【作者】 蓝丹；

【导师】 经承学； Karl Welte；

【作者基本信息】 广西医科大学 ， 儿科学， 2006， 博士

【摘要】 目的： 研究前B细胞集落促进因子(Pre-B-cell colony-enhancing factor，PBEF)诱导正常人以及重型先天性中性粒细胞减少症患者(severe congenital neutropenia，SCN)的CD34~+细胞向髓系细胞分化功能。 方法： 1、利用激光辅助骨髓涂片单个细胞采集技术及逆转录实时定量PCR方法检测正常人骨髓中不同分化阶段髓系细胞PBEF表达水平。 2、用逆转录实时定量PCR及ELISA方法检测正常人的髓系细胞经粒细胞集落刺激因子(granulocyte colony-stimulating factor，G-CSF)体内、外刺激后PBEF mRNA表达水平、细胞培养液或血浆中PBEF蛋白质水平，同时检测细胞烟酰胺腺嘌呤二核苷酸(NAD~+)合成水平的变化，研究G-CSF刺激与PBEF表达、NAD~+合成的关系。 3、用人类重组PBEF体外刺激正常人的CD34~+细胞后，通过光学显微镜观察细胞形态学的变化、流式细胞术检测表面抗原CD11b、CD15的表达以及逆转录实时定量PCR检测细胞C/EBPa、C/EBPe、PU．1和ELA2等基因mRNA的表达，研究PBEF胞外刺激与CD34~+细胞向髓系分化的关系。 4、正常人的CD34~+细胞经由Lentivirus转导PBEF基因后，光学显微镜观察细胞形态学的变化、流式细胞术检测表面抗原CD11b、CD15的表达变化以及逆转录实时定量PCR检测细胞C/EBPa、C/EBPe、PU．1和ELA2等基因mRNA的表达变化，研究PBEF胞内过度表达与CD34~+细胞向髓系分化的关系。 5、用逆转录实时定量PCR、ELISA方法以及共聚焦荧光显微镜断层扫描技术检测经长期G-CSF治疗的SCN病人髓系细胞的PBEF mRNA、胞内蛋白质水平以及血浆蛋白质水平的变化，研究G-CSF治疗后SCN病人髓系细胞“成熟障碍”的纠正与PBEF表达变化的关系更多还原

【Abstract】 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,更多还原