Human plasma specimens
In the current study, three paired plasma samples (ONFH patients and healthy individuals) were used for sequencing, an itional nine paired plasma samples were used for PCR validation. The blood specimens were placed in BD vacutainer K2 EDTA blood collection tubes and mixed lightly. Next, the anticoagulant-treated blood samples were performed to centrifuge separates at 1000 rpm for 15 min at 20 °C to removes the blood cell debris and impurities. The upper layer containing plasma was then obtained from each tube and transferred into sterile EP tubes by the pipette. Taking care not to touch the bottom sediment during this step. At last, each plasma was labeled and stored at -80 °C for subsequent experiments. None of the ONFH patients received any therapy before blood collection. Each participant was signed written informed consent before the experiment. This study was performed with the approval of the Human Ethics Committee of the First Affiliated Hospital of Fujian Medical University
Isolation and identification of exosomes
The ExoQuick™ Plasma Prep and Exosome Precipitation Kit (SBI, EXOQ5TMA-1, Japan) were used to isolate exosomes from plasma. Briefly, plasma samples were incubated for 1 h at -20 ℃ and 4 ℃, respectively. After ultra-centrifuged for 15 min (13,000 rpm), partial cells and their debris were removed from plasma samples. Add 5 µL SBI Thrombin Reagent to the supernatant and mixed. Then centrifuge isolation was conducted at 10,000 rpm for 5 min at 4 °C to help dissolve the fibrin. After refrigerated 30 min at 4°C, discarded the supernatant, the exosomes pellet were resuspended with 1X PBS (20 μg exosomes per 1 mL PBS) and stored at − 80 °C. The exosomes were negatively stained with the 3% (w/v) sodium phosphotungstate solution, and a photograph was captured using an LVEM5 TEM (Delong America, Montreal, QC, Canada). NTA (NanoSight; Malvern Panalytical, Worcestershire, UK) was carried out to the diameter and size distribution of exosomes.
Small RNA sequencing and bioinformatics analysis
Illumina sequencing was performed for plasma exosomal small RNA sequencing according to the instructions of the Multiplex Small RNA Library Prep Kit (Illumina, USA). The concrete operations were briefly described as follows. Total RNA from plasma exosomes was firstly extracted using the Trizol method (Qiagen, Germany), measured and quantified by NanoDrop 2000 (Thermo, USA). For each sample, 3’ adaptor was connected to RNA (200 ng), and reverse primer hybridization and 5' adaptor connection were disposed of orderly. Previous products were synthesized into cDNA and then enriched by PCR. The sequencing of screened DNA fragments was performed using the HiSeq platform (Illumina, USA). The original sequence data (ONFH patients and healthy participants) were filtered and mapped to reference genome, the internationally recognized algorithm DESeq2.0 was adopted to select the differentially expressed miRNAs (DEmiRNAs) with the threshold of p-value < 0.05 and |Log Fold Change| > 1. Subsequently, the hierarchical clustering of DEmiRNAs was analyzed by using MEV software and plotted the heatmap. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology terms (GO) annotation analysis were used to infer the functional roles of DEmiRNAs. Additionally, RNAHybrid and Miranda were used for the target gene prediction of miRNAs and the miRNA-mRNAs interaction network analysis was constructed using Cytoscape software 3.6.1 (https://cytoscape.org/).
Isolation and culture of cells
Bone marrow was respectively obtained from SD rat using a sterile syringe with heparin, followed by quick mixed with heparin. Next, the fat cells were removed from the BMSCs with a centrifuge separates (1000 rpm for 20 min), and then rinsed the sediment three times with Dulbecco s Modified Eagle Medium (DMEM), and cultured the BMSCs in RPMI-1640 media (GIBCO) with additional 10% fetal bovine serum (FBS). Human umbilical vein endothelial cells (hUVEC) (Procell Life Science&Technology Co., Ltd) were incubated in modified ECM medium (added 10% FBS, P/S, and 1% ECGS). All cells were incubated at 37 ℃ with 95% air and 5% CO2. After 72 h of incubation, the whole medium was replaced, and the culture medium changed every 2-3 d. When cell confluence reached about 80%, cell subculture was performed.
BMSCs were prepared in a 6-well plate for miRNA transfection. Briefly, 100 pmol of synthetic miR-150-5p mimics or negative control (NC) RNA were mixed with 250 μL serum-free Opti-MEM (Gibco, Grand Island, NY, USA) and incubated for 5 min at room temperature, respectively. Meanwhile, 5 μL Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) was diluted with 250 μL serum-free medium Opti-MEM at room temperature for 5 min. The above two reagents were mixed and incubated at room temperature for 20 min and added to the wells containing BMSCs. The 6-well plate was placed in a 37 ℃ incubators for 6 - 8 h and refreshed the medium. Subsequent tests were performed after an additional 24 - 48 h incubation.
Internalization of exosomes
The exosomes derived from BMSCs were extracted and ultra-centrifuged (120,000 x g) at 4 ℃ for 3 h after PBS dilutions using a Beckman tabletop ultracentrifuge. After washed with 100 ml medium, the exosomes’ precipitation was resuspended in 700 ml diluent C. The exosomes were labeled by PKH67 Fluorescent with PKH67 Fluorescent Cell Connection Kit（Sigma-Aldrich）according to manufacturer protocol. Exosomes solution (250 ml) was mixed with diluted PKH67 dye and incubated for 4 min, and neutralize the excess PKH67 dye with bovine serum albumin (1%, 4.2 ml). The bye-labeled exosomes were centrifuged at 120,000 x g for 3 h (4 ℃) and washed the precipitation with 1 x PBS. The HUVEC cells (1.3 x 104 cells /well) were placed on 8-well slides and cultured for 24 h. After 1 x PBS washing, the medium containing PKH67-labeled exosomes was added and incubated for 48 hours. The cells were washed and incubated with a 4% paraformaldehyde solution at room temperature for 10 minutes. After 1 x PBS washing, the nuclear staining was employed by ProLong Gold Antifade Reagent containing DAPI (Thermo Fisher Scientific, USA). The internalization of exosomes in HUVEC cells was observed with a confocal laser-scanning microscope (LSM510, Carl Zeiss, Germany).
Matrix-gel based in vitro angiogenesis assay
HUVECs were suspended in a serum-free ECM culture medium, seeded in a 24-well plate, and incubated with miR-150-5p-exosomes (miR-Exo), NC-exosomes (NC-Exo), and PBS for 24 h. The angiogenesis analysis was employed on ice, adding a 200 ul cooled Matrigel Matrix (10m /ml; Becton Dickinson, USA) and incubated at 37 °C for 1 h. HUVECs (6 × 104 per group) were seeded at coagulated Matrigel and incubated for 18 h at 37 °C. The blood vessels formation of each group was observed with an inverted microscope (NIKON, Japan).
The cells were seeded in a 6-well plate and formed adherent cells. The monolayer cell was scraped in a straight line to create a "scratch" by a p200 pipette tip when cell density reached 90–100% confluent. Then the cells incubated with BMSCs-derived exosomes (10 mg/mL). Images of the scratch were acquired at 0 h and 48 h. The migration distance of the scratched area and the node numbers were observed, and the multiple visual fields of cell migrated were randomly selected and photographed and performed a comparison between groups.
Reverse transcription-quantitative polymerase chain reaction
Total RNA was extracted as the previous method. After the detection of RNA quality, purity, and content, 1ug RNA was reverse-transcribed into cDNA with a PrimeScriptTM RT reagent kit with gDNA Eraser kit (TaKaRa, Tokyo, Japan). The Reverse transcription-quantitative polymerase chain reaction (qRT-PCR) reaction was performed with the SYBR Green PCR kit (Toyobo, Osaka, Japan) on Applied Biosystems 7300 real-time PCR system (Applied Biosystems, Foster City, CA). The reaction conditions were: 10 min of pre-denaturation at 95 ℃, 45 cycles of denaturation at 95 ℃ for 15 s, and annealing at 60 ℃ for 60 s. The internal references in qRT-PCR were β-actin and U6. Three independent experiments were conducted. The relative expression of each factor was evaluated using the 2-ΔΔCt method. The RT primer and special PCR primers were listed in Additional file 1.
All of these experiments were repeated three times. Statistical analyses were assessed with the SPSS v.21.0 software (IBM, USA). The comparisons of means among the two groups were evaluated by Student’s t-test. One-way ANOVAs were performed for multiple comparisons. For all tests, p < 0.05 was considered to be statistically significant.