| Objectives:Lower back pain has been the leading cause of YLDs(Years Lived with Disability)in modern life with increasingly large health-care and socioeconomic costs.Even though the mechanism is not very clear,but amounts of evidences suggest that the degenerative disc disease is the common reason leading to lower back pain.The essential feature of disc degeneration is lack of water and the number of active cells(mainly the nucleus pulposus cells,NPCs)decreased in the intervertebral disc.As a worldwide issue,the therapeutic methods for IVD degeneration have achieved great progress.While a number of approaches have been considered,MSC-based regenerative therapies offer huge potential.In vitro study,MSCs were co-cultured with NPCs by direct cell-cell contact or noncontact methods.They differentiated along the NP lineage and simultaneously stimulated the degenerate NP cell population to regain a normal(non-degenerate)phenotype.However,the mechanisms by which NPCs and MSCs interact in this system are currently unclear.In recent years,exosomes was wildly studied in many fields.Exosomes are nanosized membrane vesicles that originate from multivesicular endosomal and are released into extracellular environment.Exosomes as a vital paracrine mechanism in cell-cell communication have been highly focused on.The purpose of this study was to detect the role of exosomes derived from nucleus pulposus cells in inducing bone marrow mesenchymal stem cells(BMSCs)towards into NP-like cells.Methods:The NP tissue was obtained from the intervertebral disc surgery and the NPCs was isolated and amplifying cultured in vitro.To prepare the exosomes depleted medium to culture the NPCs for 48 hours and collect the supernatant.Using the method of differential ultracentrifugation to isolate and purify the NPC-exosomes.The exosomes secreted by NPCs were identified by transmission electron microscope and marker proteins immunoblot analysis.Fluorescence confocal microscope was used to examine the uptake of PKH67-labeled NPC-exosomes by BMSCs.The effects of NPC-exosomes on the differentiation of BMSCs,after stimulated for 3,7,10,14 days,were determined by the morphological change of BMSCs,Alcian blue stain,NPC marker genes expression such as Aggrecan、SOX-9、Collagen II、HIF-1α、CA12 and KRT19 by q PCR analysis.And the translational level of protein was also measured by western-blot.The differentiated efficiency was compared between the manner of indirect co-culture and NPC-exosomes stimulation.Results:1)The NPCs secrete a kind of classical extracellular vesicles,which are identified as exosomes on the basis of their morphological characteristics and marker proteins.2)PKH67-labeled NPC-exosomes can be absorbed by BMSCs after co-incubation.3)After stimulation by NPC-exosomes,the BMSCs exert an increasing in ACAN、SOX-9、Col2a1、CA12、HIF-1α and KRT19 genes compared with control group(P<0.05).The western-blot experiments support the results of q PCR.4)In the comparison of differentiated efficacy of NPC-exosomes and indirect co-culture on BMSCs,the ACAN、SOX-9、Col2a1、CA12 and HIF-1α have a significant increase in NPC-exosomes group(P<0.05),both group of genes are significantly up-regulated to control group(p<0.05).Conclusion:It is the first time that using the method of differential ultracentrifugation to isolated human NPC-exosomes.Our data indicates that the exosomes act as an important vehicle in information transfer from NPCs to BMSCs to regular the activity of BMSCs.Our study suggests that it is an optimizing way to generate NP-like cells in vitro through stimulating the BMSCs by NPC-exosomes,compared with co-culture method.That will be more simply to provide the seed cells for tissue engineering therapy in intervertebral disc diseases.Given a variety of functions and multiple advantages,the exosomes alone or loaded specific genes and drugs would be the appropriate option in cell-free therapy strategy for intervertebral disc degeneration. |
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