2.1 Cell culture
The femur and tibia of nude mice (Beijing Vital River, China) were aseptically removed, washed with sterile PBS (Shanghai Qiagen, China), and then immersed in ethanol (Shanghai Titan, China) for disinfection. A syringe was used to aspirate the basal medium (Shanghai iCell Bioscience, China) and flush the bone marrow until the bones turned white. After collecting the washing solution, it was centrifuged at 1000g for 5 min, the cells were resuspended in EPC unique medium (Shanghai iCell Bioscience, China) and then cultured in a 37°C/5% CO2 incubator (Thermo Fisher Scientific, USA).
2.2 Flow cytometry identification
Cell morphology and growth density were observed, and then the cells were resuspended in PBS and centrifuged at 300g for 5 min, suspended at density of 2×106 cells/ml, and placed in a refrigerator at 4°C for 30 min. The cell suspension was transferred to a flow test tube, and 10 µl each of fluorescently labelled anti-mouse monoclonal antibodies CD14-FITC and CD34-APC was added (Shanghai Rebiosci Biotech, China). The cells were placed in a refrigerator at 4°C and incubated in the dark for 90 min, after which 500 µl of PBS was added to each tube and centrifuged at 1000g for 10 min. The supernatant of each tube was then discarded, 300 µl of PBS was added, and the samples were stored in a refrigerator at 4°C in the dark. Flow cytometry (BD FASAria Cell Sorter, USA) was used to detect the expression of cell surface antigens.
2.3 CCK-8
EPCs were inoculated into a 96-well culture plate at a concentration of 3,000 cells/well and cultured overnight in an incubator. During 1-6 days of culture, 10 µl of CCK-8 (Shanghai Dojindo, China) was added to each well, and culture medium was used as a blank control. After mixing, the cells were incubated for 2 h then placed in a microplate reader (Thermo Fisher Scientific, USA) to measure the absorbance at 450 nm. The OD values of the test sample and blank control wells at 450 nm were recorded, and the final OD was determined using the following equation: OD value of the test sample − OD value of the control sample.
2.4 Transwell cultivation
The concentration of EPCs was adjusted to 80 000/ml. The Transwell cocultivation chamber (Corning, USA) was placed into the culture plate, 20 µl of PBS was added to immerse the chamber and the plate was incubated at room temperature for 30 min. A total of 0.1 ml of cell suspension was pipetted into the chamber, and 0.6 ml of culture medium was added to the lower well plate. The chamber was carefully placed into the well plate and incubated at 37°C. Migration was regularly observed, and cell status was recorded. After washing twice with PBS, 0.5 ml of 4% paraformaldehyde (Bio Biotech, USA) was drawn into a clean hole, placed in a small chamber and fixed for 15 min at room temperature. A Crystal Violet Staining Kit (Genemed Biotechnology, USA) was used to wash and stain the chamber, and the chamber was placed at room temperature for 20 min.One millilitre of Regent A was placed in the washing well, and the chamber was put back for washing three times until the Regent A became a colourless and transparent liquid. Images were acquired and recorded using an electron microscope (Nikon, USA).
2.5 Matrigel assay
Matrigel (B.D. Co, USA) was dissolved and added at 50 µl per well into a 96-well plate (kept on ice) and allowed to fix in a 37°C incubator for 1 h. The concentration of EPCs was then adjusted to 6×105/ml. To each well of a 96-well plate containing Matrigel, 100 µl of EPC suspension was added. The formation of blood vessels was observed via electron microscopy 24 h and 48 h after EPC addition.
2.6 Establishment of a nude mouse model of lower limb ischemia
Animal experiments were approved by the Ethics Committee of Nanchang University. Six-week-old NU/NU nude mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. Intraperitoneal injection of 4% chloral hydrate was used for anaesthesia, and the dose was 40 mg/kg per animal. The lower limbs of each nude mouse were exposed, and the skin was gently cut with a sterile scalpel. Tissue blood vessels were separated under a microscope at 15× magnification, the femoral artery of the lower limbs was exposed and the tissue ligaments between the femoral artery, femoral vein and nerves of the lower limbs were separated. The two ends of the femoral artery were ligated with 10/0 nylon thread, the femoral artery was cut off in the middle the skin of the lower extremity was intermittently closed with 3/0 nylon thread; the incision was sterilized with iodophor. A laser Doppler blood flow metre (Beijing Jiandel Technology Co., Ltd.) was used to check the blood flow velocity of the ischaemic limb.
2.7 Cell transplantation
Endothelial progenitor cells were collected at a density of 15×106/ml. After 48 h of ischemia, 200 µl of EPCs was injected into the tails of nude mice. The control group was injected with the same amount of NS, and the normal control group was not treated. On the third day and the seventh day, laser Doppler was used to check the blood flow recovery of the lower limbs. The gastrocnemius muscle was used for the next experiment.
2.8 HE staining
The gastrocnemius muscle was placed in 10% paraformaldehyde fixative (Beijing Solarbio Co., China) for more than 24 h, and the edges were trimmed smoothly and rinsed with double-distilled water. After 24 h, conventional gradient ethanol dehydration was applied. The tissue was embedded in paraffin to form a wax block. Slices were cut to a thickness of 4-5 µm, attached to the slide and placed in a 65°C constant-temperature drying oven for 2 h. The baked slides were then dewaxed, placed in hematoxylin dye solution for 10 min and rinsed with running water. The sections were placed into 0.5% eosin solution for staining for 1 min and then rinsed with running water for 1-2 min. The slides were dehydrated and mounted with different concentrations of ethanol successively and then cleared with xylene. Finally, the slides were dried at room temperature and observed with neutral gum.
2.9 RT-PCR
After weighing the gastrocnemius muscle tissue, RNA was extracted by the TRIzol method (Beijing TransGen Biotech, China), and 1 µl was used to determine the OD value with a nucleic acid analyser (G.E. Medical Company, USA); the remainder of the sample was stored at −80°C. A cDNA reverse transcription kit (Beijing TransGen Biotech, China) was used in the PCR amplification instrument (Thermo Fisher Scientific, USA) to synthesize cDNA as follows: 42°C for 15 min, 85°C for 5 s, 4°C ∞ and storage at −20°C until further use. A two-step cycle protocol was used to measure mRNA expression, and a PCR amplification kit (Beijing TransGen Biotech, China) was used for amplification on a fluorescent quantitative PCR instrument (ABI 7500, ABI Inc., USA). The data were analysed and the relative TUG1 and miR-320 levels calculated using the comparison 2 − ΔΔCq method. Primers used were as follows: TUG1 forward 5’-CTGAAGAAAGGCAACATC-3’, reverse 5’-GTAGGCTACTACAGGATTTG-3’, miRNA320 forward 5’-AAAAGCUGGGUUGAGAGGGCGA-3’.
2.10 Western blot analysis
Gastrocnemius muscle tissue was cut into small pieces, and the protein components were extracted with a protein extraction kit (Beijing TransGen Biotech, China) and centrifuged at 14,000g for 10 min at 4°C. The supernatant was carefully collected. Samples were incubated in a water bath at 99°C for 10 min and then stored at −20°C for later use.
The SDS-PAGE gel was prepared, wrapped with plastic wrap to exclude exposure to the air, and placed in a refrigerator at 4°C for later use. The prepared SDS-PAGE gel (Beijing Boster Biological Technology, China) was placed into the electrophoresis tank, and the 50-µl protein samples were added to the gel lanes in the electrophoresis tank. After electrophoresis, the glass plate was transferred to a NC membrane (Thermo Scientific Company, USA). The NC membrane was removed after electroporation and sealed at room temperature for 1 h. The membrane was washed once with PBST (Beijing Solarbio Co, China) for 10 min, after which the following corresponding primary antibodies were added: anti-VEGFR-2 (CST, USA, 55B11), anti-Wnt-5a (Novus Biologicals, USA, NBP2-24752), anti-STAT3 (CST, USA, 79D7), anti-β-catenin (CST, USA, D10A8), and anti-β-tubulin (CST,USA,9F3). The membrane was placed in a refrigerator at 4°C overnight. The membrane was washed three times with PBST, and secondary antibody (CST, USA) was added. The ratio of exposure liquid (Beijing Solarbio Co, China) was A:B = 1:1, and an appropriate amountwas placed on the film so as to completely cover it. Proteins were detected based on the intensity of grey colour, as determined using Image J software.
2.11 Statistical analysis
The experimental data were analysed using GraphPad Prism 7.0 statistical software, and the values are expressed as the mean ± standard deviation (SD). ANOVA was used to compare data between multiple groups, with p<0.05 indicating that a difference was statistically significant. A t-test was used for comparisons between two groups, and p<0.05 was considered statistically significant.