| The treatment of large bone defects caused by trauma,degenerative diseases,tumors,infections and surgery is a great challenge in clinical practice.There are many problems in autologous and allogeneic bone transplantation which restrict its wide application in clinic.Tissue engineering provides a new idea for bone defect repair and reconstruction.Tissue engineering usually consists of cells,growth factors,and material scaffolders,among which an important component is the seed cell with the potential for multiple differentiation.Bone marrow mesenchymal stem cells(BMSCs)are often used in the treatment of bone defects as ideal bone tissue engineering transplantation cells.Improving the osteogenic differentiation ability of BMSCs will better stimulate the formation of new bone.Long non-coding RNA(lnc RNA)plays an important role in cell metabolism regulation,but the role of most lncrnas in BMSCs osteogenic differentiation is not well understood.In this study,lncrna highly expressed in osteogenic differentiation of m BMSCs were screened as research objects by high-throughput sequencing,and the regulatory role of target lncrna in osteogenic differentiation was systematically studied,providing a new idea for faster and better promotion of bone formation.The main research contents are as follows:(1)Screening of differentially expressed lncrnas during osteogenic differentiation of m BMSCs.In this study,the osteogenic differentiation of m BMSCs was induced firstly,and the m BMSCs induced produced more mineralized nodules and alkaline phosphatase,and the expression of osteogenic genes was increased,indicating that the osteogenic differentiation of m BMSCs was successfully induced.Then RNA samples of m BMSCs induced by osteogenic differentiation for 14 days were subjected to high-throughput sequencing to screen the differentially expressed lncrnas of m BMSCs during osteogenic differentiation.There were1,933 differentially expressed lnc RNA,among which 1,260 genes were up-regulated and 673 genes were down-regulated.Gm16263,Gm35782 and Lncpint were selected as the initial research objects,and the effects of these three lnc RNA on osteogenic differentiation were explored by using overexpression eukaryotic vectors.The overexpression of Gm16263 significantly promoted the expression of osteogenic genes OPN,OCN,Runx2,ALP and Col-I,while the overexpression of Gm35782 and Lncpint had no obvious influence on the expression of osteogenic genes.(2)Study on the regulation of Gm16263 on osteogenic differentiation.After screening,Gm16263 was confirmed as the research object.The expression of Gm16263 was higher on day 7 of osteogenic differentiation,and it was located to play a role in the cytoplasm.Continuous overexpression of Gm16263 resulted in the secretion of more mineralized nodules and alkaline phosphatase on days 7 and 14,and the expression levels of osteogenic related proteins Col-I and Runx2 increased.On day 7,the expression levels of osteogenesis related genes OPN,Runx2,ALP and Col-I were significantly increased,and the expression level of the early marker enzyme ALP gene was increased by about 9.4 times.On day 14,the expression levels of osteogenic related genes OPN,OCN and Runx2 were significantly increased,among which the expression level of OCN gene,a landmark protein in late osteogenic differentiation,was increased by about 2.3 times.When Gm16263 was knocked down,the expression levels of osteogenic related genes Runx2,ALP,Col-I and Osx were significantly decreased,and the expression levels of osteogenic related proteins Col-I and Runx2 were decreased.Then,the effect of Gm16263 on target genes was analyzed according to the prediction results of target genes in high-throughput sequencing.When Gm16263 was overexpressed,the gene expression level and protein expression level of Taok3 increased.When the expression of Gm16263 was knocked down,the gene expression level and protein expression level of Taok3 decreased.Gm16263 can promote osteogenic differentiation of m BMSCs by promoting the expression of Taok3.(3)The effect of F127 coated with Gm16263 m BMSCs on bone formation.F127 was selected as the carrier to encapsulate cells.F127 is injectable,has a reversible gelation mechanism,and has good biocompatibility.The osteogenic ability of Gm16263 in vivo was evaluated using a mouse skull 4.4 mm critical defect animal model.The F127 group with Gm16263 m BMSCs overexpressed generated more new bone.In this study,Gm16263,which was highly expressed during the osteogenic differentiation of m BMSCs,was screened by high-throughput sequencing.Gm16263 promoted the osteogenic differentiation of m BMSCs by promoting the expression of Taok3,and its application in bone defect repair promoted bone formation,which is expected to provide a new idea for bone defect repair. |
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