Related Research On MiR-127-5p-mimic And MiR-99a-inhibitoror Coordinately Promote Chondrogenic Differentiation Of Adipose-Derived Stem Cells

Objective: 1.Isolation and culture of brown adipose stem cells(BADSCs)and white adipose stem cells(WADSCs),identify two cells and study the growth dynamics of both cells.2.To compare the in vitro osteogenesis and cartilage ability of BADSCs and WADSCs,and provide evidence for further research and clinical application of adipose-derived stem cells.3.To study the role of different miRNAs in cartilage differentiation of ADSCs,and the synergistic effects of different miRNAs on the chondrogenic differentiation of ADSCs,and to explore its mechanism of action.Methods: 1.The brown fat under the scapula of SD rats and the white adipose tissue at the groin were isolated to obtain BADSCs and WADSCs.The morphology of the cells was observed by an inverted microscope,the cell count was used to detect the proliferative ability,and the cell surface antigen marker was identified by flow cytometry.2.The passaged cells are subjected to adipogenic,osteogenic and chondrogenic induction.BADSCs and WADSCs were induced by oil red-O staining after osteogenic induction.After osteogenic induction,alkaline phosphatase(ALP)activity was detected and alizarin red staining was performed.After cartilage induction,acin blue staining was performed.qRT-PCR further detected the expression of genes related to adipogenesis,osteogenesis and cartilage.3.Time-dependent expression of miR-99 a and miR-127-5p-mimic was detected by qRT-PCR in the cartilage differentiation of ADSCs.Subsequently,miR-99a-inhibitor and miR-127-5p-mimic were delivered to ADSCs via lipofectamine 2000,and ADSCs delivering miR-99a-inhibitor were designated as M group.ADSCs delivering miR-127-5p-mimic were designated as N group,and ADSCs delivering miR-99a-inhibitor and miR-127-5p-mimic were designated as M+N group.Cells that were not treated served as a negative control(NC group).The amount of miR-99 a and miR-127-5p in ADSCs was detected by qRT-PCR 24 hours later,and the proliferation ability of the four groups of cells was examined by MTT assay.4.Four groups of ADSCs were induced by cartilage-inducing medium,and qRT-PCR was used to detect the expression levels of chondrogenic markers type II collagen(COL2A1)and aggrecan(ACAN)mRNA.Western Blot was used to detect the expression levels of COL2A1 and ACAN proteins.Results: 1.The BADSCs and WADSCs were isolated and cultured.After the cells were initially attached to the wall,the volume of WADSCs was larger than that of BADSCs.The surface antigen markers were identified by flow cytometry.Both of them were consistent with the characteristics of mesenchymal stem cells.The ability indicates that WADSCs are stronger than BADSCs.2.The ALP activity of BADSCs was greater at the same time point after osteogenic induction.And the area of calcium nodules formed at 5 weeks was larger than WADSCs.The expression of the osteogenic marker genes Runx2 and OCN is also relatively high.Both BADSCs and WADSCs have the ability to differentiate into cartilage.There was no significant difference in the depth of the acin blue staining between the two cell sections and the expression levels of the cartilage marker genes COL2A1 and ACAN were not significantly different.3.MiR-99a-inhibitor and miR-127-5p-mimic were delivered to ADSCs via lipofectamine2000.After 24 hours,qRT-PCR detected that the amount of miR-99 a was lower than that of NC group and miR-127-5p was higher than that of NC group.There was no significant difference in the proliferative capacity of the four groups of ADSCs detected.4.After induction of chondrogenic medium,the expression levels of COL2A1,ACAN gene and COL2A1,ACAN protein in miR-99a-inhibitor group and miR-127-5p-mimic group were higher than those in NC group.The expression levels of COL2A1,ACAN gene and COL2A1,ACAN protein in miR-99a-inhibitor+miR-127-5p-mimic group were higher than those in miR-99a-inhibitor group and miR-127-5p-mimic group.Conclusion: 1.This method can extract BADSCs and WADSCs more quickly and purify them.Both extracted BADSCs and WADSCs have strong proliferation ability and can be expanded into a large number of cells for experimental research.2.Both BADSCs and WADSCs have adipogenic differentiation ability,but WADSCs have strong adipogenic differentiation ability;BADSCs and WADSCs have osteogenic differentiation ability,but BADSCs have strong osteogenic differentiation ability;BADSCs and WADSCs have the potential of chondrogenic differentiation.And there is no significant difference in the ability of the two to differentiate into cartilage.3.MiR-99 a and miR-127-5p-mimic can play a regulatory role in the cartilage differentiation of ADSCs.MiR-99 a can inhibitor or negatively regulate the cartilage differentiation of ADSCs.miR-127-5p-mimic can promote positive regulation in cartilage differentiation of ADSCs.MiR-99a-inhibitor attenuates the negative regulation of miR-99 a and synergizes with the positive regulatory factor miR-127-5p-mimic to promote cartilage differentiation of ADSCs.