| Objective:To investigate the effect of shear stress mediated M2 type macrophage exosomes mi R-423-5p on osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs).Methods:1.IL-4 was used to induce macrophages into M2 type macrophages.Flow cytometry and q RT-PCR were used to detect the specific markers of M2 type macrophages Arg-1,CD206,TNF-α.2.M2 type macrophages were treated with shear stress loading technology for 24h,and the shear stress was 12dyn/cm~2.Exosomes(Exo)were extracted from the supernatant,and were divided into shear stress group(AExo)and normal exosomes(NExo).The exosomes were identified by transmission electron microscopy and Western blotting.3.BMSCs were determined by flow cytometry,osteogenic staining,lipogenic staining and chondrogenic staining.4.BMSCs were interfered with different concentrations of M2 type macrophage exosomes,and the best concentration of intervention was screened,and BMSCs were treated with AExo and NExo of this concentration.5.AExo and NExo interfere with BMSCs respectively,Western blotting and q RT-PCR experiments verify the effect of exosomes on the osteogenic differentiation of BMSCs,and detect the expression level of osteogenic differentiation related factors.6.Sequencing and bioinformatics analysis were performed on the exosomes to find the differential mi RNAs related to osteogenesis.7.The binding sites of osteogenesis related differential mi RNA and BMP-2 were verified by Luciferase report experiment.8.The transfection efficiency was detected after transfection of differential mi RNA.9.The expression level of BMP-2 was detected by immunofluorescence staining after the transfection experiment.10.The effect of differential mi RNA on the migration effect of BMSCs was verified by scratch test.11.The effect of differential mi RNA on the differentiation of BMSCs into osteoblasts was verified by q RT-PCR,Western blotting and alizarin red staining.Results:1.The up-regulation of Arg-1,CD206,TNF-αexpression in macrophages treated with IL-4 suggests that macrophages are polarized towards M2 type macrophages.2.The exosomes is a 30-150nm vesicle,cup-shaped or globular.The exosomes specific proteins Alix,Tsg101,and CD63 are highly expressed,suggesting that the exosomes has been successfully collected.3.The surface markers CD73 and CD105 of BMSCs are positive,while CD19,HLADR,CD45 and CD34 are negative,and BMSCs have the ability to differentiate into bone,fat and cartilage.This BMSCs can be used for subsequent experiments.4.Compared with the PBS group,While the concentration is60μg/m L,the uptake efficiency of BMSCs in exosomes was the highest(P<0.001),and the uptake efficiency of BMSCs in AExo was significantly higher than that of NExo(P<0.01).5.Compared with NExo group,the levels of bone related factors ALP,RUNX2,BMP-2protein and m RNA of AExo were significantly higher(P<0.05),suggesting that AExo could regulate and control the differentiation of BMSCs into osteoblasts.6.The Phred score of mi RNA base mass fluctuates in the range of 33-36,and the length of mi RNA is 18-26.The original data conforms to the mi RNA sequencing standard.7.A total of 42 differential mi RNAs were expressed,of which 36 were down-regulated in AExo and 6 were up-regulated.The GO results showed that the differential mi RNAs were significantly enriched in"protein binding","ATP binding","transferase activity"and other items,and the differential mi RNAs were mainly enriched in"MAPK signal pathway","Wnt signal pathway",and"Ras signal pathway".8.Compared with NExo group,the expression of mi R-423-5p in AExo group was lower(P<0.01).Bioinformatics software bibiserve analysis showed that there was a binding site with mi R-423-5p on the 3’UTR of BMP-2.9.The results of Luciferase report experiment showed that the activity of Luciferase in the mi R-423-5p mimics+p GL6-mi R-BMP-2-WT group was significantly lower than that in the p GL6-mi R-BMP-2-Mut-3’UTR group(P<0.05),indicating that mi R-423-5p could bind to the binding site on the 3’UTR of BMP-2.10.After mi RNA transfection,compared with the mimic NC group,the mi RNA expression level of mi R-423-5p in the mi R-423-5p mimics group was significantly higher(P<0.001).Compared with the inhibitor NC group,the mi RNA expression level of mi R-423-5p in the inhibitor group was significantly decreased(P<0.001),indicating that mi RNA transfection was successful.11.After the transfection experiment,the results of BMP-2 immunofluorescence staining showed that the expression of BMP-2protein in the mi R-423-5p inhibitor group was significantly higher than that in the mi R-423-5p mimics group(P<0.001).12.In the scratch test,the degree of cell migration in the mi R-423-5p inhibitor group was higher than that in the mi R-423-5p mimics group(P<0.001).13.Compared with mi R-423-5p mimics group,the levels of protein and m RNA of bone related factors ALP,RUNX2 and BMP-2 in mi R-423-5p inhibitor were increased(P<0.05),the content of mineral deposits was higher,and the absorbance was higher(P<0.001),indicating that down-regulation of mi R-423-5p relieved the inhibition of mi RNA,and the expression of BMP-2 protein was increased,which induced the osteogenic differentiation of BMSCs.Conclusions:Shear stress relieves the inhibition of mi RNA by down-regulating the content of mi R-423-5p in the exosomes of M2 type macrophages,and makes BMSCs differentiate into osteoblasts. |
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