| 研究実績の概要 |
For the proteomic analysis of ROCK inhibitor-treated extracellular vesicles (ROCKin'-EVs), MagCapture Exosome Isolation Kit was used to prepare Control extracellular vesicles (Control-EVs) and ROCKin'-EVs from the culture medium of MC3T3-E1 cells. Mass spectrometry was performed to elucidate the changes in the proteomic composition. We detected there were changes ROCKin'-EVs protein composition. We identified that a number of Rab GTPase (known as the key regulators of intercellular vesicle transport) were upregulated in ROCKin'-EVs.
For the analysis of osteoblastic mineralization regulated by ROCKin'-EVs, we isolated the Control-EVs and ROCKin'-EVs again from the culture medium of MC3T3-E1 cells on Day3, 6 and 9 of osteoblastic differentiation using the culture filtration device. We measured the insolated proteins in the Control-EVs and ROCKin'-EVs. The protein concentration of the ROCKin'-EVs were 5.9times more than the Control-EVs. The ROCKin'-EVs were diluted to the same concentration as the Control-EVs, and MC3TC-E1 cells were treated with dimethyl sulfoxide (DMSO) (Control), 5 μM ROCK inhibitor (ROCK), 5 μg/ml Control-EVs, and ROCKin'-EVs, with induction of osteoblast differentiation. For the evaluation of mineralization activity, ALP staining and Picrosirus Red staining were performed. ALP and picrosirus red activity were significantly increase in ROCK inhibitor treated cells (ROCK) than in Control, as expected. However, the ALP staining and Picrosirus Red staining of Control-EVs and ROCKin'-EVs treated cells didn’t show significant difference from the Control.
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