The Effects Of SGPL1 Protein On T Cell Development In Thymus Organs

PART Ⅰ DISTRIBUTION OF SGPL1 IN MOUSE THYMUSObjective: To investigate the distribution of sphingosine-1-phosphate lyase 1(Sphingosine-1-phosphate lyase 1,SGPL1)in mouse thymus.Methods: 4～6 weeks C57BL/6 mouse were sacrificed to get the thymus;Immunohistochemical staining was used to detect the distribution of SGPL1 in the structure of thymic cortex and medulla.Results: The results of immunohistochemical staining showed that SGPL1 was mainly expressed in sub-capsular zone and medulla.Conclusion: SGPL1 was unevenly distributed in mouse thymus,and was highly expressed in subcapsular zone and medulla.This result laid a foundation for further investigating the role of SGPL1 and S1 P signals in T cell development.PART Ⅱ CONSTRUCTION OF THYMIC ORGANS TO INDUCE T CELL DEVELOPMENT IN VITROObjective: Designed thymic organoids simulating the three-dimensional structure of thymus tissue in vitro,to induce hematopoietic stem progenitor cells(HSPCs)differentiate into T cells.Methods: The retroviral vector expressing the Delta-like ligand 1(Delta Like Canonical Notch Ligand 1,DLL1)of NOTCH1 and green fluorescent protein(GFP)were constructed,and the OP9-DLL1 cell line was established in OP9 cells with the aid of retroviral infection.The m RNA and protein level of DLL1 in OP9-DLL1 cells was validated by Quantitative Real-time PCR and Western blot respectively.Immunofluorescence assay was used to detect the DLL1 protein expression and distribution in OP9-DLL1 cells.HSPCs were extracted from E13.5fetal liver and bone marrow of C57 BL/6 mouse,and mixed with OP9-DLL1 cells in an appropriate ratio respectively,compacted by centrifuging and cultured at the air-liquid interface in medium.Fluorescence microscope was used to observe the growth of thymic organoids.Flow cytometry was used to detect the expression of T cell surface markers,CD3,CD4,CD8,CD25,CD44,CD45,CD117 and TCRβ.Immunofluorescence cytochemical staining was used to observe the distribution of hematopoietic cells in thymic organoids.Results: The retroviral vector expressing DLL1 and GFP was successfully constructed.The OP9 cells were infected with the retrovirus we constructed,and OP9-DLL1 cells were selected by GFP level.The m RNA and protein level of DLL1 in OP9-DLL1 cells were significantly increased,and DLL1 was expressed in the membrane OP9-DLL1 cells.Within 40 days of culture with induction of three-dimensional structure,the thymic organoids were in good condition and gradually increased in volume.The thymic organoids induced programmed differentiation of T cells,and HSPCs differentiated into CD3+ T cells.Conclusion: OP9-DLL1 cells are successfully used to construct thymic organoids and induce differentiation of HSPCs into T cells in vitro.PART Ⅲ THE EFFECT OF OVEREXPRESSION SGPL1 ON T CELL DEVELOPMENT IN VITROObjective: To study the effect of overexpression SGPL1 on T cell development in vitro.Methods: The retroviral vector expressing SGPL1 was successfully constructed;The OP9-DLL1 cells were infected by retroviral to get the OP9-DLL1-SGPL1 cells.Real-time fluorescence quantitative PCR(RT-PCR)and Western blot method were used to detect the m RNA and protein expression of SGPL1 in OP9-DLL1-SGPL1 cells respectively;HSPCs were extracted from C57 BL/6 bone marrow,and then mixed with OP9-DLL1 cells or OP9-DLL1-SGPL1 cells in appropriate proportion respectively,compacted by centrifuging and cultured at the air-liquid interface in medium.Fluorescence microscope was used to observe the growth of thymic organoids.Flow cytometry was used to detect the expression of T cell surface markers,CD3,CD4,CD8,CD25,CD44,CD45,CD117 and TCRβ.Results: The retroviral vector expressing SGPL1 was successfully constructed.The OP9-DLL1 cells were infected with the constructed retrovirus,and OP9-DLL1-SGPL1 cells were selected by hygromycin resistance.The m RNA and protein level of SGPL1 in OP9-DLL1-SGPL1 cells were significantly increased.The ratio of TCRβ cells was lower in the thymic organoid based on OP9-DLL1-SGPL1 cells than that of OP9-DLL1 cells on day 13 to 16 of culturing,by inducing with 3D structure of OP9-DLL1 cells.Conclusion: The OP9-DLL1-SGPL1 cells were successfully constructed by SGPL1 expression of retrovirus,and the thymic organs constructed by OP9-DLL1-SGPL1 cells were used to induce the development of T cells in vitro,and SGPL1 overexpression was observed to lead to a decrease the in ratio of TCRβ cells.