Experimental animals: 5 Bama miniature swine, weighing 1200-1500g, 3-5 days old, male or female, provided by Wujiang Tianyu Biotechnology Co., LTD., License Number SCXK 2016-0006.
Experimental reagents and instruments: Up-regulated and silenced expression of KDM5B and negative control lentivirus vectors were purchased from Nanjing Shengji Biotechnology Co., LTD. (with EGFP fluorescent markers); GAPDH, KDM5B, H3K4me3, α-actin and cTnT antibodies were purchased from Abcam company in the United States. HCN4 antibody was purchased from Nanjing Baode Biotechnology Co., LTD. Fetal bovine serum (Gibco, USA); DMEM/F12 medium (Gibco, USA); Polybrene (Sigma, USA); RT-PCR kit (Dalian Bao Bioengineering Co., LTD.); Fluorescence quantification Kit (QIAGEN, Germany); Western Bolt kit (Wuhan Boshide Co., LTD.); Patch-clamp amplifier (AXon-700B, USA); Signal acquisition application digital-to-analog Converter (Digidata 1440A, USA); Processing software (PCLAMp 10.4, USA); Microelectrode drawing instrument (P-83, Japan).
Experimental methods
1. BMSCs isolation, culture and lentivirus transfection Small swines were killed by intravenous injection of euthanasia agents. The bone marrow cavity of bilateral femurs and tibia was rinsed with complete medium (DMEM/F12 medium containing 10% fetal bovine serum). Then cells in the rinse solution were inoculated in 25cm2 flask at a density of 2×106/cm2. All cells were cultured in DMEM/F12 complete medium containing 10% fetal bovine serum, 100kU/L penicillin and 100mg/L streptomycin at 37 ℃ with 5% CO2. The cells were divided into four groups: The up-regulated expression group (KDM5B-ov), silenced expression group (KDM5B-si), negative control group (KDM5B-nc, lentivirus without vector) and Blank control group (no transfection). BMSCs were transfected with the corresponding virus vector in the presence of 6µg/mL Polybrene for 12 h, and the stable transfected cells were cultured for subsequent experiments.
2. Immunofluorescence detection of virus transfection efficiency the third generation BMSCs cultured 72h after transfection were digested by pancreatin and inoculated in a confocal small dish incubator with a cell concentration of 5×106/L. After 24h, the expression intensity of EGFP was observed by confocal microscope to calculate the transfection efficiency.
3. BMSCs induced differentiated into myocardium BMSCs in good condition were inoculated into 12-well plates. When the cell proliferation reached 80% of the area, induction solution (10umol/L 5-AZA + 0.1ug /L CT-L serum-free medium) was added for 24 h. After that, DMEM/F12 medium including 10% fetal bovine serum was used for culture at 37℃ and 5% CO2, and the cell morphology was observed every another day.
4. Immunofluorescence Staining The isolated BMSCs also known as treated cells were rinsed 3 times with PBS for 10 min each. 4% paraformaldehyde was then added to the cells for a 30-minute fixation before paraformaldehyde was absorbed and discarded and rinsed 3 times with PBS. 0.2% TritonX-100 was added to the membrane for permeabilization for 20 min. This was then followed by rinsing 3 times with PBS and treatment with 10% FBS for blocking for 2 hours. Blocking solution was extracted and discarded before the addition of primary antibodies α-actin and cTnT proteins in different wells, respectively, overnight. Subsequently, the corresponding secondary antibody was added and incubated for 2 hours in the dark, rinsed in PBS for 3 times, and stained with DAPI for 2 min.
5. RT-PCR evaluation of gene expression levels At 7d, 14d and 21d of induced differentiation, logarithmic growth cells were taken for trypsin digestion. Cells were collected after 1200r/min centrifugation for 5min. Total RNA was extracted by Trizol method and cDNA was synthesized by reverse transcription for RT-PCR. Amplification conditions: 50℃ for 2min, 95℃ for 10min, 95℃ for 30s, 60℃ for 30s, 40 cycles. Finally, the expression of KDM5B, Nanog and HCN4 in each group were calculated. The experiment was repeated three times. Primer sequences of RT-PCR were shown in Table 1.
Table 1
Primer sequences used for RT-PCR
|
Primer sequence
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GAPDH-F
|
5’-GGA GTC AAC GGA TTT GGT-3’
|
GAPDH-R
|
5’-GTG ATG GGA TTT CCA TTG AT-3’
|
KDM5B-F
|
5’-GAA TTC GGG AAT CTT AAA TTT G-3’
|
KDM5B-R
|
5’-TAT CTC GAG TTC CTG TTC GGA ATA GG-3’
|
Nanog-F
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5’-GGT TGA AGA CTA GCA ATG GTC TGA-3’
|
Nanog-R
|
5’-TGC AAT GGA TGC TGG GAT ACT C-3’□
|
HCN4-F
|
5’-GTA CTC CTA CGC GCT CTT CA-3’
|
HCN4-R
|
5’-GCT CTC CTC GTC GAA CAT CT-3’
|
6. Western blot evaluation of protein expression levels 21 days after induced differentiation, cells in each group were collected, RIPA cell lysate was added, supernatant was centrifuged, concentration was determined by BCA protein concentration assay box and quantitative analysis was performed. Sample loading was 5µg for each sample. After separation by 10% SDS-PAGE electrophoresis, the protein bands were transferred to PVDF membrane and sealed with 5% skim milk powder for 2h. The primary antibody (1:500) was incubated at 4℃ overnight, and the secondary antibody (1:2000) was incubated at room temperature for 2h. After cleaning, the protein bands were colored.
7. Flow cytometry Subsequent to treatment with 5‑AZA, the expression of α-actin and cTnT was detected in three groups on different days. Cells were incubated overnight at 4˚C with anti‑swine α-actin and cTnT. Then, swine anti‑swine fluorescein isothiocyanate (PE)‑labeled mouse anti‑swine IgG was used as the secondary antibody and incubated with the cells for 1 h at room temperature. The percentage of fluorescent protein‑positive cells was detected by flow cytometry using a BD FACSCanto™ II flow cytometer (BD Biosciences). The results were analyzed and processed by FlowJo version 10.0 (FlowJo LLC).
8. Tyrode's solution was composed of NaCl 135mmol/L, KCl 5.4mmol/L, NaH2PO4 0.33mmol/L, MgCl2 1.0mmol/L and HEPES 5.0 Mmol /L, CaCl2 1.8mmol/L, Glucose 10.0mmol/L, and NaOH were used to adjust pH to 7.3. Calcium-free Tyrode solution and 0. 20mmol /L Ca2+ Tyrode solution were Tyrode solution without CaCl2 and 0. 20mmol /LCaCl2, respectively.
The electrode fluid was CsCl 133.0mmol/L, NaCl 5.0mmol/L, TEACl 20.0mmol/L, EGTA 10.0 mmol/L, HEPES 10.0 mmol/L, MgATP 5.0mmol/L, CsOH was used to adjust pH to 7.25-7.30;
The extracellular fluids were NaCl 135.0mmol/L, CsCl 5.4mmol/L, MgCl2 1.0mmol/L, CaCl2 1.8mmol/L, HEPES 5.0mmol/L, Glucose 10.0mmol/L and CdCl2 0.1mmol/L, adjust pH to 7.30-7.40 with NaOH.
9. Whole-cell Patch Clamp Detection Glass microelectrodes with tip diameters of about 1.5-2µm were prepared by p-83 programmed horizontal drawing instrument. The inlet resistance is maintained at 2-4Mω, and the series resistance compensation is 30%-50%. The cells were placed in extracellular solution and left for 10 min. After adherence, extracellular solution was applied to remove the remaining KB solution. Cells with good condition, smooth edge, integrity, clear horizontal lines, strong three-dimensional sense and no shrinkage were selected for the experiment, and the changes of Na current intensity on cell membrane surface were recorded. In order to minimize the experimental error caused by cell size, current density was used to represent the current value, in pA/pF.
10. Observation indicators ① Differences in BMSCs lentivirus transfection efficiency and expression levels of KDM5B, H3K4me3, H3K4, Nanog, HCN4, α-actin and cTnT in each group; ② Differences in cell membrane surface current density of BMSCs in each group.
Statistical Analysis
SPSS 21.0 statistical software was used for statistical analysis. Data were expressed as X±S. One-way anOVA was used for comparison of cell subsets, one-way ANOVA was used for comparison of mean values between groups 3 or more, and LSD-T test was used for intra-group multiple comparison. All the statistical hypothesis tests were two-sided hypothesis tests, and P < 0.05 was considered as significant difference.