1.7 Isolation and Culture of BMSC Cells
Take the femur and tibia of mice, cut off both bone ends, and use a syringe to extract DMEM-F12 culture medium without FBS. Insert a syringe from the other end to repeatedly rinse the bone marrow cavity into a 50ml centrifuge tube until the bone marrow cavity turns white. Then, repeatedly blow the bone marrow cells to prepare a single cell suspension. Cultivate and pass through in a T25 culture bottle.
Take the logarithmic growth phase BMSC cells for digestion and count, inoculate them with a 24 well plate, 1x10 5/well, and add different concentrations of MRSA bacteria (0, 1.5625 moi, 3.125 moi, 6.25 moi, 12.5 moi, MRSA 25 moi, MRSA 50 moi, MRSA 100 moi, MRSA 200 moi, MRSA 400 moi, and MRSA 800 moi) to the cell wall. After 24 hours, CCK8 activity testing is performed. Add CCK-8 reagent and incubate at 37 ℃ for 2 hours. Measure the OD value at 450nm and calculate IC50. Moi=number of MRSA/BMSCs (multiple infections=number of bacteria/number of bone marrow stem cells)
By adding osteogenic inducers (100 mmol/L dexamethasone, 0.05 mmol · L-1 ascorbic acid, and 10 mmol · L-1) to BMSCs cells β- Sodium glycerophosphate for osteogenic differentiation induction.
1.8ALP staining
Take each group of cells, clean them with PBS, add ALP fixative and fix for 3 minutes. Add the prepared ALP incubation solution dropwise, place it in a wet box, incubate in dark for 15-20 minutes, and re stain with nuclear fixation red staining solution or methyl green staining solution for 3-5 minutes. Clean the cells with PBS and perform microscopic examination.
1.9 IF experiment
Each group of cells was incubated at room temperature in a 4% PFA solution for 30 minutes and fixed. Then, the samples were sealed in PBS containing 2% Triton X-100 (PBST) and 5% goat serum for 1 hour. They were incubated overnight at 4 ° C with the following primary antibodies: Runx2, Osterix (OSX), ALP, Osteocalcin. Then, incubate the cells with the corresponding secondary antibody at 37 ° C for 1 hour. Imaging and analyzing cells under a fluorescence microscope.
1.10 Construction of MRSA infected osteomyelitis model
Male mouse aged 6-8 weeks were randomly divided into 7 groups, with 10mouse in each group; Drill a hole in the right tibia, inject sterile physiological saline into Group A, and inject 1 into Groups B, C, D, E, F, and G, respectively × 109, 1 × 108, 1 × 107, 1 × 106, 1 × 105 and 1 × 104 CFU/mL MRSA was used for 2 weeks to observe the wound healing, skin temperature, and the presence of redness, swelling, pus discharge, and sinus formation. Take the right tibia and secretions for subsequent experiments.
1.11 HE staining
After decalcification, bone tissue was fixed overnight in 4% paraformaldehyde, dehydrated in ethanol gradient, and embedded in paraffin μ Slice m thick. Seal the film after staining with hematoxylin and eosin, and randomly select areas for optical microscopy observation.
1.12 IHC staining
After decalcification, bone tissue was fixed overnight in 4% paraformaldehyde, dehydrated in ethanol gradient, and embedded in paraffin μ Immunohistochemical staining was performed on m thick sections. The primary antibodies used for immunohistochemistry include Runx2, OSX, ALP, and Myh7.
1.13 Transcriptome sequencing
1.3.1 Experimental process
Use TRIzol (Thermofisher, 15596018) to separate and purify the RNA of the total sample according to the operating plan provided by the manufacturer. Then, NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA) was used to control the total RNA content and purity of three duplicate bone tissue samples from the WT group, MRSA group, KO sdrc group, and MRSA+KO sdrc group of mice, and the RNA integrity was tested using Bioanalyzer 2100 (Agilent, CA, USA); Concentration>50ng/ μ L. RIN value>7.0, total RNA>1 μ G satisfies downstream experiments. Using oligo (dT) magnetic beads (Dynameads Oligo (dT), cat.25-61005, Thermo Fisher, USA), the mRNA carrying PolyA (polyadenylate) was specifically captured through two rounds of purification. The captured mRNA was fragmented using the NEBNextR Magnesium RNA Fragmentation Module (cat. E6150S, USA) under high temperature conditions, at 94 ℃ for 5-7 minutes. Fragmented RNA is synthesized into cDNA by reverse transcriptase (Invitrogen SuperScriptTM II Reverse Transcriptase, cat. 1896649, CA, USA). Then use E Coli DNA polymerase I (NEB, cat. m0209, USA) and RNase H (NEB, cat. m0297, USA) undergo two-strand synthesis, converting the composite double stranded DNA and RNA into DNA double stranded. At the same time, dUTP Solution (Thermo Fisher, cat. R0133, CA, USA) is added to the double stranded DNA to make the ends of the double stranded DNA flat, and an A base is added to each end to connect it to the junction with a T base at the end, And use magnetic beads to screen and purify its fragment size. Using UDG enzymes (NEB, cat. m0280, MA, US) to digest the two strands, PCR pre denaturation was performed at 95 ℃ for 3 minutes, followed by 8 cycles of denaturation at 98 ℃ for 15 seconds each. Annealing was performed at 60 ℃ for 15 seconds, stretching at 72 ℃ for 30 seconds, and finally extending at 72 ℃ for 5 minutes to form a library (chain specific library) with a fragment size of 300bp ± 50bp. Finally, we used Illumina NovaseqTM 6000 (LC Bio Technology Co., Ltd. Hangzhou, China) to perform double ended sequencing according to standard procedures, with sequencing mode PE150.
1.3.1 Data quality control
The raw data generated by sequencing is preprocessed and filtered (usually genes are screened from both the difference multiple and significance level, with the difference multiple FC>=2 or FC<0.5 (i.e. the absolute value of log2FC>=1) and q value<0.05 (| log2fc |>=1&q<0.05) as the threshold standard (multiple groups are compared with no difference multiple, and genes with q<0.05 are screened as genes with statistical differences between multiple groups) to obtain valid data (Clean Data), Compare with the reference genome again to obtain comprehensive alignment information. At the same time, calculate the gene location information specified in the genome annotation file gtf separately: 1) Compare the sequencing data with the reference genome for reads statistics; 2) Regional distribution statistics of sequencing data compared to reference genomes. Genome: ftp://ftp.ensembl.org/pub/release-101/fasta/mus_musculus/dna/ )
1.4 Construction of Myh7 Interference Stable Transgenic Plants
The Myh7 interference plasmid was purchased from the Ono gene. A 2ul plasmid was added to 100ul of Escherichia coli, and a single colony was smeared onto 20ml of liquid culture medium. It was incubated overnight on a shaker at 37 ° C and 200r. Extract plasmid RT-PCR to screen the optimal interfering vector, transfect it into 293T cells for packaging, and collect cell culture supernatant 48 hours after transfection. Centrifuge 500g for 10 minutes to remove cell fragments and perform virus titer measurement or virus concentration. Transfected mouse osteoblasts were continuously screened with 2.5ug/ml puromycin for one month.
1.5 WB experiment
After cracking the sample on ice for 10 minutes, centrifuge 14000 g at 4 ℃ for 15 minutes. Measure the protein concentration using the BCA protein quantification kit and take 80 μ L protein plus 20 μ Mix the 5 x protein loading buffer well and cook in a boiling water bath for 5 minutes before performing SDS-PAGE electrophoresis. After the membrane transfer is completed, remove the PVDF membrane and place it in 1 × The strips after TBST soaking and cutting are placed in a sealing solution (5% skimmed milk powder) and sealed on a shaking table at room temperature for 40 minutes. First antibody (Myh7, Myl2, Tnnc1, Runx2, OSX β- Actin, ALP, and osteocalcin were incubated overnight at 4 ℃, and the secondary antibody was incubated for 40 minutes, developed, and photographed for preservation.
1.6 Alizarin Red Staining
Clean the cell sample with PBS, fix it with 10% formalin for 10-30 minutes, remove the fixative, clean the sample twice with PBS, add alizarin red staining solution dropwise, cover the sample, stain for 1-5 minutes, remove the staining solution, wash with PBS twice, observe and take photos under the microscope.
1.7 Statistical Analysis
Statistical analysis was conducted using Prism graphpad, and the experimental data were presented in the form of mean ± standard error (Mean ± SEM). The comparison of inter group measurement data was conducted using T-test, and the comparison of multi group measurement data was conducted using one-way analysis of variance. Setting p<0.05 has statistical differences: p<0.01 has significant statistical differences; P<0.001 has a highly significant statistical difference.