Animals and sample collection
From the Guangzhou Purebred Pig Farm in Guangzhou City, Guangdong Province, China, four male Landrace pigs that were three days old and in good health were chosen. The pigs were anesthetized via electrical stunning and sacrificed by exsanguination. The longissimus dorsi (LD) muscle and subcutaneous adipose tissue (SAT) were dissected and taken to the lab to be transferred to a DMEM-F12 medium (Gibco, New York, NY, USA).
The isolation and culture of porcine MuSCs
Small sections of porcine muscle samples were cut and then moved into DMEM-F12. The trimmed tissues were digested with 0.2% collagenase type II for one hour. Subsequently, the treated tissue was centrifuged at 1500 g for 10 min at a temperature of 4 °C. The precipitate was resuspended in DMEM-F12. The sediment was reconstituted in DMEM-F12 and underwent three rounds of centrifugation at 800 g, 4 °C, for 10 min. Afterward, the suspension of cells was passed through a cell strainer with a pore size of 75 μm. After filtration, the supernatant was subjected to centrifugation at a speed of 800 g and a temperature of 4 °C for 5 min. The lower pellet was suspended in DMEM-F12 solution and placed in a cell culture flask for one hour at a temperature of 37 °C and a CO2 concentration of 5%. Skeletal muscle progenitor cells stay suspended in the supernatant, whereas fibroblasts rapidly attach to the base of the cell culture flask. In the end, porcine MuSCs were cultivated in DMEM-F12 (20% FBS) at a temperature of 37 degrees Celsius with a 5% concentration of CO2.
Isolation and culturing of porcine primary adipocyte
The subcutaneous fat tissue was divided into sections measuring 1 mm3 and then moved to the DMEM-F12 medium. The tissue sample was treated with 0.2% collagenase type II (Gibco, New York, NY, USA) for 2 h at a temperature of 37 °C while being agitated. After passing through a 150 μm mesh, the digested tissue was filtered, and the resulting liquid was then centrifuged at 600 g for 10 min. The pellet was mixed with Erythrocyte Lysis Buffer (Sangon Biotech, Shanghai, China) and left undisturbed for 10 min to break down the red blood cells. Afterward, the blend was subjected to centrifugation at a force of 800 g for 10 min. Afterward, the pellet was suspended again using DMEM-F12. After passing through a 40 μm mesh, the liquid that had been resuspended was subjected to centrifugation at 800 g for 5 min. The adipocyte pellet was suspended again and grown in DMEM-F12 medium with 10% fetal bovine serum (FBS, Gibco, New York, NY, United States) at a temperature of 37 °C and a CO2 concentration of 5%. Mature adipocytes are formed by induction, which consists of 10% FBS, DMEM-F12, 50 μM oleic acid, 0.5 M octoic acid, 50 nM insulin, and 50 nM dexamethasone. In the course of the induction procedure, 10 μg of small extracellular vesicles were introduced into each well on day 0 and subjected to treatment for 24 h. The primary Porcine adipocytes were seeded in 12-well plates with a density of 1.0 × 105 per well. Transfection with miR-146a-5p inhibitor (40 nM) (GenePharma, Shanghai, China) was initiated using lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA) when the cell density reached 70%. Further analysis was conducted on cells harvested on day 8 of differentiation.
Isolation of small extracellular vesicle
Upon achieving 80% confluence, the cells were rinsed thrice with phosphate buffer saline (PBS, Sangon Biotech, Shanghai, China) and subsequently cultured in a fresh DMEM-F12 medium for 48 h. Ultracentrifugation was employed to isolate small extracellular vesicles from the culture supernatant. The particular procedures were as follows. After subjecting the culture supernatant to centrifugation at 2000 × g for 10 min and 12,000 × g for 30 min, the large fragments and deceased cells were eliminated. The supernatant was subjected to an ultracentrifuge at a speed of 100,000 × g for 70 min. In the end, the cells were washed in 38 ml of PBS and subjected to ultracentrifugation at 100,000 × g for 70 min. The pellets were resuspended in 100 μL of PBS. Carefully remove the supernatant, then resuspend the pellet containing small extracellular vesicles in PBS and store it at − 80 °C. The protein concentration of small extracellular vesicles was determined using the BCA Protein assay kit (Bioteke, Beijing, China).
Transmission electron microscopy analysis
After incubating 10 μL of small extracellular vesicles on formalin-coated copper for 5 min, the surplus liquid was removed. Apply uranyl acetate onto the grid for negative staining and leave it for 1 min, then remove any extra liquid. The specimens were analyzed utilizing a transmission electron microscope with an acceleration voltage of 100 kV.
Nanoparticle tracking analysis
Small extracellular vesicles were diluted with PBS to the appropriate concentration. Nanoparticle tracking analysis (NTA) was used to measure the dimensions of small extracellular vesicles obtained from porcine primary skeletal muscle stem cells. To obtain detailed guidance on how to operate the device, which includes concise explanations on how to load samples, capture photographs, and analyze results, please consult the manual.
MTT assays
MTT assays were employed to assess cell proliferation. The optimized cell number (5000 cells/well) was used to seed the cells in a 96-well plate at a density of one well per cell. Following 24 h of seeding, the cells were subjected to a dosage of 10 μg/mL of small extracellular vesicles. Each well received twenty microliters of MTT solution (5 mg/ml), which was incubated for 4 h. After that, the reaction was stopped by adding 100 μl of dimethyl sulfoxide (DMSO) and incubating at 37 °C in the absence of light for 10 min. Cell growth was evaluated by quantifying the optical density at wavelengths of 570 nm and 630 nm (Absorbance, OD570-630 nm).
Measurement of triglyceride content
The quantity of TG in adipocytes was measured utilizing an assay kit as previously explained25. In short, the cells were washed and lysed using the provided lysis buffer. Subsequently, the TG assay reagent was added as per the instructions given by the manufacturer. A spectrophotometer plate reader was used to measure the optical density of the solution at 510 nm. The TG concentration was determined using the standard curve for each assay and the results were adjusted based on the total amount of cellular protein (Biovision, Milpitas, CA, USA).
Staining with oil red O
Porcine primary adipocyte was washed with PBS, followed by fixation in 4% formaldehyde in PBS for 30 min at room temperature, and subsequently washed three times with PBS. Afterward, the cells were stained with Oil Red O (#O1391; Sigma-Aldrich, St. Louis, MO, USA) for 15 min. Following three rinses with water, the microscope was used to observe and capture images of the lipid droplets (TE2000-E; Nikon, Tokyo, Japan).
Quantitative PCR in real-time
Total mRNA was extracted using Trizol reagent (15596-026, Thermo Fisher Scientific, Shanghai, China). Following DNase I treatment (2270A, Takara Bio, Kusatsu, Shiga, Japan), 1 μg of total RNA was converted into cDNA using MLV Reverse Transcriptase (M1705, Promega, Madison, WI, USA) and either oligo (dT) 18 primer or a dedicated stem-loop primer for miR-146a-5p. The STRATAGENE Mx3005P Sequence Detection System and SYBR Green Master Mix (Promega) were utilized for conducting real-time PCR. The findings were standardized to the levels of the GAPDH and U6 by employing the 2-ΔΔCt technique. Supplement Table 1 displays the primer sequences utilized.
Western blot analysis
The cells were disrupted using RIPA buffer which included a protease inhibitor (P7626, Sigma) at a concentration of 1 mmol/L PMSF. The BCA Protein Assay kit (Thermo Fisher Scientific, 23,227) was used to determine the concentration of the protein. According to the instructions provided by the manufacturer, the primary antibodies used were rabbit anti-Alix (1:1000, #D262028; Sangon Biotech), rabbit anti-TSG101 (1:2000, #381,538; ZEN BIO), rabbit anti-CD9 (1:1000, #AP68-965-100; Abcepta), rabbit anti-CD63 (1:2000, #D160973; Sangon Biotech), rabbit anti-PPARγ (1:1000, #2443; CST), and rabbit anti-GAPDH (1:5000, #BS65529; Bioworld). A blocking buffer was used to incubate primary antibodies overnight at 4℃. Secondary antibodies from Life Technologies were then added for 1 h. Capture detection by chemiluminescent imaging system. The Image J software was used to quantify and analyze the protein grayscales.
Statistical analyses
For one-way ANOVA, stand-alone sample t-test analysis, and plotting the statistical analysis was performed using SPSS software (version 25) and GraphPad Prism (version 9.0). The mean ± SEM was used to express all of the results. The importance of the distinction was evaluated based on a level of * p < 0.05 or ** p < 0.01. The levels of statistical significance of the difference between the groups are represented by the letters a, b, and c. Distinct letters indicate a substantial disparity, while identical letters indicate insignificance in the disparity.
- SEO Powered Content & PR Distribution. Get Amplified Today.
- PlatoData.Network Vertical Generative Ai. Empower Yourself. Access Here.
- PlatoAiStream. Web3 Intelligence. Knowledge Amplified. Access Here.
- PlatoESG. Carbon, CleanTech, Energy, Environment, Solar, Waste Management. Access Here.
- PlatoHealth. Biotech and Clinical Trials Intelligence. Access Here.
- Source: https://www.nature.com/articles/s41598-024-77110-5