Abstract:

Objective     To study the features such as the diameter distribution and average radius of the hypoxia preconditioned bone marrow mesenchymal stem cells (BMSCs)-derived exosomes; to elucidate whether the exosomes are able to target cardiomyocytes efficiently; to explore the feature of time-dependent uptake of exosomes by H9C2. Methods      We collected exosomes from the supernatant of hypoxia preconditioned BMSCs by the combination of step-by-step centrifugations and ultracentrifugation, characterized exosomes by transmission electron microscopy (TEM) and western blotting, statistically analyzed the messages of the diameters from results of TEM by image analysis software Image Pro Plus 6.0, obtained the distribution of diameter and average radius of exosomes by excel and statistical software Graph Pad 5.0, tracked exosomes with PKH-67, incubated PKH-67-dyed exosomes with H9C2 to observe whether rat BMSCs-derived exosomes could be uptaken by H9C2. Further, we incubated PKH-67-labeled exosomes with H9C2 for different periods to explore the relationship between uptake and time. Results   We observed that the extract was of micro-capsule structures, approximately spherical or ellipsoidal in shape and homogeneous in size. They scattered neatly in the vision, and positively expressed both CD63 and CD9 molecules. The diameters of exosomes approximately ranged from 20nm to 60nm, and the radius was (17.03±0.40) nm. In the incubation experiment of exosomes with H9C2, we could only observe green fluorescent-flashing exosome pellets within the cytoplasm of H9C2 in the dyed positive group. The uptake of exosomes by H9C2 was not different between four subgroups of the control group. For the PKH-67-dyed exosomes within H9C2 cytoplasm 1h after the co-culture, during the period of 1.5h to 2.5h, the numbers of green fluorescent-flashing exosome pellets were more and the fluorescence intensity was stronger than any other experimental subgroups. Conclusion    We successfully isolated hypoxia preconditioned rat BMSCs-derived exosomes, and proved that cardiomyocytes H9C2 were the biological targets of such exosomes. The uptake of exosomes by H9C2 showed significant changes with the extension of incubating time.

Key words: Hypoxia preconditioning; BMSCs; Exosome; H9C2; Heart failure; Ischemia-reperfusion injury