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.