Reagents and antibodies
Human bone tissue specimens were taken from the femoral bone marrow of patients undergoing hip and knee replacement surgery at the Third Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine. They were collected aseptically during the operation and stored in liquid nitrogen for later use. The study patients all gave informed consent to this experiment, signed an informed consent form, and the study was approved by the medical ethics committee of our hospital. This experimental study was registered by China Clinical Trial Research, registration number: CHiCTR2000037969.
DMEM high sugar medium(Gibco,#11965092), Penicillin-Streptomycin(Gibco,#15070063), fetal bovine serum (FBS,Gibco,#10099133),Tissue ROS detection kit(Beibo Biological,#BB-470532),cell reactive oxygen detection kit(Biyuntian,#S0033S),cell apoptosis detection kit (KGI Bio, #KGA108-1), CCK8 reagent (Dongren Chemical, #CK04), superoxide Substance detection kit (Biyuntian,S0060), autophagy staining detection kit (Solebao, #G0170-100T), Lyso-Tracker Red(Solebao,#L8010-50μl); Goat Anti-Rabbit IgG H&L (HRP)(Jackson,#111-035-003),Beclin-1 (D40C5) Rabbit mAb(CST,#3495T), mTOR (7C10) Rabbit mAb(CST, #2983T), Phospho-mTOR (Ser2448) (D9C2) Rabbit(CST, #5536T), LC3A/B (D3U4C) XP® Rabbit mAb(CST,#12741T), Cleaved Caspase-3 (Asp175) Antibody(CST, #9661T), 3-MA(MCE, #HY-19312), Endonuclease SgrAI(NEB, #R0603S), Endonuclease EcoRI(NEB, #R3101V).
Isolation, culture and grouping of hBMSC
Remove the bone marrow specimen from the liquid nitrogen, thaw it, pipette the bone marrow evenly to make a cell suspension, add 25 mL of IMDM culture medium containing 10% FBS, and inoculate it in a 175 cm2 culture flask at 37℃, 5% CO2 incubator. nourish. Collect logarithmic growth phase human bone marrow mesenchymal stem cells (hBMSCs) cells, count them, resuspend the cells in DMEM low-sugar complete medium, adjust the cell concentration to 1×105 cells/ml, inoculate a 6-well plate, add 2ml cell suspension to each well Incubate overnight at 37°C and 5% CO2.Discard the old culture medium and proceed as follows: blank group: add 2ml fresh DMEM low sugar complete medium; 3-MA pretreatment group: add 2ml DMEM low sugar complete medium containing 5mM 3-MA to pre-condition for 30 minutes, discard the culture Add 2ml DMEM low sugar complete medium; H2O2 intervention group: add 2ml DMEM low sugar complete medium containing 0.05 mM H2O2; 3-MA pretreatment group + H2O2 group: add 2ml DMEM low sugar complete containing 5mM 3-MA The medium was pretreated for 30 minutes, the medium was discarded, and 2ml of DMEM high-glycosylated complete medium containing 0.05mM H2O2 was added. Cells in each group were cultured at 37°C and 5% CO2 for 48 hours.
CCK-8 analysis detects the effect of different concentrations of H2O2 on the proliferation of hBMSCs
Collect hBMSCs cells, count them, resuspend the cells in DMEM high glucose complete medium, adjust the cell concentration to 1×105 cells/ml, inoculate in 96-well plates, add 0.1 ml cell suspension to each well, and incubate overnight at 37°C, 5% CO2 . Discard the old medium, add 0.1ml of DMEM high-sugar complete medium containing 0, 0.05, 0.1, 0.2, 0.4 mM H2O2, and continue culturing at 37°C and 5% CO2. After 24h, 48h, 72h, discard the culture medium in the well, add 0.1ml DMEM high glucose complete medium containing 10% CCK8, incubate for 2-3h at 37℃, 5% CO2, measure OD450 on the microplate reader, and Draw cell growth curve.
DCFH-DA detects cellular ROS level
Dilute DCFH-DA (2,7-Dichlorodihydrofluorescein diacetate) with serum-free medium at 1:1000 to make the concentration 10μM. After the hBMSCs cells were treated with H2O2 for 48h, the cells were collected and suspended in DCFH-DA at a cell concentration of 106-2×107/ml, and incubated in a cell incubator at 37°C for 20 min. Mix by inversion every 3-5 min to make the probe and the cells fully contact. The cells were washed three times with serum-free cell culture medium to fully remove the DCFH-DA that did not enter the cells. Flow cytometry detection, 488nm excitation wavelength, 525nm emission wavelength, real-time detection of fluorescence intensity.
Autophagy staining detection(MDC)
After 48 hours of induction treatment of the above groups of cells, the culture medium was discarded, and the cells were washed with Wash Buffer. Add monodansylcadaverine(MDC) staining solution and incubate in the dark at 37 ℃ and 5% CO2. Add Wash Buffer to wash the cells 2-3 times. Observe under a fluorescence microscope (Ex/Em=355nm/512nm), count and take pictures.
Autophagy Lysosome Red Fluorescent Probe (Lyso-Tracker Red)
Take a small amount of Lyso-Tracker Red and add it to the DMEM high-glycemic medium at a ratio of 1:20000. After each group of cells is induced for 48 hours, the culture medium is discarded, and an appropriate amount of Lyso-Tracker Red working solution is added at 37 ℃, 5% CO2 Incubate for 30-120 min under dark conditions. Discard the cell surface staining solution and add fresh DMEM high-sugar complete medium. Observe under a fluorescence microscope. Lysosomes are stained with bright and strong fluorescence.
Immunofluorescence staining to detect LC3A/B
After the above groups were induced and cultured for 48 hours, the cell culture medium was discarded, and the cells were fixed with freshly prepared 4% paraformaldehyde for 10 minutes after washing with PBS three times. Wash with PBS three times, 5min each time. Permeabilize the cells with 0.2% triton X-100 (prepared in PBS) for 10 minutes. Wash with PBS three times, 5min each time. Blocked with 2% BSA for 30 min, washed twice with PBS. Add rabbit anti-LC3A/B monoclonal antibody (1:400) and incubate at room temperature for 1h. Wash with PBS three times, 5min each time. Add FITC-labeled secondary antibody (1:500) and incubate at room temperature for 30-45min. Wash with PBS four times, 5min each time. Add 0.5μg/mL DAPI (prepared in PBS) for staining for 10 minutes. Wash three times with PBS to remove excess DAPI. Observe with a fluorescence microscope and take pictures and record.
Annexin V-FITC/PI cell apoptosis detection
After H2O2 treatment of hBMSCs cells for 48 hours, the cells were trypsinized without EDTA, centrifuged at 2000 rpm for 5 min at room temperature, and 1-5×105 cells were collected and washed twice with PBS. Add 5μl PI dye solution to 50μl Binding Buffer and mix well. Add PI staining solution to the cell pellet, mix well, and react for 5-15 min in the dark at room temperature. After the reaction, add 450μl of Binding Buffer and mix well. Add 1μl Annexin V-FITC and mix well, and react for 5-15 min in the dark at room temperature. Detect with flow cytometer, excitation wavelength Ex=488 nm; emission wavelength Em=530 nm, Annexin V-FITC fluorescence signal is green, use FL1 channel detection; excitation wavelength Ex=488nm, emission wavelength Em≥630 nm, PI red Fluorescence is detected by the FL3 channel.
Gene expression profiling chip to detect autophagy-related proteins
After the above groups of cells were cultured for 48 hours after intervention, the cells were collected, and gene expression profiling chips were performed to detect autophagy-related proteins.
Western blot detection of Beclin1, mTOR, p-mTOR (Ser2448), LC3A/B, Cleaved caspase-3 protein expression
After the cells of each group were treated with conditioned medium and hydrogen peroxide, the protein was extracted from the phosphorylated protein lysate, electrophoresed, transferred and blocked after quantification, and Beclin1, mTOR, p-mTOR, LC3A/B, Cleaved caspase-3 were added. Incubate the antibody overnight at 4°C, wash with TBST, incubate the secondary antibody for 2 hours at room temperature, develop and expose the eECL, and use Image J software to calculate the band gray value. Use Beta-action as an internal reference to calculate the relative protein amount.
The above experiments were repeated three times
SPSS 20.0 software was used for statistical analysis. Mean±SD was used to represent measurement data. All data were tested for normality and homogeneity of variance. The comparison between groups was tested by t, and the non-parametric test was used when the analysis of variance was not satisfied. The detection level was α= 0.05, P<0.05, the difference is statistically significant.