Investigation The Function Of PBEF Induces Myeloid Differentiation Of CD34～+ Cells From Healthy Individuals And Patients With Severe Congenital Neutropenia

Objective:To investigate the function of PBEF induces myeloid differentiation of CD34+ cells from healthy individuals and patients with severe congenital neutropenia.Methods:1.Using laser-assisted single-cell picking and quantitative real-time PCR technique, we analyzed the expression of PBEF gene in different hierarchy myeloid cells (myeloblasts, promyelocytes, myelocytes,, metamyelocytes) and mature granulocytes from bone marrow of healthy individuals.2.To evaluate the relationship between PBEF production and the stimulation of G-CSF in healthy individuals . We analysed PBEF mRNA and protein (ELISA method) expression in CD33+ bone marrow myeloid progenitors and polymorphonuclear leucocyte(PMNs) from healthy volunteers treated with G-CSF or their cells treated in vitro experiments, and also measured NAD+ in the cells.3. The ability of PBEF to induce myeloid differentiation of healthy individuals was assessed by in vitro experiments:①. Treatment of CD34+ bone marrow progenitors with recombinant human PBEF, FACS analysed CD11b and CD15 expression on the cells , quantitative real-time PCR analysed C/EBPa, C/EBPε, PU.1 and ELA2 mRNA expression of the cells, analysed cells morphology using cytospin's staining with hematoxilin-eosin, and also measured NAD+ in the cells.②. After lentiviral transduction of CD34+ cells from the healthy individuals,...