| Background:Tumor,trauma,infection,congenital deformity and other reasons can cause jaw defect,which not only adversely affects the patient’s chewing,speech and other functions,but also damages the beauty of the face,and gives patients psychological and social adaptation Caused obstacles[1],currently the main way to repair jaw defects in clinic is bone grafting,but the effect is not good,the amount of bone is in short supply and the side effects are large[2].Therefore,it is urgent to find a better repair method.Let-7a is a member of the let-7 family of micro RNAs.It has been extensively studied.Studies have shown that it can regulate the immunomodulatory function of BMMSCs[3],but there is no report on its regulation of osteogenic differentiation of stem cells at home and abroad.Objective:This experiment aims to explore whether let-7a can regulate the osteogenic differentiation of BMMSCs cell aggregates and participate in the repair of rat jaw bone defects.Methods:1.Cultivation and identification of BMMSCs and culture of cell aggregates:BMMSCs were isolated from the bone marrow cavity of SD rats and passed down after the density grew to 80-90%in complete medium.P3-P5 cells were used for identification and subsequent experiments;Identification includes flow cytometric identification of cell surface antigens,proliferation capacity,adipogenic osteogenic differentiation identification;cell aggregates are obtained after 7 days of culture in vitamin C complete medium,and gross and HE staining observations are performed.2.The effect of Let-7a on the osteogenic differentiation ability of BMMSCs cell aggregates in vitro:Divide into three groups（mi,in and nc）,respectively transfect BMMSCs cell aggregates with let-7a mimic,inhibitor and negative control at a certain concentration,Perform osteogenic induction and then perform in vitro osteogenic differentiation detection,such as real-time quantitative RT-PCR,Western blot detection of osteogenic related gene and protein expression,ALP staining and activity detection of early osteogenic differentiation,Alizarin red staining to observe the results The formation of mineralized nodules in the late stage of bone differentiation.3.The regulatory effect of Let-7a on the subcutaneous ectopic osteogenesis of BMMSCs cell aggregates in nude mice:Divide into three groups（mi,in and nc）,culture BMMSCs into cell aggregates,and perform cell transfection in the same way as in Method 2.The polymer and HA-TCP were combined and transplanted under the skin of nude mice.After 4 weeks,the materials were taken and fixed,decalcified,and sliced for HE staining for observation.4.Let-7a regulate BMMSCs cell aggregates to participate in the repair of rat jaw bone defects:Divide into four groups（mi,in,nc and blank control）,culture BMMSCs into cell aggregates,perform cell transfection in the same way as Method 2,and then The polymer is combined with HA-TCP to make a graft for later use.The rat jaw defect model was established,and the graft was implanted into the defect area,and the blank control group was not transplanted.At 1w,4w,and 6w,samples were taken and fixed,and micro-CT scans were performed,followed by decalcification and staining of sections for observation.Results:1.Successfully extract and complete the cultivation and identification of BMMSCs;2.Successfully cultivated BMMSCs cell aggregates;3.After let-7a mimic,inhibitor and negative control were transfected with BMMSCs aggregates,the Fas m RNA and protein expression levels were compared between the groups,the mi group was the lowest and the in group was the highest;the let-7a expression was compared between the groups,and the mi group was higher than the others The two groups rose significantly.It indicates that the transfection is successful and effective.4.Comparing the expression of osteogenic genes after transfection,the m RNA levels of OCN,OPG and RUNX2 in the mi group increased significantly,and the protein levels of OSX and RUNX2 increased significantly;the comparison of ALP staining and activity and Alizarin Red S staining between the groups showed that The mi group had the deepest ALP staining and the highest activity,and the in staining was the lightest,and the activity was the lowest,suggesting that let-7a can promote early bone formation in vitro;the mi group had the most mineralized nodules stained with alizarin red,and the in group had the least mineralized nodules.It is suggested that let-7a can promote late osteogenesis in vitro.5.The expression of extracellular matrix-related genes was compared between the groups after transfection.The m RNA expression of Col-1,Fibronectin and Laminin in the mi group increased significantly,the expression of Col-1 protein in the mi group increased significantly,and the expression of integrinβ1 protein increased significantly compared with the in group.let-7a promotes the expression level of extracellular matrix-related genes and proteins;there is no significant difference between the HE staining groups,suggesting that let-7a has no significant effect on the extracellular matrix of BMMSCs.6.Let-7a was transfected with BMMSCs polymer and implanted subcutaneously in nude mice.The comparison between HE staining groups showed that the mi group had the most osteocytes and bone lacunas,the new bone formation was the most,and the in group had the least effect,and the difference was significant;7.After Let-7a processed BMMSCs aggregates to repair rat jaw defects,the comparison between micro-CT and HE staining groups showed that the mi group had the highest repair completion rate than the other three groups,and the repair was basically completed at 6 weeks,followed by the nc group,in The group is only higher than the blank control group without grafts,the difference is significant.Conclusion:Let-7a can promote the osteogenesis of BMMSCs cell aggregates in vivo and in vitro,has a certain promotion effect on the gene and protein levels of extracellular matrix related genes of cell aggregates,and can improve the effect of in situ repair of rat jaw bone defects.The molecular mechanism remains to be further studied. |
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