Fig. 2

Identification of MSCs and exosomes (EXs), PC12 cells engulfed EXs and expressed netrin-1 protein. (A) Cell surface markers (CD34, CD45, CD73, CD70 and CD105) of MSCs was detected by flow cytometric analysis. (B-C) Typical images of EXs morphology were captured by transmission electron microscope (TEM). Scale bar: (B) 200 nm (C)100 nm. (D) Particle size distribution of EXs was measured by nanoparticle trafficking analysis (NTA). (E) Protein markers of EXs were detected by western blot analysis in EXs and MSCs. EX-MSCs: Exosomes derived from MSCs without mRNA transfection; EX-NC: Exosomes derived from MSCs transfected with negative control mRNA; EX-netrin1: Exosomes derived from MSCs transfected with netrin-1 mRNA. (F) The relative level of netrin-1 mRNA in three different EX was detected by qRT-PCR. (G) A fluorescence microscope was used to observe the changes in the uptake of fluorescently labelled EXs by PC12 cells. Scale bar = 20 μm. (H) Percentage of EXs absorbed by PC12 cells at different time points. (I) A cell injury model was constructed by treating PC12 cells with lipopolysaccharide (LPS). qRT-PCR was used to detect the expression of netrin-1 mRNA in PC12 cells. (J-K) Western blot was used to detect the expression of netrin-1 protein in PC12 cells. (L) The levels of netrin-1 in the supernatants of PC12 cells treated with three different EXs at different time points were identified by ELISA. (Error bars showed means ± SD; n = 3; ^￥￥￥p < 0.001; *P < 0.05, ***P < 0.001, vs. LPS group; ^###P < 0.001, vs. LPS+EX-MSCs group; ^^^P < 0.001, vs. LPS+EX-NC group; ^$P < 0.05, ^$$P < 0.01, ^$$$P < 0.001, vs. EX-MSCs group; ^&P < 0.05, ^&&P < 0.01, ^&&&P < 0.001, vs. EX-NC group)