Effect Of Mimic-micro503 And Inhibitor-micro144 Combined Transfection On Osteogenic Differentiation Of Adipose-derived Stem Cells

Objective1.Primary cell bone marrow mesenchymal stem cells and adipose stem cells were compared by proliferation ability,osteogenesis and adipogenesis differentiation ability,selecting suitable stem cells as the research cells of this experiment.2.The expression of miR503 and miR144 in adipose stem cells was observed under osteogenesis induction environment.3.Detection of mimic-miR503,inhibitor-miR144 promoted osteogenesis differentiation of adipose-derived stem cells,and mimic mimic miR503,combined with inhibitor-miR144,has a synergistic effect on osteogenesis differentiation of adipose-derived stem cells.MethodSD rat primary BMSCs and ADSCs were extracted,the morphology of the cells was compared by microscope;cell phenotype was detected by flow cytometry;P3 generation cell count was used to detect cell proliferation ability and growth curve was drawn;osteogenesis induction of ALP staining and alizarin Red staining and adipogenesis induction were induced by oil red O staining.qRT-PCR was used to detect the osteogenesis and adipogenic differentiation of the two groups of cells.P3 generation ADSCs osteogenesis induction group and blank control group were used to detect the relative expression of miR503 and miR144 by qRT-PCR.The miRNA database Target Scan(http://www.target scan.org)predicts miRNA503-5p and miRNA144-5p related targets,and miRNA503-5p targets osteogenesis genes including: WNT3?,ATK3,SMAAD7,WNT4,miRNA144-5p target The osteogenesis genes mainly include: TFG,SMAD6,SMAD5,SMAD1,WNT2,WNT5?,WNT7?,BMP10,MMP13 and the like.In vitro transfection of Mimic-miR503(group M),Inhibitor-miR144(group I),Mimic-miR503 and Inhibitor-miR144 mixture(M+I group),and experimental control group(NC group)to achieve over expression of miR503 and low miR144 Expression;cell flow cytometry was used to detect the cell phenotype of ADSCs;MTT was used to detect the proliferation ability of four groups of ADSCs and the relevant growth curves were drawn;ALP staining,qRT-PCR and Western Blot were used to detect the osteogenic markers of each group.ResultsBoth BMSCs and ADSCs could be stably passaged to P10 generation,and the cell morphology was uniform and stable.Both of them had osteogenesis and adipogenesis differentiation ability.Cell proliferation experiments showed that the proliferation of P3 ADSCs was stronger than that of BMSCs(P<0.05).QRT-PCR showed that the expression of miR503 was increased by 50-fold and the expression of miR144 was decreased by 2 fold compared with the blank control group.The intensity of ALP staining was stronger in M group,I group and M+I group than in NC group(116.623±3.02%,117.9753±2.97%,141.2263±3.59%).Compared with group M and group I,the ALP staining intensity of M+I group was stronger(25.7230±2.73%,24.5267±2.53%).qRT-PCR detected osteogenic related genes,and the expression levels of all osteogenic related genes(RUNX2,BSP,OCN)in M group,I group,and M+I group were significantly higher than those in NC group,M+ The expression levels of osteogenic related genes(RUNX2,BSP)in group I cells were significantly higher than those in group M and group I,and there was no significant difference between group M and group I.ConclutionsCompared with BMSCs,ADSCs have a wide range of sources,strong proliferative capacity,and osteogenic differentiation ability.ADSCs can be the research cells for osteogenic differentiation.miR503 and miR144 may be involved in the regulation of osteogenic differentiation of ADSCs.The M+I composition was stronger than the M group and the I group,and the ability to transfect the bone was stronger than that of the negative control NC group.Mimic-miR503 activates the canonical Wnt/?-catenin pathway and Inhibitor-miR144 activates the SMAD signaling pathway to promote osteogenic differentiation of ADSCs.Mimic-miR503 and Inhibitor-miR144 synergistically promote bone differentiation.