The Interactive Dialogue Between Notch And MTOR Promotes The Study Of The Mechanism Of Thymic Epithelial Cell Aging

Objective: The mouse thymic epithelial cell line 1(mTEC1)was used to investigate the role and mechanism of Notch signaling and mTOR signaling in thymic degeneration and senescence.It may be helpful for the further elucidation of the mechanism of thymic aging and the prevention and treatment of T cell immunodeficiency caused by thymus degeneration and senescence.Methods:1.Bone marrow-derived dendritic cells(BMDCs)were induced by GM-CSF+IL-4,and activated by LPS.The Jagged1 and maturation surface markers were detected by flow cytometor.The mTEC1 cells were co-cultured and transwell cultured with activated BMDCs.The cell senescence markers,including ?-galactosidase stainning and p21 were used to evaluate the senescence of mTEC1.2.The mTEC1 cells were co-cultured with the Jagged1 recombinant protein and treated with the Notch inhibitor DAPT,or transfected with the Notch3 intracellular activation fragment NICD3 lentiviral vector.The cell senescence markers,including ?-galactosidase stainning and p21 were used to verify that the activation of Notch signaling during the process could promote senescence of mTEC1.3.In the aging system induced by Jagged1/Notch signaling activation,shRNATSC2 lentivirus infection was used to activate mTORC1 signaling or shRNA-Raptor and Rapamycin were used to inhibit mTORC1 signaling in mTEC1 cells.The changes of Notch signal,Foxn1 and senescence markers,including ?-galactosidase stainning and p21 were detected.4.The shRNA-Rictor lentiviral vector transfecting mTEC1 cells were used to verify whether the mTORC2 signaling was involved in the Jagged1-induced mTEC1 senescence.5.The changes of mTOR signaling,Foxn1 and senescence markers,including p21 and ?-galactosidase staining were detected after the mTEC1 cells were transfected with NICD3 lentiviral vector or treated with DAPT.Results:1.The senescence of mTEC1 cells were induced when co-cultured with activated BMDCs but not in the transwell culture system.2.The p21 level of mTEC1 was increased after Jagged1 recombinant protein cocultured with mTEC1.It was demonstrated that the senescence of mTEC1 could be induced by the activation of the Jagged1/Notch signaling.The senescence of mTEC1 was reversed by Notch inhibitor DAPT.The p21 level of mTEC1 was also increased after transfected with NICD3 lentiviral vector.3.In the Jagged1/Notch activation-induced senescence system,the expression of Hes1 and p21,and the positive rate of ?-galactosidase staining were increased after the mTORC1 signaling of mTEC1 activated of by shRNA-TSC2.The senescence of mTEC1,induced by Notch activation,was reversed by suppressing the mTORC1 signaling of mTEC1 via transfected with shRNA-Raptor or treated with rapamycin.4.There was no effect on the Jagged1/Notch-induced senescence of mTEC1 when the mTORC2 signaling of mTEC1 was inhibited by shRNA-Rictor.5.The mTEC1 cells were senescent significantly after the Notch singaling activated via transfected with NICD3 lentiviral vector and the phosphorylation levels of mTOR,p70s6 k of mTEC1 were also increased.It suggested that Jagged1 could induce mTEC1 senescence by activating Notch signaling by binding to Notch3 ligand.The cell senescence was reduced by treated with DAPT,and the phosphorylation levels of mTOR and p70s6 k were down-regulated.It suggested that the mTORC1 signaling was also regulated by activation of Notch signaling.Conclusion: The Notch signaling,activated by Jagged1/Notch,induces cell senescence of the thymic epithelial cells,and mTORC1 and Notch signals have positive regulation with each other during this process.