Separation, culture of PDLSCs
The culture procedure of periodontal stem cells was performed according to the previous research method of our group [18] and was approved by the ethics committee of Nantong University Hospital. Ten orthodontically decompensated premolar and interrupted teeth from patients aged 14–22 years were collected from the outpatient clinic of the Department of General Dentistry, Nantong University Hospital with the informed consent of so adolescent patients. All participants rinsed their teeth with sodium bicarbonate solution before extraction, and immediately after extraction the teeth were placed in phosphate-buffered saline (PBS) containing 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen, Life Technologies, Carlsbad, CA), rinsed twice to remove blood, and treated with a sterile surgical blade in an ultra-clean The periodontal membrane tissue was scraped from the middle third of the tooth surface in the ultra-clean table and digested in 3 mg/ml type I collagenase (Worthington Biochem, Freehold, NJ) and 4 mg/ml dispase (Roche, Mannheim, Germany) for 30 min. Cell suspensions of periodontal membranes were inoculated into 25 cm cell culture dishes and suspended in complete medium containing low glucose Dulbecco's modified Eagle's medium (GIBCO BRL, Grand Island, NY) with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were inoculated in tissue culture dishes and cultured at 37°C with 5% CO2, and the medium was changed every 3 days. When the cells in the culture dish reached 80%, they were cultured in a 1:3 ratio of passages. So the experiments were performed using the fourth generation of periodontal stem cells.
Osteogenic differentiation
The osteoblast differentiation process was as described in a previous study by our group [19]. The P4 generation of PDLSCs was used as the study subject and inoculated in 6-well culture plates at a density of 3,000 cells/cm2, and the osteogenic differentiation induction medium (50 µg/mL ascorbic acid, 10 mM sodium glycerophosphate, and 1.0 µg/mL dexamethasone) was configured with different glucose concentrations of complete culture medium (5. 5 mMol/L, 11 mMol/L, 25 mMol/L, 44 mMol/L), 11 mMol/L and 44 mMol/L glucose concentrations were configured from L-DMEM with appropriate amount of medical 5% sterile glucose solution, and differentiation induction cultures were incubated for 7, 14 and 21 days, with fluid changes every 1 day.
Alizarin red staining and ALP staining
Cells were co-cultured with osteogenic medium for 21 days and stained to detect the mineralization potential of the cells. The medium in the 6-well culture plate was first discarded, the cells were washed 3 times with appropriate amount of 0.01 M PBS, inverted to drain the remaining water, and the cells were fixed with 4% paraformaldehyde for 30 minutes. The cells were then washed 3 times with an appropriate amount of 0.01 M PBS and the remaining water was drained. The cells were then incubated with 40 mm Alizarin Red S solution (Solarbio) for 15 min according to the manufacturer's instructions for Alizarin Red staining and with 40 mm Alkaline Phosphatase Assay Kit (Beyotime) for 12 h for Alkaline Phosphatase staining.
Western blot
The cellular protein extraction process was performed as described in a previous study by our group [20]. Cells cultured in osteogenic induction medium with different glucose concentrations were first fully lysed using PMSF solution and RIPA solution (Beyotime), followed by protein collection and centrifugation to extract the supernatant. Membrane, Nuclear and Cytoplasmic Protein Extraction kit (Sangon Biotech, C510002) was used to extract membrane and nuclear proteins from cells cultured in osteogenic medium with different glucose concentrations according to the product instructions, and then the protein concentration was measured by BCA method. The protein concentrations were then measured by BCA to sample the calculated amounts. After 90 min, the PVDF membrane was placed in the closure solution and gently shaken on a shaker for 2 h at room temperature. The primary antibody was incubated overnight at 4℃ and the secondary antibody was incubated for 2 h at room temperature. The following primary antibodies were used: rabbit anti-SLUG(1:1000,Cell Signaling Technology), rabbit anti-RUNX2 (1:1000, Sangon Biotech), rabbit anti-IGF-1R (1:1000, Abcam), rabbit anti-GAPDH (1:1000, Abcam), mouse anti-SUMO1 (1:1000, Santa Cruz),mouse anti-PCNA (1:1000, Santa Cruz),mouse anti-PI3K (1:1000, Santa Cruz), mouse anti-pAKT (1:1000, Santa Cruz), rabbit anti-AKT (1:1000, Proteintech), and rabbit anti-Na+-K+-ATPase (1:1000, Abbkine). The second antibodies were goat-anti-rabbit or goatanti-mouse horseradish peroxidase-conjugated IgG (1:1500, Abcam).
Quantitative real -time reverse transcription-polymerase chain reaction
According to the manufacturer's instructions (Invitrogen), total RNA was extracted from cells using Trizol reagent. Complementary DNA (cDNA) miRNA transcripts were amplified using Maxima ™ H Minus cDNA Synthesis Master Mix, with dsDNase (Thermo Fisher Scientific, Waltham, MA). The polymerase chain reaction (PCR) mixture was prepared using LightCycler 480 SYBR Green (Roche Applied Science, Penzberg, Germany). For GADPH, RUNX2 and SLUG detection, cDNA was synthesized using RevertAid RT Reverse Transcription Kit (Thermo Fisher Scientific). AceQ qPCR SYBR Green Master Mix (without ROX) (Vazyme) was then used for quantitative PCR of these genes. GAPDH was used for normalization. We use a Light Cycler 480 Real-Time PCR System (Roche Diagnostic, Mannheim, Germany) to test these levels. The primer sequences used in the experiment were as follows:
GAPDH:5’-GAAGGTGAAGGTCGGAGTC-3’,5’-GAAGATGGTGATGGGATTTC-3’;RUNX2:5’-TCAACGATCTGAGATTTGTGGG-3’,5’-TCAACGATCTGAGATTTGTGGG-3’;SLUG:5’-TGTGACAAGGAATATGTGAGCC-3’,5’-TGAGCCCTCAGATTTGACCTG-3’.
The ratio obtained by comparing the target gene with the CT value is the relative expression of the gene. Three replicates were set up three times for each experiment.
Co-Immunoprecipitation
Co-immunoprecipitation protocol was performed using a Pierce co-IP kit (Thermo Scientific™ Pierce™ Classical magnetic bead immunoprecipitation/immunocoprecipitation Kit, 88804). Incubate the cell lysate with the rabbit anti-IGF-1R (Abcam) for 1–2 hours at room temperature, or overnight at 4 ºC. Bind the antigen/antibody complex to the protein A/G magnetic beads for one hour at room temperature. Wash the beads twice with immunoprecipitation lysis/rinse buffer, followed by one wash with pure water. Elute the antigen/antibody complexes. Finally, immunoblotting experiments for immune complexes were performed.
Immunofluorescent staining
PDLSCs were planted with a density of 1×105cells/ mL. After 24 h of cell attachment, PDLSCs were fixed by 4% paraformaldehyde was fixed for 1 h. The primary antibody was diluted in proportion with Immunostaining Blocking/Primary Antibody Dilution Buffer (Sangon Biotech) and incubated overnight at 4 ºC. PDLSCs were incubated with the corresponding secondary antibody for 2 h at room temperature. Nuclei were stained with DAPI (1:1000; Santa Cruz) and the cells were mounted on an inverted fluorescent microscope and photographed.
Cell viability assay
PDLSCs were seeded on 96-well plates at a cell density of 1×103 cells/well. 0, 0.5, 1, 2, 5, 10, 20, 50umol/L IGF-1R inhibitor NVP-ADW742 (Beyotime, Shanghai, China) were add into Osteogenic Induction Solution and were used to culture PDLSCs for 24h, 48h, 72h. Then we washed the wells, added 100 uL L-DMEM and 10 uL CCK8 (Beyotime, Shanghai, China) per well, and incubated the cells at 37 ℃ for 2 h. The optical density was measured down at 450 nm.
Statistical analysis
The experimental data were analyzed by the GraphPad Prism software. All experiments were repeated at least three times, with three replicate wells per design and the data shown as mean ± standard deviation. Statistical significance was assessed by independent sample t test, p < 0. 05 was considered statistically significant.