Résumé :
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Exosomes are nanovesicles of endocytic origin secreted by different cell types including epithelial, hematopoietic, and some tumor cells. They are also present in some biological fluids such as serum, urine, breast milk, and bronchoalveolar lavage fluid. Here, we demonstrate that exosome-like vesicles are present in the culture medium of C2C12 myoblasts and myotubes. These vesicles have been isolated by differential centrifugation and are characterized further by granulometry, immunoblotting, electron microscopy. The genomic information of these vesicules have been characterized by proteomic and microarray analysis, and TaqMan Low Density arrays for microRNA characterization. C2C12 myoblast or myotube vesicles exhibited characteristic exosomal size (30-100 nm) with typical surface, cytoskeletal, and cytoplasmic proteins characteristic of exosomes, including the multivesicular and late endosomal membrane markers Tsg101 and CD63. Respectively, 476 and 428 proteins were identified in myoblast or myotube exosomes, and 291 were found in both. Specific proteins were also identified. For exemple, myotube exosomes specifically exhibited the Acetylcholine receptor subunit beta involved in muscle terminal differentiation and contraction, which was not found in myoblast exosomes. In addition, 4000 different mRNA were identified in myotube exosomes. From them, 182 mRNA were associated with the corresponding proteins in myotube exosomes. We have also analyzed the microRNA population both expressed in the cytoplasm of C2C12 myoblasts and myotubes, or in their secreted exosomes, respectively. We found that only 50% of the microRNA expressed in cells are also present in exosomes, indicating that some microRNAs are preferentially selected for secretion. When we compared the level of expression of microRNA between myoblast and myotube exosomes we found a strong enrichment for miR-1, miR-133a, miR-206 and miR-181c in myotube exosomes. These microRNAs are well-known for their involvment in muscle differentiation. Exosomes from myoblasts were enriched in miR-222 and miR-223. These 2 microRNA are involved in cellular proliferation. Taken altogether, these results indicate that during differentiation muscle cells secrete specific exosomes. As exosomes represent an important intercellular communication vehicle, muscle exosomes could represent a new signal necessary for muscle differentiation. We are currently working on this hypothesis.
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