Effect Of Platelet-rich Plasma On Osteogenic Differentiation Of Human Adipose Stem Cells And Its Mechanism

Objective: Human adipose stem cells(hADSCs)are ideal seed cells for the regeneration of alveolar bone defects due to their easy access and high proliferation ability.Platelet-rich plasma(PRP),rich in growth factors,could promote tissue repair.The purpose of this study was to observe whether PRP could promote the osteogenic differentiation of hADSCs and to carry out high-throughput sequencing in order to explore the mechanism.Methods: 1.The PRP was prepared,and the immunophenotype of hADSCs was identified by flow cytometry,and multiple differentiation abilities of hADSCs were identified.2.HADSCs were divided into three groups for culture:negative control group(CON group,Serum-free Cell Culture Medium containing 10%FBS),positive control group(OM group,human adipose-derived stem cell osteogenic differentiation medium)and experimental group(PRP group,human adipose-derived stem cell osteogenic differentiation medium containing 5%PRP).3.CCK-8 method was used to detect the proliferation ability of hADSCs in the three groups at 24,48 and 72 h.4.On the 14 th day,the osteogenesis was detected by alizarin red staining,and the expression of the osteogenic transcription factor Runx2 was detected by qRT-PCR.5.On the 14 th day,total RNA of the OM group and PRP group was extracted for high-throughput sequencing.Target gene prediction and miRNA/mRNA combined analysis were performed for the differentially expressed osteogenic related miRNAs,and qRT-PCR was used to verify the expression of differentially expressed osteogenic related genes.Results: 1.The hADSCs used in this experiment met the identification criteria.2.The results of CCK-8 method showed that compared with the CON group,both OM group and PRP group could enhance the proliferation ability of hADSCs,and the differences at48 h and 72 h were statistically significant(P<0.05),while there was no significant difference between OM and PRP groups(P>0.05).3.Alizarin red staining showed that more calcium nodules were formed in PRP group than in CON group.4.QRT-PCR results showed that the expression level of Runx2 in PRP group was significantly higher than that in OM group,and the difference was statistically significant(P<0.05).5.Through miRNA sequencing and mRNA sequencing analysis,the differential miRNA expression profiles and the differential mRNA expression profiles of PRP promoting osteogenic differentiation of hADSCs were obtained.6.Mi RNA/mRNA combined analysis and qRT-PCR verification results showed that compared with the OM group,"hsa-miR-212-5p was down-regulated while CNR1 up-regulated" and "hsa-miR-495-3p＿r +1 was down-regulated while CNR1 up-regulated" in the PRP group.Conclusions: 1.PRP could promote the osteogenic differentiation of hADSCs.2.The down-regulation of hsa-miR-212-5p,hsa-miR-495-3p＿r +1 and up-regulation of CNR1 may be involved in the process of PRP promoting the osteogenic differentiation of hADSCs.