Construction Of Lentivirus Expression Vector Of KyoT2 The Effect Of Flt3L On DC Drived From Peripheral Blood Mononuclear Cell

The Notch signaling pathway was originally discovered through genetic studies in the fruit ?y Drosophila melanogaster in 1919, and the name derives from the effect that some Notch alleles create notched wings. Notch can be found in all metazoans ,including vertebrates and invertebratas. And it is remarkably conserved in evolution. Notch signaling plays a critical role in cells divisions, differentiation, and growth in many tissues,it controls cell's fate. It appears reasonable to assume that deregulation of Notch function is involved in the genesis of different types of cancers.There is a close relationship between the over-activation in Notch and many malignancy neoplastic hematologic disorders'happened. Human T-cell lymphoblastic leukemias and lymphadenoma are one of them.Activating mutations NOTCH1 are found in over 50% of human T-cell lymphoblastic leukemias (T-ALLs). These mutations are caused by the t(7;9)(q34;q34.3) chromosomal translocation. The result of this chromosomal translocation is some of the heterodimerization domain mutations potentiate the dissociation of an engineered soluble form of the heterodimer, mimicking the biological effects of ligand-in-duced Notch signaling, these imply that heterodimer dissociation leads to ligand-independent, constitutive cleavage of NTM to produce active NICD.So theγ-secretase inhibitor has no effect on some T-ALL cases. But the LIM domain protein KyoT2 could negatively regulates the Notch signaling pathway through interaction with RBP-J, which is the core element of the Notch signaling pathway in the nucleus.We assummed that if we could suppresse the growth and survival of NOTCH1-mutation T-ALL cells through blockade of NOTCH pathway with KyoT2.We constructed the lentivirus expression vector of KyoT2,wish to escape from corruption practicein the conventional transfection methods to suspend cells.These provided new vectors for further studies on the relationship between T-cell-type acute lymphatic leukemia and Notch signaling pathway.Flt3L is a transmembrane protein similar to SCF and M-CSF, both in size and structure. It is ligand for tyrosine kinase receptors and plays a role in regulating the proliferation and differentiation of cells in the hematopoietic system by binding to and activating the FLT3 receptor (FLT3R). It was confirmed that Flt3L played a notable effect on the generation, differentiation, mature of dendritic cell ,natural killer cell and cytotoxic T lympho-cyte.And it also has an important influence in hematopoiesis and immunomodulation. DC are reported to be an important potent of APC, characterized by apparent constitutive ability to present Ag to naive T cells in an immunostimulatory manner.Studies have found numbers of dendritic cells (DC) in various organs increased after injection administration of ?t3L into mices, including the absolute number of CD11c+ DC in spleen and lymphonode. there are also studies show, a local application on bronchus resulted a significant increase of DC, which had a stimulatory effect on local Ab production.But many studies about FLT3 was done in vivo or effected on the hematopoietic stem cell, there are few studies have been reported in the effect of Flt3L on differentiation and maturation of DC derived from peripheral blood mononuclear cell.My experiments were included as follows:One Construct lentivirus expression vector of KyoT2 and make elementary identification.Two Disassociation peripheral blood mononuclear cells from human fresh blood with Lymphocyte Separation Medium,cultured with GM-CSF,IL-4,flow cytometry analysis of cell surface antigen CD11c, CD83,HLA-DR expression.Conclusion:1 We constructed the lentivirus expression vector of KyoT2,which provide new vectors for further studies on the relationship between T-cell-type acute lymphatic leukemia and Notch signaling pathway.2 With flow cytometry analysis of dendritic cell surface antigen expression,we found Flt3L can enhance the expression of cell surface antigen CD11c, CD83,HLA-DR ,and promote the maturation of DC, but it could not affect the proliferation of DC.we think these could provide some references for tumour'biotherapy.