Serum-Derived Exosomes From Healthy Women Promote Osteogenic Differentiation Of BMSCs In Postmenopausal Osteoporosis(PMOP) Patients By Transferring ILK

Aims: To investigate the effect of serum-derived exosomes from healthy women on the osteogenic differentiation ability of bone marrow stromal cells(BMSCs)in postmenopausal osteoporosis(PMOP)patients and the underlying mechanism.Methods: BMSCs were isolated from the vertebra-derived bone marrow of PMOP patients(T ≤-2.5SD)undergoing posterior lumbar interbody fusion(PLIF)and confirmed by flow cytometry.Serum samples from healthy female volunteers(aged 20-35 years)were obtained and isolated by differential centrifugation to extract exosomes,which were characterized by dynamic light scattering(DLS),transmission electron microscope(TEM)and Western Blot.Laser confocal microscope was used to determine the uptake of serum-derived exosomes by BMSCs.BMSCs of PMOP patients were treated with serum-derived exosomes from healthy women.After 14 days of osteogenic induction,the osteogenic differentiation level of BMSCs was analyzed by Western Blot,RT-q PCR and Alizarin red staining.Proliferation assay confirmed BMSCs proliferation level after serum-derived exosomes treatment of healthy women.Proteomic analysis was performed to screen the functional molecules involved in regulation of osteogenic differentiation biological processes in serum-derived exosomes of healthy women and determine the effects of candidate protein on osteogenic differentiation of BMSCs.Results: The serum-derived exosomes exhibited a cup-or sphere-shaped morphology with a mean diameter of 50-100 nm and positive for TSG101 and CD9,and the purity of BMSCs extracted in this study was high.Laser confocal microscope confirmed that serum-derived exosomes could be transferred to BMSCs.Serum-derived exosomes(50μg/ml)from healthy women could promote the proliferation of BMSCs in PMOP patients.Under the osteogenic induction,BMSCs of PMOP patients were treated with healthy female serum-derived exosomes(50μg/ml),the expression of osteogenic differentiation genes increased,and the formation of calcified nodules also increased.Proteomic analysis revealed that serum-derived exosomes of healthy women were enriched in the proteins involved in regulation of osteogenic differentiation.Particularly,Integrin-linked kinase(ILK)was highly expressed in serum-derived exosomes of healthy women.The effect of serum-derived exosomes transferred into ILK inhibitors(QLT0267)on promoting osteogenic differentiation of BMSCs is weakened,and expression of BMP and Wnt/β-catenin pathway genes was down-regulatedConclusions: Serum-derived exosomes from healthy female can promote osteogenic differentiation and proliferation of BMSCs in PMOP patients,ILK transferred by serum-derived exosomes from healthy female may contribute to this effect through regulation of the BMP,Wnt/β-catenin signaling pathway.