Ighthroughput sequencing of RNA (RNAseq) was performed to detect the genes differentially expressed under the

Ighthroughput sequencing of RNA (RNAseq) was performed to detect the genes differentially expressed under the influence of exosomes derived from hWJMSCs. A GIONFH rat model was produced to investigate the pathogenesis of GIONFH. Additionally, the protective effect with the exosomes derived from hWJMSCs was investigated, which was located to be mainly mediated by exosomal miR21, which inhibits PTEN in osteocytes.Supplies and methodsCell culture and treatmentshWJMSCs have been cultured as previously reported8, and these cells had been identified by flow cytometry. Positive markers (CD13, CD73, CD90, and CD105) and unfavorable markers (CD34 and CD45) were analysed for identification from the cells (Supplementary Fig.1). Murine osteocytelike MLOY4 cells had been kindly offered by Prof. Lynda Bonewald (University of MissouriKansas City, Kansas City, MO, USA). MLOY4 cells (grown in culture dishes coated with 0.15 mgmL rat tail sort I collagen) were cultured in MEM (Hyclone, UK) with 2.5 of foetal bovine serum, two.5 of calf serum, one hundred UmL penicillin, and one hundred UmL streptomycin at five CO2 and 37 11. To investigate the effects of exosomes, MLOY4 cells had been treated with dexamethasone (Dex), exosomes, or each. The concentration of Dex was ten M for four days for the CCK8 analysis, and 10 M for 24 h for 5ethynyl2deoxyuridine (EdU) staining. Subsequent, 10 M Dex was applied for 21 days in an osteogenic differentiation assay. Apart from, one hundred M Dex was utilized for 24 h in an apoptosis experiment including a terminal deoxynucleotidyl transferase dUTP nick finish labelling (TUNEL) assay, western blotting, and flow cytometry. The concentration of exosomes was 50 mL.Exosome isolation, purification, and identificationFirstly, exofree foetal bovine serum was prepared as previously reported12,13. The culture supernatant from hWJMSCs or MLOY4 cells was collected after 48 h cultivation. Then, the supernatant was centrifuged at 4000 rpm for 15 min to take away the cells, followed by filtration through a 0.22 m filter to eliminate cell debris. Exosomes in the medium were precipitated together with the exoEasy Maxi Kit (D-Isoleucine Epigenetic Reader Domain Qiagen) in accordance with the manufacturer’s instructions. The isolated exosomes had been stored at 0 for later use. Transmission electron microscopy (TEM) micrographs (Hitachi HT7700 transmission electron microscope, Tokyo, Japan) have been analysed to determine the diameter with the exosomes. The size distribution of exosomes was calculated by the NanosizerTM technology (Malvern, UK). The exosomes have been diluted in the ratio of 1:1000 with 1 mL of PBS. The control medium and filtered PBS servedhttp:www.ijbs.comInt. J. Biol. Sci. 2019, Vol.as controls. In addition, western blotting was performed to examine specific exosome biomarkers CD9, CD63, CD81, and TSG101.M miR21 Agomir or miR21 Antagomir (Sangon Biotech, Shanghai, China) have been injected intramuscularly as soon as a week into GIONFH rats.Exosome labelling with PKHExosome labelling with PKH26 (Sigma) was performed following the manufacturer’s directions. Briefly, 100 of isolated exosomes had been labelled with 40 of PKH26, after which 500 of dilution buffer was added. The mixture was incubated within the dark for 20 min at area temperature. Subsequent, 500 of 10 BSA was added to quit the staining reaction. Ultracentrifugation was performed at 100000 g for 1 h at four , then the supernatant was aspirated, plus the exosomes had been resuspended in PBS.A cell viability assayWe performed a Cell Counting Kit8 assay (CCK8) to estimate the cell proliferation price. A total of 5000 MLOY4.