSCs was detected by oil Red O and Alizarin red staining. Western blotting and immunofluorescence methods were used to detect the positive expressions of the ADSCs markers CD29 and CD44； the ADSC-Exos were extracted by Exos isolation kit， and the morphology， size， and distribution of particle size of the ADSC-Exos were examined by transmission electron microscope and nanoparticle tracking analyzer； the expression levels of exosome-specific markers CD9 and TSG101 proteins in the ADSC-Exos were detected by Western blotting method； the uptake of ADSC-Exos by the macrophages was observed by tracing method. The macrophages RAW264.7 were divided into control group， 10 mg·L^-1 ADSC-Exos group， 20 mg·L^-1 ADSC-Exos group， and 40 mg·L^-1 ADSC-Exos group. The activities of the macrophages in various groups were detected by 5-ethynyl-2'-deoxyuridine （EdU） staining； the number of migration macrophages in various groups was detected by Transwell chamber assay； the adhesion of macrophages in various groups was observed by fluorescence microscope. Results After 24 h of primary culture， the ADSCs adhered to the wall and exhibited scattered， elongated shapes； after 7 d of culture， the adherent cells showed a comb-like， vortex-like orderly arrangement， resembling fibroblasts； after 10 passages， the irregular morphology of the ADSCs and decreased proliferation rate were found. The isolated ADSCs showed potential for the osteogenic and adipogenic differentiation， and the expressions of CD29 and CD44 proteins were positive.The transmission electron microscope observation resuls showed that the ADSC-Exos appeared disc-shaped， and the main peak of particle size distribution was around 132 nm. The CD9 and TSG101 proteins were positively expressed in the ADSC-Exos， indicating successful extraction. The fluorescence microscope results showed red fluorescence signals around the nuclei of the RAW264.7 cells， indicating the uptake of ADSC-Exos by the macrophages. Compared with control group， the rates of EdU positive cells in 10，20， and 40 mg·L^-1 ADSC-Exos groups were significantly increased（P<0.05）； compared with 10 mg·L^-1 ADSC-Exos group， the rate of EdU positive cells in 20 mg·L^-1 ADSC-Exos group was significantly increased （P<0.05）. Compared with control group， the numbers of migration cells in 10， 20， and 40 mg·L^-1 ADSC-Exos groups were significantly increased （P<0.05）； compared with 10 mg·L^-1 ADSC-Exos group， the numbers of migration cells in 20 and 40 mg·L^-1 ADSC-Exos groups were significantly increased （P<0.05）. Compared with control group， the numbers of the adherent macrophages in 10， 20， and 40 mg·L^-1 ADSC-Exos groups were significantly increased （P<0.05）； compared with 10 mg·L^-1 ADSC-Exos group， the number of adherent macrophages in 20 mg·L^-1 ADSC-Exos group was significantly increased （P<0.05）. Conclusion The ADSC-Exos can be internalized by the macrophages and they can enhance the migration ability of the macrophages by affecting the cell adhesion.

Key words: Adipogenic stem cells, Exosomes, Macrophages, Cell migration, Cell adhesion