Human CD34+ hematopoietic stem/progenitor cells express high levels of FLIP and are resistant to Fas-mediated apoptosis
Human CD34+ hematopoietic stem/progenitor cells express high levels of FLIP and are resistant to Fas-mediated apoptosis
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DOI：
10.1634/stemcells.20-2-174
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发表时间：
2002-01-01
期刊：
影响因子：
5.2
通讯作者：
Civin, CI
中科院分区：
文献类型：
--
作者：
Kim, H;Whartenby, KA;Civin, CI
我们试图确定来自人胎盘/脐带血(CB)或成人动员血(PBSC)的淋巴造血干/祖细胞(HSC)对Fas诱导的细胞凋亡是否敏感。体外培养人CD34(+)细胞，加入或不加入造血生长因子(FKT：Flt-3配体[FL]、KIT配体[KL]、血小板生成素[TPO])，加入或不加入可溶性Fas配体(SFasL)或激发型抗Fas抗体。培养5~48h后，检测细胞活力，Annexin V和7-氨基放线菌素D染色，荧光激活细胞分选分析细胞凋亡。采用体内非肥胖型糖尿病/重症联合免疫缺陷小鼠移植潜能(SEP)实验和体内亚硝基造血祖细胞集落形成细胞(CFCs)实验对培养细胞进行鉴定。实时定量聚合酶链式反应检测CD34(+)细胞中Fas、FLICE抑制蛋白(FliP)和Caspase8mRNA的表达水平。Western blotting证实了FliP的表达。在加入sFasL或激发型抗Fas抗体的条件下，脐血或外周血CD34(+)细胞的存活率、CFC值或SEP均未见下降。人脐血和动员的PBSC CD34(+)细胞表达高水平的FliP，低比例的Caspase8：FliP和低水平的Fas。因此，人脐血和外周血CD34(+)HSC对Fas途径激动剂具有耐药性。FliP的高水平表达可能在一定程度上保护CD34(+)细胞免受Fas介导的细胞凋亡。
We sought to determine whether lympho-hematopoietic stem-progenitor cells (HSC) from human placenta/umbilical cord blood (CB) or adult mobilized blood (PBSC) are sensitive to Fas-induced apoptosis. Human CD34(+) cells from CB or PBSC were cultured in serum-free medium, with or without hematopoietic growth factors (FKT: FLT-3 ligand [FL], KIT ligand [KL], and thrombopoietin [TPO]), and with or without soluble Fas ligand (sFasL) or agonistic anti-Fas antibody. After 5-48 hours of culture, cells were assessed for viability and stained with Annexin V and 7-Aminoactinomycin D for apoptosis analysis by fluorescence-activated cell sorting. Cultured cells were also assessed by in Nitro hematopoietic colony-forming cell (CFC) and in vivo nonobese diabetic/severe combined immunodeficient mouse engraftment potential (SEP) assays. Levels of Fas, FLICE inhibitory protein (FLIP), and Caspase 8 mRNA in CD34(+) cells were determined by real-time quantitative polymerase chain reaction. Expression of FLIP was confirmed by Western blotting. No decrease in viability, CFC, or SEP was observed in CB or PBSC CD34(+) cells cultured in the presence of sFasL or agonistic anti-Fas antibody. Human CB and mobilized PBSC CD34(+) cells expressed high levels of FLIP, low ratios of Caspase 8:FLIP, and low levels of Fas. Thus, human CB and PBSC CD34(+) HSC were resistant to Fas pathway agonists. High-level expression of FLIP likely provides one level of protection of CD34(+) cells from Fas-mediated apoptosis.