SEL1L, The Negative Regulating Factor Of Notch Signaling, Might Affect The Apoptosis Of Mouse Incisor Cervical Loop Epithelial Cells

After the accomplishment of crown development, the tooth root begin todevelop because of HERS. The specific molecular mechanisms of tooth rootdevelopment is still under research. The incisors’ labial root apical of murineanimals form cervical loop stem cell niches. The epithelial stem cells candifferentiate into ameloblasts and form enamel which make the incisorspersistently grow during the whole life. The molars of murine animals formHERS and tooth root identical with the men’s teeth. So murine animals’ incisorsare often used to investigate the molecular mechanisms of cervical loop stemcell niches maintenance and the initiation of tooth root development.The Notchsignaling contribute to the maintenance of cervical loop stem cell niches. SEL1L,the negative regulating factor of notch signaling,was strongly expressed in themolars but weakly expressed in the incisors. We conclude that SEL1L is animportant influencing factor of tooth root development.In this study, we used primary cell culture, immunohistochemistry,celltransfection and RT-PCR to identify the SEL1L expression in primary culturedincisor cervical loop cells and its effect on cell apoptosis.Then we can demonstrate that whether SEL1L inhibits the maintenance of cervical loop stemcell niches by enhancing cell apoptosis and promotes the formation of HERSand tooth root.Part1: Primary culture of mouse incisor cervical-loop epithelial cellsby explants with enzymatic digestionWe isolated the labial cervical loops of PN7-8d mouse incisors. Afterenzymatic digestion with collagenaseⅠand dispaseⅡ the tissue masses wereused to culture primary cells. The fibroblast cells were removed by digestingwith trypsin. Immunostaining with antikeratin antibody is used to characterizethe cell lineage. The primary cells consisted of spindle fibroblast cells andpolygonal epithelial cells. After trypsin digestion we can obtain purifiedepithelial cells which were positive for cytokeratin. The successful culture ofincisor cervical loop epithelial cells is very important for the followingexperiments.Part2: The identification of pEGFP-N2-SEL1L recombinant plasmidand cervical loop cells transfectionpEGFP-N2-SEL1L recombinant plasmid was transformed into competentcell of DH5α. The target gene PCR, restriction enzyme digestion and genesequencing were used to identify the plasmid. Then the pEGFP-N2-SEL1Lplasmid was transfected into the cervical loop cells by lipofectamine2000.Culturing for16-18h we can get single bacterial colony, they were selected outand cultured with LB medium containing kanamycin. The extracted-plasmidwas appeared as corresponding DNA band(s) by PCR and restriction enzyme digestion and was also identified by DNA sequencing. After24h posttransfection we can detect the expression of green fluorescence by invertedphase contrast microscope.Part3: The identification of pEGFP-N2-SEL1L recombinant plasmidtransfected cervical loop cells and cell apoptosisThe expressions of SEL1L,Notch1and Hes1were detected with RT-PCR tojudge if the SEL1L of transfected cervical loop cells could successfully function.Effects of SEL1L on cell morphology changes and expressions of Caspase-3andBcl-2were investigated by DAPI staining and RT-PCR. The recombinantplasmid group show more apoptosis cells, higher expression of Caspase-3andlower expression of Bcl-2mRNA compared with the blank plasmid group.In the end we can conclude that SEL1L may inhibit maintenance of stemcells niches and initiate the tooth root development by promoting the apoptosisof cervical loop cells.