Effect Of Plexin-A1 On Neural Differentiation Of Dental Pulp Stem Cells

Objectives:Plexins,are membrane proteins,are major receptors of signaling hormone,which play an important role in multiple systems.Plexin-A1,one of the plexins,plays a key role in nervous system,immune system,bone homeostasis,cardiovascular system,etc.,but few reports have been made on its oral studies.Our previous studies have found that plays a positive role in the odontoblastic differentiation of human dental pulp stem cells,Plexin-A1 is spatio-temporal specifically expressed in the process of mouse tooth development.This study aims to investigate whether Plexin-A1 plays a regulatory role in the neural differentiation of human dental pulp stem cells through in vivo and in vitro experiments in order to lay a certain foundation for the realization of clinical dental pulp repair and regeneration.Materials and Methods:1.Hematoxylin-eosin staining was used to observe the histological morphology of molars at different time points after birth,and immunofluorescence double staining was used to investigate the expression of Plexin-A1 and Nestin,a nerve marker,during the development of pulp nerve in mice.2.Human pulp cells were isolated and cultured by tissue block-enzyme digestion.Human dental pulp stem cells were obtained by monoclonal method.After induction,the lipid differentiation ability was detected by oil red O staining.After osteogenic induction,alizarin red staining was used to detect osteogenic differentiation ability.Stem cell surface markers were detected by flow cytometry.3.Human dental pulp stem cells were cultured in complete medium and neural induction medium for 3 days,and their morphological changes were observed by microscope.After 3,7 and 14 days of culture,mRNA expression levels of neuroassociated markersβⅢ-Tubulin and glial fibrillary acidic protein were detected by Quantitative real-time polymerase chain reaction,and after 3 and 7 days of culture,protein expression levels of βⅢTubulin were detected by Western blot.The selected neural induction method was preliminarily verified.4.Human dental pulp stem cells were transfected with lentivirus to endogenously inhibit the expression of Plexin-A1,and the transfected human dental pulp stem cells were cultured in nerve induction for 3,7,14 days.mRNA and protein expression levels of neuro-associated markers βⅢ-Tubulin,GFAP and Nestin were detected by qRT-PCR and Western blot,respectively.To explore the regulatory effect of Plexin-A1 on neural differentiation of human dental pulp stem cells.Results:1.HE staining showed the histological structure of mouse molars,and immunofluorescence results showed that Plexin-A1 was co-expressed with Nestin during the development of dental pulp nerve in mice.2.The primary human dental pulp cells were successfully cultured by tissue block-enzyme digestion.The cells adhered to the wall and showed a spindle shape.Human dental pulp stem cells were obtained by monoclonal method.After 28 days of lipid induction,oil red O staining showed the formation of orange-red lipid droplets.After 21 days of osteogenic induction,alizarin red staining showed obvious mineralization nodules.Stem cell markers CD44 and CD90 were detected by flow cytometry.3.After neural induction of human dental pulp stem cells,the morphology of the cells changed from long spindle to multipolarization,and immunocytochemical staining showed positive expression of nerve related markers GFAP.Compared with the normal culture group,mRNA expression levels of βⅢ-Tubulin and GFAP were significantly increased after 3,7,and 14 days of nerve induction,and protein expression levels of βⅢ-Tubulin were significantly increased after 3,7 days of nerve induction.4.Lentivirus transfected human pulp stem cells inhibited the endogenous expression of Plexin-A1,and the mRNA and protein expression levels of transfected human pulp stem cells decreased after 3,7,and 14 days of culture in the nerve induction solution,includingβⅢ-Tubulin,GFAP,and Nestin.Conclusion:1.Plexin-A1 may be related to the development process of dental pulp nerve in mice.2.The human dental pulp cells with stem cell characteristics were successfully cultured by tissue block-digestion.3.The neural induction method selected in this study has a good induction effect on human dental pulp stem cells.4.Lentivirus transfection successfully inhibited the endogenous expression of Plexin-A1 in human dental pulp stem cells,and Plexin-A1 played a positive regulatory role in neural differentiation of human dental pulp stem cells.