| Objective: Previous work has shown that running training promotes bone growth and bone remodeling in mice,and differential expression of mechanoresponsive micro RNAs(miRNAs)has been found in bone tissue.Exosomes,on the other hand,are important media involved in intercellular communication,and miRNAs secreted by bone cells may be transported via exosomes to osteoblasts to function.In this study,mice were trained to run,extracting mouse bone exosomes,exosomes were sequenced with miRNAs to screen for differentially expressed miRNAs associated with osteogenesis or bone metabolism,setting the stage for subsequent studies of how these miRNAs are transported via exosomes to osteoblasts to regulate osteogenesis.Methods: 1.Using the small animal treadmill,8 week C57 BL / 6J mice were divided into control group and running group for 6 weeks of treadmill exercise(13 m / min,40 m / day,uphill 9°,5 days a week for 6 weeks).2.Serum NOS and ALP levels were measured in the kit after the running session.WB protein imprinting was used to detect Col-1 and OCN protein expression in mouse bone tissue,and fluorescence quantitative PCR was used to detect Col-1and OCN gene expression in mouse bone tissue.HE staining,micro CT detection of bone remodeling index levels,and calcium chlorophyll fluorescence markers were used to detect bone growth.3.Exosomes from bone tissue were extracted by ultrafast centrifugation and examined by electron microscopy,particle size and nanofrequency fluorescence analysis.4.Exosomes were labeled with PHK67 fluorescent labeling dye,and osteoblasts were cultured with labeled exosome medium.5.High-throughput exosomal miRNAs were sequenced,differentially miRNAs were screened,and literature studies were combined to further screen for target genes associated with osteogenesis differentiation or bone metabolism,and these miRNAs were validated by fluorescence quantitative PCR.Results: 1.Compared to the control group,serum NOS and ALP levels were increased in the running group,Col-1 and OCN protein expression were both upregulated in the running group,and OCN and Col-1 gene expression were both upregulated in the running group.Compared with the control group,the bone density of the running group increased significantly,the bone trabeculae grew,thickened and the reticular structure became denser3.Electron microscopy,particle size and nanofluidic fluorescence analysis showed that exosomes were successfully expressed in both control and running groups.4.fluorescence results showed that the exosomes could be endocytosed by osteoblasts and enter into the interior of osteoblasts.5.There were 788 miRNAs differentially expressed in bone tissue of mice before and after treadmill training,of which 10 were significantly different.Screening out 13 miRNAs with target genes related to osteogenic differentiation or bone metabolism.6.eight miRNAs(miR-5108,miR-2137,miR-205-5p,miR-3473 a,miR-7010-5p,miR-29b-3p,miR-615-5p,miR-706)were selected for fluorescent PCR quantitative analysis in the exosomes of bone tissue,in which miR-2137 was significantly up-regulated,miR-29b-3p and miR-615-5p were significantly down-regulated,consistent with the sequencing results.Conclusions: 1.C57BL/6J mice showed a significant increase in osteogenesis differentiation and bone remodel-related markers after running.2.Bone tissue exosomes were successfully extracted and second generation exosomes miRNA sequencing was performed.3.Screening for differentially expressed miRNAs in bone exosomes predicts target genes associated with osteogenesis differentiation,laying the groundwork for subsequent studies of how these miRNAs are transported via exosomes to osteoblasts to regulate osteogenesis differentiation. |
