Sprague-Dawley male rats randomly divided into MI group and control group were obtained from the experimental animal center of Chongqing Medical University. All animals in this study received humane care in compliance with the “Guide for the Care and Use of Laboratory Animals”. All animal experiments were approved by the Institutional Animal Care and Use Committee of Chongqing Medical University.
30 male rats aged 6-8 weeks were randomly divided into sham operation group and MI group and then anesthetized intraperitoneally with pentobarbital sodium (30~50mg/kg). A catheter was intubated into the trachea and a small animal ventilator provided positive pressure ventilation at 1~2 ml/cycle and a respiratory rate of about 60 breaths per minute. After the thoracic cavity at the level of the fourth rib and along the left sternal border was opened, the left anterior descending coronary artery (LAD) was ligated with a 6-0 silk suture 3 mm from the tip of the left auricle. The chest wall was closed with a continuous 5-0 prolene suture, followed by a 4-0 polyester suture in order to close the skin. The procedure of sham operation was the same as the above-mentioned MI induction but without LAD ligation. After LAD ligation in rats, ST segment elevation in the chest leads checked by an electrocardiogram (ECG) was the sign of a successful MI surgery and the success rate was approximately 70%.
Human Umbilical Vein Endothelial Cells (HUVEC) were purchased from the company and cultured in RPMI-medium 1640 supplemented with penicillin, streptomycin and 10% FBS, and used to the 10th generation. The stable-growing HUVEC was seeded in 6 six-well plates at a density of 2×104 cells/well, and placed in a normoxic cell incubator overnight. These cells were taken out next day and placed in a hypoxic cell incubator (5% O2) for 1 hour, 2 hours, 4 hours, 12 hours, 24 hours, 48 and 72 hours.
The pre-experiment result showed that the most suitable MOI was10, and the formal experiment of lentiviral transfection is carried out according to the instructions. Inoculate HUVEC in advance with a 6-well plate at a concentration of 3~5×104/well. According to the control group, vehicle group and Foxp1 RNAi group, complete medium, 1×108 TU/ml negative control virus and 1×108 TU/ml Foxp1 interfering RNA lentivirus were added to the inoculated HUVEC respectively. The infection system was 2 ml. The optimal conditions are used for infection conditions. Change the fluid one day after infection. Three days after infection, the infection efficiency of lentiviral and cell status was observed with a fluorescence microscope.
Before the experiment, use a marker and a ruler to draw 6 horizontal lines on the back of the 6-hole plate, with an interval of about 0.5 to 1 cm, to make the horizontal lines evenly pass through the holes. Add about 5×105 HUVEC cells to the six-well plate and place them in the incubator for overnight culture; the next day, use a 200uL pipette tip and a ruler perpendicular to the horizontal line on the back of the orifice to make a scratch on the cells in the orifice; wash the cells 3 times with sterile PBS buffer, and add 2 mL of serum-free simple medium to each well; Enter the 37°C 5% CO2 incubator to continue the cultivation. Observe and take pictures under an inverted phase-contrast microscope at 0, 6, 12, and 24 hours after the scratch.
Tubule formation experiment
Spread the pre-chilled Matrigel polycarbonate glue on a 96-well plate, 50μl per well, put it in a 5% CO 2 incubator at 37°C, let it stand for 60 min, and combine to solidify; Add each group of HUVEC with a density of 1.5×105/mL, add 100uL cell suspension to each well, and continue to culture; After 2 hours, the number of cells in each group was observed under an inverted phase-contrast microscope. Obvious tubular structures were visible in 2.5 hours; 6 fields of view were randomly selected for shooting, and then image J software was used for analysis and processing.
The three groups cells were respectively seeded in a six-well plate at a density of 5*104 and cultured overnight; the floating cells were collected in a centrifuge tube, and then the cells were digested into single cells with 0.25% pancreatin; The cells were collected in a centrifuge tube, added with an appropriate amount of PBS buffer, centrifuged, and the supernatant was discarded. After two times centrifugation, resuspend the cells in 100uL PBS buffer, and slowly add 900uL pre-cooled 75% ethanol. Or collect the cells in each well in several centrifuge tubes, add an appropriate amount of PBS buffer, centrifuge at 1000 r/min for 5 minutes, and discard the supernatant. Centrifuge twice in the same way; finally 1×106 cells were resuspended in PBS (PH=7.2) 500uL buffer, placed in a 1.5mL EP tube, and then sent to the School of Life Sciences, Chongqing Medical University for cell cycle and cell apoptosis detection.
Western blot analysis
For western blot analysis of FoxP1 expression, cultured cells were lysed in RIPA buffer on ice and protein concentration was quantified using the Bradford method. The equal amount of total protein (15μg) was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% milk in Tris-buffered saline Tween (TBST) (50 mM Tris, 150 mM NaCl, 0.5 mM Tween-20, pH=7.5), and then were incubated over night with antibodies directed against human FoxP1. Beta-actin was used as loading controls.
The rat hearts from each group were fixed in 4% PFA and then stained with haematoxylin-eosin (n=3 for each group at every time point). Pictures were taken using microscope at different magnification. And then after dehydration with 70%, 80%, 90%, 100% gradient alcohol and transparent xylene, the whole embryo was embedded with paraffin and sliced with a thickness of 10um. Pictures were taken using microscope at different magnification.
The cells, which were grown on cover slips without coated, were fixed with 4% paraformaldehyde at room temperature for 15 min, and the heart tissue sections followed by permeabilization with 0.25% Triton X-100 in PBS (0.01 mol/l) at 37℃for 10 min. Cells or sections were blocked with 10% goat serum (Boster Biological Technology, Pleasanton, CA, USA) at 37˚C for 10 min, and then incubated with the following primary antibodies at 4˚C overnight. The cells were then incubated with cyanine 3-conjugated goat anti-rabbit immunoglobulin G (CWBIO, Beijing, China) at 37˚C for 45 min, followed by DAPI at room temperature for 10 min. Subsequently, the cells were subjected to confocal microscopy imaging (original magnification, x400). Imaging conditions for each antibody were kept consistent across all samples.
RNA extraction and quantitative real-time PCR (RT‑qPCR)
Cells were collected and lysed with TRIzol (Takara Bio Inc., Otsu, Japan) to extract total RNA, which was reverse-transcribed to complementary DNA using a Prime Script Reverse Transcriptase reagent kit (cat. no. RR047A; Takara Bio Inc.) according to the manufacturer's protocols. β-actin was used as an internal reference for each gene. The relative gene expression levels were calculated using the 2-∆∆Cq method and normalized to β-actin.
All experiments were repeated three times. Values are expressed as the mean ± standard deviation.The data were analyzed using SPSS (version 20.0; IBM Corp., Armonk, NY, USA), and one-way analysis of variance followed by a Tukey's test was applied for comparison between groups. P<0.05 was considered to indicate a statistically significant difference.