Proteome Analysis Of Serum Extracellular Vesicles And Its Application In Osteoporosis

Extracellular vesicles(EVs)are membrane vesicles derived by cells,which could specifically carry bioactive substances such as proteins and nucleic acids.EVs could change the cell fate through delivering the content into recipient cells and widely present in human biofluids.Studies indicate that EVs have close relationship with the occurrence and development of diseases and play unique roles in the clinical diagnostics and therapeutics.Osteoporosis is a type of bone metabolic disease caused by decreased bone mineral density with a high incidence in the elder.Due to the deterioration of bone microstructure,osteoporosis can lead to higher risk of fragility fracture.Diagnostic technologies with high specificity are urgently needed to foretell osteoporosis in early stage.In this study,two subtypes of EVs,exosomes and microvesicles were isolated and characterized comprehensively in human serum.Label free quantification was utilized to further study the comparative proteomics from normal,osteopenia and osteoporosis group in order to discover EVs proteins for bone disease evaluation.In this project,exosomes and microvesicles were isolated with differential centrifugation,which were further characterized with TEM,NTA and WB.Proteome result showed that 162 and 179 proteins were identified in serum microvesicles and exosomes,respectively.GO(Gene Ontology)report indicated that identified proteins were participated in important biological processes such as vesicle-mediated transport,response to stress and defense response,protein activation cascade,immuno response.Quantitative proteomics analysis identified 229 and 194 proteins in serum exosomes and miceovesiles,including 17 exosomal candidate proteins and 24 microvesicles candidate proteins,which were significantly regulated in osteopenia and osteoporosis group compared to normal group.Our results suggest that the serum EVs proteome were systematic changed during the human bone loss process.Considering both differential expressed level and relationship with bone homeostasis,we selected three candidates named Filamin A,Vinculin and Profilin 1 to further verify in serum microvesicles with immunoblot.Western blot result showed that expression of Profilin 1 was consistent in immunoblot with mass result,continuously up-regulated in the normal group,osteopenia group and osteoporosis group(p < 0.05).Profilin 1 was further prevalidated with another larger sample set with commercial ELISA kit.Results showed that the expression level of Profilin 1 in the osteoporosis group(96.22 pg/mL)was two times of that in the normal human group(46.37 pg/mL,p=0.018)and osteopenia group(47.82 pg/mL,p=0.027),which is capable of differentiating osteoporosis from normal and osteopenia.In summary,serum EVs were isolated with differential centrifugation.Comparative proteomics analysis discovered many candidates related to bone loss process,which were potential targets for bone homeostasis and bone loss mechanism research.Key candidate proteins,which were initially verified and further validated,are expected to be used as biomarkers for clinical screening and molecular diagnosis of bone diseases.