Study Of Satb2 On Promoting The Neuronal Differentiation Of Rat Bone Mesenchymal Stem Cells

Research background and objective:Periodontitis is a common disease induced by microbial infection,which results in destruction of periodontal support tissue.Alveolar bone resorption is an important pathological manifestation and is the major cause responsible for loss of teeth.The treatment of periodontitis is mainly through the removal of plaque,tartar to achieve the purpose of controlling inflammation.But this method can not effectively achieve functional periodontal tissue regeneration,especially the regeneration of alveolar bone.Currently,periodontal tissue regeneration engineering is persistently developed and has attained certain achievement.However,there are still obvious disparity of tissue engineered bones compared with the periodontal bone tissue.The nerve plays an important role in the regeneration of bone tissue.There is a wide distribution of nerves in normal bone tissue,especially in the osteogenic active area.And a growing evidence has indicated that,the nervous system is closely correlated with bone regeneration,repair and remodeling.SATB2 is a nuclear matrix binding protein,thus playing a vital role in integrating genetic and epigenetic regulation.Research has verified that SATB2 plays a critical part in osteoblast differentiation and bone tissue development.In addition,it is also the major regulator of callosal neurons in the middle-layer of cerebral cortex,which exerts important function in brain neuronal axon development.Research on SATB2 in the nervous system development has aroused increasing attention from scholars.Base on the important role of SATB2 in skeletal and neural development,we aimed to study the influence of SATB2 on the neural differentiation of rat bone mesenchymal stem cells(BMSCs),and to further determine the role of SATB2 in neural differentiation,which could provide theoretical foundation for comprehensively understanding the role of SATB2 in osteogenic and neural differentiation.Materials and methods:Part 1.BMSCs were isolated from the femurs and tibiae of Wistar rat,and cell culture and passage were performed through adherent isolated culture.The MSC surface markers on the P3 generation cells were detected through flow cytometry,and the multipotential differentiation of BMSCs was detected through osteogenic and adipogenic induction.Part 2.Construct the lentiviral vector pLVX-IRES-PURO-satb2 and finally obtaining bone marrow mesenchymal stem cells stably expressing satb2.Afterwards,SATB2 expression in BMSCs was detected using RT-PCR and Western blotting.Part 3.Fibroblast growth factor(bFGF)was used to induce the neural differentiation of BMSCs,meanwhile,the mRNA and protein expression of neuroblastic related indexes neuron-specific enolase(NSE)and nerve growth factor(NGF)in BMSCs with high SATB2 expression was detected on day 3 and day 5.Results:1.Rat BMSCs cultures were adherent,displayed a spindle-like shape and grew in a vortex manner.Flow cytometry discovered that,CD29(98.6%),CD44(98.8%)and CD90(99.0%)were expressed on P3 generation cell surface,while CD45(0.16%)and CD11b(0.12%)were not expressed.7 days after osteogenic induction,ALP staining suggested that,bluish violet granulate region could be observed under microscope,and red-stained round nodules occurred 28 days after induced alizarin red staining.Besides,red-stained lipid droplet vesicles could be observed under microscope upon oil red O staining 14 days after adipogenic induction.2.The lentiviral vector pLVX-IRES-PURO-SATB2 was successfully constructed,and rat BMSCs stably expressing SATB2 were constructed by means of lentiviral infection.The SATB2 protein and mRNA levels in experiment group were markedly higher than those in control group upon Western blotting and RT-PCR.3.Obvious neuronal morphological changes could be seen in the rat BMSCs on day 2,4 and 7 after bFGF induction;in addition,the cell bodies shrunk and axon-like analogues appeared.NGF and NSE protein expression in the induction group on day 4 and 7 was distinctly higher than that in the wild type group.Moreover,it was found during the induction of rat BMSCs with high SATB2 expression that,the high expression group was associated with a higher proportion of cells with neuronal changes and more distinct cell morphological changes.Furthermore,NGF and NSE expression on day 3 and 5 detected by RT-PCR and Western blotting suggested that,the expression of related indexes in the experiment group was higher than that in the control group.Conclusion and significance:Our pilot study finds that,the expression levels of nerve-related indexes NGF and NSE in the high SATB2 expression group are higher than those in the control group.Meanwhile,cell morphological changes in the former are more distinct,and SATB2 can promote the neuronal differentiation of rat BMSCs.SATB2 plays a positive role in the neural differentiation of rat BMSCs,which can provide the theoretical foundation for comprehensively understanding the role of SATB2 in bone regeneration.