cell culture
Mycoplasma-negative umbilical cord-derived mesenchymal stem cells (MSCs) were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco, 11885092) supplemented with 10% fetal bovine serum (Gibco, 10099141), 100 U/mL penicillin, and 100 μg/mL. I did. mL streptomycin (Gibco, 15070063). Mycoplasma-negative RAW 264.7 cells were cultured in RPMI-1640 (Sigma, R2405) supplemented with 10% FBS (Gibco, 10099141), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, 15070063). Both cell types were maintained at a temperature of 37°C in 5% CO .2. RAW 264.7 cells (ATCC) were purchased from Central South University Cell Bank (Changsha, China).
Preparation of PDMS substrates
PDMS substrates with different levels of stiffness were prepared according to previous studies. [27]. Components A and B of SYLGARD (Sylgard 184; Dow Corning, Midland, MI, USA) were mixed in ratios of 50:1 and 100:1. The mixture was then spread into cell culture dishes (10 or 15 cm diameter dishes) and cured overnight at 70°C. Sulfo-SANPAH solution (0.1 mg·mL-One, 22 589; Thermo Scientific, Waltham, MA, USA) was dropped on the PDMS substrate surface and exposed to UV light for 10 min. After removing the Sulfo-SANPAH solution, it was further investigated for 5 minutes. The PDMS substrate was then coated with collagen I (25 μg·mL).-One) After washing twice with phosphate-buffered saline (PBS). The mechanical properties of PDMS substrates were evaluated using a rotational rheometer (HAAKE MARS III, Thermo Scientific) in oscillatory mode (0.1–10 Hz, 25 °C) using a parallel plate as described in a previous study.[27]. The data obtained were analyzed using RheoWin Data Manager software (Thermo Scientific).
EV Collection
MSCs were cultured on substrates of different stiffnesses for 12 h, washed twice with PBS, and replaced with serum-free medium. After incubation for an additional 48 h, the supernatant was collected. The collected supernatants were then centrifuged at 1500 g for 20 min at 4°C to remove cell debris. The supernatant was then filtered through a 0.22 μm filter (SLGP033RB, Millipore). The filtered supernatant was centrifuged at 150,000 g for 2 h at 4°C. The resulting EV pellet was resuspended in PBS for further use.
Characteristics of EV
The size and number of EV particles were measured using Nanoflow (Apogee Flow Cytometer A50-Micro) according to the instructions provided in the user manual. Data obtained from EVs were collected and analyzed using Apogee Histogram software. The hydrodynamic size and concentration of the samples were measured using nanoparticle tracking analysis (NTA) using a Zeta View PMX 110 (Particle Metrix, Meerbusch, Germany) and the corresponding software Zeta View 8.04.02.
Transmission electron microscopy imaging
EVs dissolved in PBS were aspirated, and 10 μL of solution (200 ng/μL) was added dropwise to the copper mesh. The mixture was allowed to stand for 1 minute and the excess liquid was absorbed using filter paper. Next, 10 μL of phosphotungstic acid was added dropwise to the copper mesh and left to stand for 1 minute, and then the floating liquid was absorbed using filter paper. The sample was then dried at room temperature for a few minutes. Electron microscopy imaging (HT-7700, Hitachi) was performed at 80 kV.
Fluorescent labeling using EVs
DiO (1 μM) was incubated with EVs (20 μg/μL) for 30 min, followed by ultracentrifugation at 12,000 xg for 15 min to collect EVs. The DiO-labeled EV pellet was then resuspended in PBS buffer. The concentration of DiO-labeled EV particles was characterized using Nanoflow and adjusted to ensure consistency before being used in subsequent EV uptake experiments.
RAW 264.7 Cell Imaging
RAW 264.7 cells were plated at 60%–70% confluency in 35 mm culture dishes for 24 h. Afterwards, the cultured cells were washed twice with PBS (pH 7.4) and then imaged. After incubation with DiO-labeled EVs (50 μg/mL) on various substrates for 6 h, the medium was removed. Treated RAW 264.7 cells were washed with PBS. Before imaging, cells were stained with DAPI. All images were obtained using a laser confocal microscope (Leica TCS SP8). The wavelength set was 488 nm excitation (Ex)/510-650 nm emission (Em) for DiO-labeled EVs. Cellular uptake of EVs was quantified by determining MFI values. Quantization by plot was performed using a software package provided by Leica Instrument. Each experiment was performed at least three times.
RNA-seq data analysis
High-throughput sequencing of miRNAs in exosomes. Total RNA was extracted and purified from EVs using the miRNeasy Serum/Plasma Advanced Kit (Qiagen, catalog number 217204) according to the kit instructions. RNA concentration and purity were assessed using the RNA Nano 6000 Assay Kit on the Agilent Bioanalyzer 2100 System (Agilent Technologies, CA, USA). For small RNA libraries, a total of 3.0 ng RNA per sample was used as input material for RNA sample preparation. Sequencing libraries were generated using the QIAseq miRNA library kit (Qiagen, Frederick, MD) according to the manufacturer's recommendations, and index codes were added to the attribute sequences of each sample. Reverse transcription (RT) primers with unique molecular index (UMI) were introduced to analyze quantification of miRNA expression during cDNA synthesis and PCR amplification. Finally, library quality was assessed on Agilent Bioanalyzer 2100 and qPCR. Clustering of index-coded samples was performed on the acBot cluster generation system using the TruSeq PE Cluster Kitv3-cBot-HS (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. After cluster generation, library preparation was sequenced on an Illumina HiSeq2500 platform and purchased from EchoBiotech Co., Beijing, China. Ltd. Paired-end reads were generated.
Differential expression analysis. The Bowtie tool was used to align clean reads with various databases, including Silva, GtRNAdb, Rfam, and Repbase. This alignment helped filter out ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), other non-coding RNA (ncRNA), and repetitive sequences. We then used the remaining reads to detect known miRNAs and compared them with the genome and known miRNAs in miRBase to predict new miRNAs. |log2(FC)|≥ 1 and p ≤ 0.05 were defined as differentially expressed. Target gene differentially expressed miRNA prediction was performed using miRanda.[36] and RNA hybrid[37] equipment. Randfold software was utilized to predict new miRNA secondary structures.
Gene ontology (GO) enrichment analysis. To analyze differentially expressed genes (DEGs), GO enrichment analysis was performed using the topGO R package. High-throughput sequencing of miRNAs and data analysis were performed at Biomarker Inc. Three independent samples were measured from each group.
Bioinformatics analysis of proteins
Protein pretreatment. MSC and EV samples were sonicated three times on ice using a high-intensity ultrasonic processor (Scientz) in lysis buffer containing 8 M urea and 1% protease inhibitor cocktail. After sonication, the remaining debris was removed by centrifugation at 12,000 g for 10 min at 4°C. The supernatant was then collected and protein concentration was determined using the BCA kit according to the manufacturer's instructions. After extraction, protein solutions were subjected to trypsin digestion, TMT/iTRAQ labeling, HPLC fractionation, and LC-MS/MS analysis.
Differential expression analysis. A total of 5130.0 proteins were identified, of which 4471.0 were quantifiable after searching a theoretical protein data library using mass spectrometry secondary spectra (quantifiable proteins had quantitative information for at least one of the comparison groups). indicates that is available). |log2(FC)|≥ 0.58 and p ≤ 0.05 were defined as differentially expressed.
Clusters of Orthologous Groups (COG) classification. To gain a comprehensive understanding of the proteins identified and quantified in the data, we used GO to analyze differentially expressed protein functions and properties. GO annotated proteomes were obtained from the UniProt-GOA database (http://www.ebi.ac.uk/GOA/). Additionally, COG functional classification was performed by comparing differentially expressed proteins to the database.
Enhanced GO analysis. A two-tailed Fisher's exact test was used to assess the enrichment of differentially expressed proteins within these categories. GO terms corrected with p < 0.05 were considered significant in terms of enrichment. Proteomics detection and data analysis were completed by PTM Bio Inc. Three independent samples were measured from each group.
Bioinformatics analysis of lipids
Lipid pretreatment. MSC and EV samples were removed from the -80 °C refrigerator and thawed on ice. 1 mL of extraction solvent (MTBE: MeOH = 3:1, v/v) containing the internal standard mixture was added to the samples. The mixture was vortexed for 15 min to ensure proper extraction. 200 μL of water was added to the mixture. The mixture was vortexed for an additional minute. Centrifugation was performed at 12,000 rpm for 10 minutes. 500 μL of the upper organic layer was carefully collected. The collected organic layer was evaporated using a vacuum concentrator. The resulting dried extract was reconstituted using 200 μL of mobile phase B. The reconstituted extract was then prepared for LC-MS/MS analysis.
Differential metabolites were selected. Lipid metabolites were qualitatively analyzed based on the retention time RT (Retention time) and secondary spectrum data of the detected substances and the self-constructed target sample database MWDB (metware database). Metabolite quantification was performed using a multiple reaction monitoring assay. |log2(FC)|≥ 1 and VIP ≥ 1 were defined as differential metabolites.
KEGG annotation and enrichment analysis. Identified differential metabolites were annotated using the KEGG compound database (http://www.kegg.jp/kegg/compound/). Afterwards, the annotated metabolites were mapped to the KEGG Pathway database (http://www.kegg.jp/kegg/pathway.html). To identify significantly enriched pathways, we performed a hypergeometric test using p for a given list of metabolites.
Lipomics detection and data analysis were completed by PTM Bio Inc. Three independent samples were measured from each group.
statistical analysis
Data are expressed as mean ± SD. Differences between groups were determined using two-factor analysis of variance for repeated measures.