Doxycycline Up-regulates The Expression Of IL-6 And GM-CSF In Mouse Thymic Epithelial Cells And Preparation Of Monoclonal Antibody Against The Fragement Of Delete Rich Proline Domain Of Human FOXP3

PartⅠObjective To reveal the mechanism of doxycycline(Dox) up-regulating the expression of IL-6 and GM-CSF in mouse thymic epithelial cell line 1(MTEC1),and to build the foundation for the further exploration of its effect on immune reconstitution.Methods The effect of Dox on the mRNA and protein level of IL-6 and GM-CSF in MTEC1 was measured by Real-time PCR and ELISA assay.Then the protein expression of phosphorylated p65(p-p65) which is the key transcriptor of IL-6 and GM-CSF genes in MTEC1 cells was analyzed by western blot.To further evaluate the contribution of the p65 and MAPK/ERK signaling pathway to the up-regulated expression of IL-6 and GM-CSF by Dox,the cells,prior to the exposure to Dox,were pretreated with the p-p65 inhibitor BAY11-7082 and the ERK inhibitor U0126.The expression of IL-6,GM-CSF and p65 phosphorylated level of MTEC1 cells were analyzed by ELISA,Real-time PCR,and western blot respectively.Results The mRNA and protein level of IL-6 and GM-CSF were found to be up-regulated in a time- and dose-dependent manner in MTEC1 cells treated by Dox.Furthermore,the level of p-p65 in MTEC1 cells was up-regulated by Dox,but it was largely abolished by p-p65 inhibitor and the ERK inhibitor.These data suggest that Dox could be able to up-regulate IL-6 and GM-CSF expression via p65 and ERK pathways in MTEC1 cells,at least partly.Conclusion Dox up-regulates the expression of IL-6 and GM-CSF via NF-κB and MAPK/ERK pathways in MTEC1.Thymus is an important organ responsible for the generation and maturation of T cells. Thymic epithelial cells(TECs) support T cell development by production of cytokines and chemokines that regulate the proliferation and migration of thymocytes in a differentiation-dependent manner.In a word,our present study discoves another bioactivity of Dox and it may have some contribution to the T cells immune reconstitution.PartⅡObjective To develop monoclonal antibodies(mAb) against human delete rich proline domain of FOXP3 protein(ΔPRD-hFOXP3) and for the further research about the function of FOXP3.Methods Balb/c mice were immunized with purified recombinantΔPRD-hFOXP3 protein which expressed by E.coli Rosetta(DE3).Once the antibody titer in serum bleeding from mouse tail vein reached 1:10~4,a fusion of mouse splenocytes with Sp2/0 myeloma cells were performed. Further,the antibodies againstΔPRD-hFOXP3 protein secreted by hybridoma cell lines were screened by indirect enzyme linked immunosorbent assay(ELISA),and cloned by limiting dilution method. In the end,the specificity and titers of mAb againstΔPRD-hFOXP3 protein were identified and tittered by ELISA and Western blot.Results After cell fusion and subclone,the two hybridoma cell lines were established and could stably secrete mAb againstΔPRD-hFOXP3 protein and were named after 1A2和3A11 which isotypes are IgG1.The mAb titers from the mouse ascites were more than 1:10~5 with ELISA assay.Using SDS-PAGE and western blot,the mAbs were confirmed by theΔPRD-hFOXP3 protein.Conclusions Two mAbs can recognize theΔPRD-hFOXP3 protein specifically.The two antibodies can be a useful tool for the further research of the biological properties of human FOXP3 protein.