| Background and Objectives: Bone marrow is an endocrine organ of varieties of complex functions,such as hematopoiesis,immune,defense and trauma repair.Bone marrow microenvironment is of cellular heterogeneity,which is composed of a variety of cells,facilitating bone formation during development and maintaining bone homeostasis during adulthood.Bone marrow mesenchymal stem cells(BMSCs)can differentiate into the lineages of osteoblasts,lipoblasts,myoblasts and chondroblasts to maintain bone homeostasis.With aging,the osteoblastic differentiation of BMSCs is down-regulated and capacity of bone formation is decreased,whereas the adipogenic differentiation of BMSCs is up-regulated and the formation of adipose is increased,leading to increased risks of bone fracture.This implies that increased adipogenic differentiation and decreased osteogenic differentiation of BMSCs in bone marrow is one of the main causes of osteoporosis.Therefore,it is important to study on the osteogenic and adipogenic differentiation of BMSCs within bone marrow microenvironment and the molecular mechanisms,which can provide new therapeutic approaches for bone related diseases,e.g.osteoporosis.Due to the complex components of bone marrow microenvironment and various media of intercellular communication,the molecular mechanisms of age related changes of BMSCs is not clear.Exosomes,as the important molecules of intercellular interactions,play an important role in maintaining bone homeostasis in the bone marrow microenvironment.Therefore,the purpose of this study was to investigate the effects of bone marrow-derived exosomes on the osteogenic and adipogenic differentiation of BMSCs.Method: Exosomes were extracted from young(4 weeks)and old(24 months)mouse bone marrows using an exosome isolation kit,and characterized by transmission electron microscope(TEM),nanoparticle tracking analysis(NTA)and western blot.We added the two kinds of exosomes into in vitro cultured BMSCs respectively,to observe the osteogenesis and adipogenesis of BMSCs by ALP and oil red O staining.Moreover,we also extracted the total RNAs from exosomes of young and old mice,and performed quantitative RT-PCR to evaluate the expressions of mi RNAs related with osteogenic and adipogenic differentiation.Results: TEM images showed that the extracted exosomes were round and uniform in size,which were about 100 nm in diameter,.NTA results showed that the exosomes were about 30~50 nm in diameter.Western blot results showed that the membrane marker protein CD63 was highly expressed in exosomes.The above results proved that we had successfully extracted exosomes with appropriate concentration,and could be used in the subsequent experiments.Then,we added the exosomes from young and old mouse bone marrows into the in vitro cultured mouse BMSCs.After osteogenic induction of 7 d,14 d and 21 d,ALP staining showed that there was no significant difference between the experimental groups and the control group.While,after adipogenic induction of 7 d,oil red O staining showed that the experimental groups produced more lipid droplets than the control group(P<0.05),however,there was no significant difference between the two experimental groups.Quantitative RT-PCR analyses revealed that there was no significant difference in the expressions of mi R-21a-5p and mi R-214-3p between young and old mouse bone marrowderived exosomes,while the expressions of mi R-22-3p and mi R-335-5p were increased in old mouse bone marrow-derived exosomes,compared with exosomes from young mouse bone marrow(P<0.05).Conclusions: Exosomes derived from young and old mouse bone marrow could promote adipogenic differentiation of BMSCs,while the effects on osteogenic differentiation was not obvious.Micro-RNAs in bone marrowderived exosomes may be involved in the changes of BMSC differentiation related with age. |
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