| Objective : Fractures are common diseases,and with the deepening of aging,the prevalence of fractures gradually increases,and in about 10% of fracture cases,bone healing takes a long time and easily causes complications such as nonunion.At present,the mechanism of bone tissue repair and self-healing has not been fully revealed,miRNA as a good biomarker has played a great role in disease progression and disease treatment,and has also attracted great attention.This study investigated the differentially expressed miRNAs derived from bone marrow stem cell secretory vesicles(B-EVs)in fracture animal models,and studied their mechanism of action in fractures in order to provide instructions for fracture healing and treatment.Methods: 1.Isolation and identification of EV and construction of fracture mouse model: 1.The study animals were C57BL/6 mice.C57BL/6CD9-/-genotype mice engineered in the laboratory and bred normally were used to construct the fracture model.The cells used were mouse bone marrow stem cells(BMSCs).2.B-EVs were extracted from BMSCs cultured for 4 – 6 passages,and the morphology of E-BVs,the expression of specific proteins(CD11b,CD45,CD29,and CD90),and particle size were identified by transmission electron microscopy,Westernblot,and nanoparticle size analyzer;3.Transverse femoral shaft fractures were produced by three-point bending using C-shaped tools in wild-type(WT)and CD9-/-C57BL/6 mice,and bone healing in mouse fracture models was analyzed at 0,1,3,4 weeks after the end of modeling,and in vivo microimaging analysis and computed tomography bone mineral density were performed by high-resolution SKYSCAN to verify mouse fracture models.2.Identification of differential miRNAs in the fracture mouse model under the intervention of EVs: 1.The mouse model constructed in the first chapter was used for the study,and the mice were further divided into groups,which were WT + NC group(WT mice injected with EV-NC after GW4869 intervention),WT + EVs group(WT mice injected with B-EVs),WT + EVs-Mock group(WT mice injected with B-EVs were pre-transfected with miR-335 inhibitor NC),and WT + EVs-Inhibitor group(WT mice injected with B-EVs were pre-transfected with miR-335itor);CD9-/-mice were divided into: CD9-/-+ NC group(EV-NC after GW4869 intervention in CD9-/-mice),CD9-/-+ B-EVs group(CD9-/-mice injected with B-EVs),CD9-/-+ EVs-Mock group(CD9-/-mice injected with B-EVs were pre-transfected with miR-335 itor NC),and CD9-/-+ EVs-Inhibitor group(CD9-/-mice injected with miR-335 inhibitor).2.Injection time and dose of EVs: Mice in all groups were injected with 100 μL of EVs or 100 μL of EV-NC in response to the exosome inhibitor GW4869 at the fracture site on days 1 and 8 after fracture;Stable bone cell lines overexpressing miR-355-inhibtor and EVs-Inhibitor were established by lentiviral transfection using the p CDH plasmid;3.Femurs of WT mice and CD9-/-mice were harvested 2 weeks after fracture modeling,and the tissues were fixed in 4% buffered paraformaldehyde for 48 h,followed by decalcification treatment with buffered EDTA.4.Immunohistochemistry(IHC)to detect bone morphogenetic protein 2(BMP2);5.Use RT-q PCR to detect the expression level of differentially expressed miRNAs;6.Radioimmunoprecipitation assay analysis to extract total protein;7.Microarray to detect differentially expressed miRNAs in 3 WT + PBS-treated mice and 3 WT + B-EV-treated mice;8.Statistical analysis by SPSS 21.0.Kolmogorov – Smirnov test whether the data were normally distributed.Measurement data are presented as mean ± standard deviation.T-test was used for comparison between two groups,one-way or two-way analysis of variance(ANOVA)was used for multiple groups,and Tukey’s multiple comparison test was used for pairwise comparison after ANOVA analysis.3.MicroRNA-335 promotes fracture recovery through Vapb(Vesicle associated membrane protein)and Wnt/β-catenin(Canonical Wnt/β-catenin pathway)pathways: 1.MTT assay was used to detect the viability of osteoblast-like cells MC3T3 and MG63;2.Tunel and Alizarinred assay were used to detect apoptosis of osteoblast-like cells MC3T3 and MG63 cells,undifferentiated ADSCs(without extracellular calcium deposition)were reddish,while ADSC-derived osteoblasts(with extracellular deposition)were bright orange-red;3.Alkaline phosphatase staining was used to detect cultured mouse osteocytes,AP staining solution was used for staining,red-stained cell colonies and colorless colonies were counted using a light microscope,and cells were observed under a fluorescence microscope;4.According to the instructions of Lipofectamine2000,plasmids were co-transfected with mimic NC or mimicmiR-335 into HEK293 T cells,respectively.Dual-Glo ? dual-luciferase reporter assay system was used to verify the binding relationship between miR-335 and Vapb;5.RNApulldown was used to detect the protein interaction relationship,primary antibody recognizing RBP was used to detect the target RBP incubated with appropriate secondary antibody to recognize the primary antibody,and enhanced chemiluminescence was used to detect the signalResults: 1.Isolation and identification of EV and construction of fracture mouse model: 1.First of all,we identified the purchased murine BMSCs cells,and the correctness of the cells was confirmed by fluorescence microscopy analysis of osteoblast marker enzymes and flow cytometric analysis of specific antibodies;2.EVs were extracted from BM-MSCs cells,and the correctness of the isolated B-EVs was demonstrated by nanoparticle size analysis,transmission electron microscopy analysis and Western blot analysis results,which could be used in this study;3.When the two groups of mice could be detected before modeling,there was no significant difference in the body weight and bone mineral density of wild-type mice,the width of the tibial growth plate of CD9-/-mice was significantly reduced,wild-type mice showed endochondral ossification through callus formation at 2 weeks after fracture,3 bone healing was significant,and CD9-/-mice showed a significant delay in fracture healing compared with wild-type mice.At 3 weeks after fracture,the bone healing rate of CD9-/-mice was 25%,which was significantly lower than that of wild-type mice,indicating that the healing ability of CD9-/-mice was decreased compared with wild-type mice;2.Identification of differential miRNAs in fracture mouse models under the intervention of EVs: 1.HE staining showed that B-EV treatment promoted the formation of bone tissue,toluidine blue staining showed that B-EV treatment increased cartilage tissue,BMP2 immunohistochemistry showed that B-EV treatment promoted osteoblast differentiation and fracture recovery;the therapeutic effect of EVs on CD9-/-mice was significantly lower than that of WT mice;2.Compared with WT mice,73 miRNAs were down-regulated and 104 miRNAs were up-regulated in cartilage tissue of CD9-/-mice after EV treatment,and the most significantly differentially expressed miR-335,miR-136,miR-125 a,miR-217,miR-487 a,miR-339,miR-298 and miR-133 a were selected for subsequent experiments;3.The distribution of miR-335 was analyzed on the EvimiRNA online prediction website,and the results revealed that miR-335 was abundant in bone marrow stem cells,and we speculated that miR-335 may play a role in the repair of fractures.4.GW4869 had no significant effect on the secretion of B-EVs;5.HE staining,toluidine blue staining and BMP2 immunohistochemistry results showed that miR-335 in B-EV promoted fracture recovery in mice;3.MicroRNA-335 promoted fracture recovery through Vapb and Wnt/β-catenin pathways: 1.MTT assay detected the proliferation rate of MC3T3 and MG63 cells,and the results revealed that the treatment of EVs promoted cell proliferation(4A).TUNEL staining results showed that EV treatment inhibited apoptosis;2.Immunofluorescence staining of F-actin observed the cell adhesion of MC3T3 and MG63 cells,and found that B-EV treatment had no significant effect on cell adhesion;3.RT-q PCR and westernblot analysis verified that B-EV treatment increased the levels of α-SMA,OCN,GDF-10 and FGF-2;4.The mRNA and protein levels of α-SMA,OCN,GDF-10 and FGF-2 were significantly decreased after EVs inhibitor treatment;5.Vapb could promote the growth of osteoclasts 33,so we focused on Vapb,dual-luciferase reporter assay in HEK293 T cells 34 showed that miR-335 could target Vapb;6.RT-q PCR and westernblot analysis detected Vapb levels in mice and cells.The results showed that EV treatment inhibited the level of Vapb,while miR-335 inhibition could alleviate the inhibition of Vapb;7.RT-PCR and westernblot were used to identify the construction of overexpressing Vapb cells.RT-q PCR showed that the expression of Vapb mRNA in the transfected cells was significantly increased,and similar results were observed in the protein using westernblot;the results of MTT assay for cell viability and TUNEL staining for apoptosis showed that the cells overexpressing Vapb cells were accompanied by a decrease in cell proliferation rate and an increase in apoptosis,indicating that Vapb overexpression induced apoptosis;Vapb overexpression partially counteracted the promoting effect of B-EVs on osteoblast differentiation of MC3T3 and MG63 cells.8.overexpression of Vapb reversed the promotion of Wnt and β-catenin protein expression by EVs.These results suggest that miR-335 targeting Vapb in EVs may be related to the Wnt and β-catenin pathways.Conclusion: 1.In this study,we successfully constructed a mouse fracture model and successfully isolated exosomes from BMSCs,exosomes contribute to fracture healing in mice.2.MicroRNA-335 in bone marrow mesenchymal stem cell-derived exosomes promotes osteoblast differentiation and bone tissue formation.3.MicroRNA-335 targeted Vapb,and the overexpression of Vapb in the exosomes intervention group inhibited cell proliferation,promoted cell apoptosis,and weakened the role of exosomes in promoting osteoblast differentiation.4.MicroRNA-335 in bone marrow mesenchymal stem cell-derived exosomes promotes fracture healing through Vapb and Wnt/β-catenin pathways This study may provide insights into fracture treatment. |
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