Microarray data obtained:
Through the research from the GEO database, the gene expression profile, GSE 70918 (GPL 19271, Affymetrix Rat Gene 2.1 ST Array) was obtained. In this profile, there contained 4 samples, 2 of which were cultivated with PRP and the other 2 samples were cultivated with PPP. All of the 4 samples were included into our study.
Identification of differently expressed genes (DEGs)
The GEO2R tool was used to analysis the differentially expressed genes. And the threshold of the DEGs in our study was set as |logFC|>1 and P-value < 0.05.
(Protein-protein interaction) PPI network construction and module selection
The PPI network based on commonly differentially expressed genes was construted by Search Tool for the Retrieval Interacting Genes (STRING) database. The Cytoscape(version 3.8.0) was used to analyze and visualized the correlations among genes. Molecular Complex Detection (MCODE; version 1.31) was used to determine the top 3 correlated gene modules.
GO and KEGG pathway enrichment analysis construction
The Database for Annotation, Visualization and Integrated Discovery (DAVID, version 6.8) was used to analyze the GO and KEGG functional enrichment based on the DEGs. Furthermore, the up-regulated and down-regulated DEGs were seperately analyzed from the online program.
The miRNA-mRNA interaction analysis
The differentially expressed miRNA was isolated from the profile. Online toll targetscan was used to determine the target mRNA of each miRNA. The venn tool was used to determine the commonly differentially expressed mRNA between the profile and target mRNA.
Animal cares
All animals received humane care in compliance with the Guidelines for the Care and Use of Laboratory Animals by the Department of Science and Technology of the People’s Republic of China (2006). One 8-week-old healthy New Zealand male, white rabbits (Provided by Jiangsu university experimental animal center of China) were used for this experiment.
Rabbit Adipose-derived Stem Cell Culture
An eight-week-old New Zealand white male rabbit was prepared under general anesthesia, and a povidone-iodine solution was used to sterilize the hind leg before extracting the stem cells. The subpatellar fat pad was then harvested and collected in a 50 ml centrifuge tube using a series of sterile surgical instruments. Several operations were carried out on a sterilized bench, as described below. The specimen was transferred to the culture dish where it was washed 3 times utilizing sterile phosphate-buffered saline (PBS). Superfluous tissues were wiped out, and the remaining tissues were minced finely into the shape of chyle, mixed with collagenase typeⅠ whose volume was two times larger than the amount of tissue and moved to a 50 ml centrifuge tube. The tube was next placed in constant temperature, and the table was set to vibration mode for 90 minutes to digest the tissue. At the end of digestion, the tissues were transferred into a centrifuge machine, and the rotator speed was set at 1700 r/min, which lasted 10 minutes. The top content of the centrifuged tube was blended with a 5ml pipette and centrifuged at 2000 r/min for 3 minutes. The top content and supernatant were discarded, leaving the lower white block mass content in the tube.
DMEM culture medium was added to the remaining tissue content left in the tube, and the mixture was centrifuged at 1500 r/min for 10 minutes, after which the supernatant was discarded, leaving just the lower part of the solution. This remaining tissue content was filtered with a 70 nm filter screen, leaving a liquid that was collected in a 15 ml centrifuge tube. After centrifuging the liquid for 3 minutes at 800 r/min and discarding the supernatant, the lower content containing the stem cells was obtained finally. Subsequently, we inoculated the stem cells into the flask filled with 5 ml DMEM of fetal bovine serum and observed the morphological characteristics under the divert microscope. The cells were cultured in a Rsbiotech in a 5% CO2-saturated humidity pontoon at 37℃, with the DMEM of fetal bovine serum subjected to culture and change every three days.
Tetrazolium method (MTT)
The passage-3 ADSCs and the passage-5 BMSCs were digested and diluted, and the mixture was transferred to a 96-well culture plate at 100µl cell suspension per well and incubated at 37℃, in a 5% CO2-saturated humidity float tank for 24 hours. 5-Aza was then added to each well containing ADSCs and the BMSCs at concentrations of 0, 10, 20, 30, 40 µmol/l. The BMSCs groups were labeled groups A, B, C, D, and E, and the ADSCs group were labeled groups F, G, H, I, and J. Following incubation under the same conditions as before for 24 hours, we added 50 µl MTT solution to each well and incubated again under the same conditions for another 4 hours, terminating the reaction of each well by aspirating the culture medium. To each well was added 150 µl DMSO solution after terminating the 4-hours of incubation, with the plate placed on the table concentrator. Finally, the absorbance of each well was measured at 550 nm (OD) to detect the cell viability under the induction of different concentrations of 5-Aza.
Induction of differentiation and division into groups of BMSCs and ADSCs
ADSCs were induced by 0, 10, and 20 µmol/l of 5-Aza in groups A, B, and C, while ADSCs were induced by20µmol/l 5-Aza and 0, 20, 40 µmol/l PRP in groups D, E, and F, respectively. Group A served as the ADSCs control group, and group D played the role of 5-Aza control group. Groups B and C and groups D and E were ADSCs and PRP experimental groups, respectively. The morphologic characteristics of ADSCs in different groups were observed and recorded under an inverted microscope at regular intervals of 1, 3, 5, 7, and 9 days after induction of the stated concentrations of 5-Aza and PRP.
Immunohistochemical staining and Semi-quantitative analysis
First, we digested and diluted the cells from each group 9 days after induction; then we added 1 ml of the solution to each well of the 12-well culture plate that had a cell slide placed in every well in advance. We next extracted the slides from each well after culturing at 37℃ in a 5% CO2-saturated humidity incubator for 24 hours. We washed the slides once with 300 µl PBS and did not remove the liquid for 5 minutes until the cells were saturated. Next, we added 150 µl 4% paraformaldehyde fixative to every slide and left them undisturbed for 30 minutes before adding 150 µl 0.1% Triton x-100 to each and left at room temperature for 3 minutes. We washed the slide 3 times with 300 µl PBS, each washing process lasting 5 minutes. The ensuing step was to drop 200 µl 5% BSA blocking solution to each slide and incubate in a 5% CO2 incubator for 1 hour. Each slide was incubated overnight at 4℃ after being mixed with 150µl α-SMA (1:200) diluted with 1% BSA. The slides were washed with 300 µl PBS 5 times the following day, each washing process lasting 5 minutes. 150 µl of the secondary antibody (1:200) diluted with 1% BSA was then added to each slide after the slides were blocked at room temperature with 200 µl 1% BSA for 15 minutes. Next, we washed the slides 5 times with 300µl PBS for 5 minutes each time. 150 µl Hoechst33258 stain (C1011 Beyotine, China) was added to each slide in a dark environment and incubated for 30 minutes at room temperature. Finally, we observed the cells under a fluorescence microscope, photographed, and stored them.
A conventional laser scanning microscope can quantify fluorescently labeled tissues and display fluorescence changes in the direction of Z-axis. ImageJ software was used for photography and Prism Demo software for data statistics.
RT-PCR
Intracellular total RNA extraction: Pancreatic enzymes were used to digest the cells that were collected after centrifuging the liquid in the flask. Next, equal volumes of Trizol lysate were added to enable the collected cells to split and decompose. The schizolytic cells were then transferred into another tube without RNA enzymes, and 200 µl pre-cooling chloroform was added per milliliter of Trizol, and the mixture was centrifuged for 15 minutes. The supernatant was absorbed after centrifugation and transferred into another tube without RNA enzymes, where an identical volume of isopropanol was added to the tube, followed by another centrifugation. This later centrifugation yielded RNA sediments that were preserved in a -20℃ surrounding for 30 minutes. The sediments were washed with 75% ethyl alcohol and centrifuged for 5 minutes, and the supernatant was discarded after washing and centrifuging the sediments twice.
cDNA synthesis: The reverse transcription system was prepared using a reverse transcription kit according to instructions provided in the protocol of the kit.
Polymerase chain reaction:
1. Primer synthesis: The primers for reverse-transcription PCR were designed and synthesized using the Primer Premier 5.0 software. The internal reference was the housekeeping gene GAPDH.
2. Real time-PCR reaction system: the SYBR Green PCR kit was used to carry out procedures in the real-time PCR reaction system.
3. PCR amplification conditions: The real-time detector was used to detect the reaction, with the following temperature program was set: 94℃ for 10 minutes and 40 cycles of 94℃ for 20 seconds, 55℃ for 20 seconds, and 72℃ for 20 seconds.
Statistical analysis
Graphpad 8.0.2 and R 4.0.2 were used to perform statistical analysis. Expressed data were shown as mean ± SD. Student’s t test was used to evaluate the statistical significance of different 3 groups. P value less than 0.05 was considered as significant.