Abstract：
OBJECTIVE: Myeloproliferative neoplasms (MPN) are a heterogeneous group of hematopoietic stem-cell diseases with excessive proliferation of one or more blood cell lines. In this study, we evaluated the effect of different oxygen concentrations on HIF-1α and NOS3 gene expression to determine the effect of the bone marrow microenvironment on JAK2V617F positive Philadelphia chromosome negative (Ph-) MPNs.
METHODS: Peripheral blood mononuclear cells (MNC) of 12 patients with Ph- MPN were collected. The presence of JAK2V617F allele status was determined with allele-specific nested PCR analysis. MPN CD34+ and CD34depleted populations were isolated from MNC by magnetic beads. Separate cell cultures of CD34+/depleted populations were managed at different oxygen concentrations including anoxia (∼0%), hypoxia (∼3%), and normoxia (∼20%) conditions for 24 h. HIF-1α and NOS3 gene expression changes were examined in each population related to JAK2V617F status with real time RT-PCR.
RESULTS: It was revealed that relative HIF-1α and NOS3 expressions were significantly increased in response to decreased oxygen concentration in all samples. Relative HIF-1α and NOS3 expressions were found to be higher especially in CD34+ and CD34depleted populations carrying JAK2V617F mutations compared to MPN patients carrying wild-type JAK2.
CONCLUSIONS: JAK2V617F might have specific role in HIF-1α and NOS3 regulations with respect to low oxygen concentrations in Ph- MPN. Further evaluations might reveal the effect of JAK2V617F on Ph- MPN pathogenesis in bone marrow microenvironment.
METHODS: Peripheral blood mononuclear cells (MNC) of 12 patients with Ph- MPN were collected. The presence of JAK2V617F allele status was determined with allele-specific nested PCR analysis. MPN CD34+ and CD34depleted populations were isolated from MNC by magnetic beads. Separate cell cultures of CD34+/depleted populations were managed at different oxygen concentrations including anoxia (∼0%), hypoxia (∼3%), and normoxia (∼20%) conditions for 24 h. HIF-1α and NOS3 gene expression changes were examined in each population related to JAK2V617F status with real time RT-PCR.
RESULTS: It was revealed that relative HIF-1α and NOS3 expressions were significantly increased in response to decreased oxygen concentration in all samples. Relative HIF-1α and NOS3 expressions were found to be higher especially in CD34+ and CD34depleted populations carrying JAK2V617F mutations compared to MPN patients carrying wild-type JAK2.
CONCLUSIONS: JAK2V617F might have specific role in HIF-1α and NOS3 regulations with respect to low oxygen concentrations in Ph- MPN. Further evaluations might reveal the effect of JAK2V617F on Ph- MPN pathogenesis in bone marrow microenvironment.
摘要：
目的：骨髓增殖性肿瘤（MPN）是一类异质性的造血干细胞疾病，具有一种或多种血细胞系的过度增殖。在这项研究中,我们评估了不同氧浓度对HIF-1α和NOS3基因表达的影响，以确定骨髓微环境对JAK2V617F阳性费城染色体阴性（Ph-）MPN的影响。
方法：收集12例Ph-MPN患者的外周血单个核细胞（MNC）。用等位基因特异性巢式PCR分析确定JAK2V617F等位基因状态的存在。通过磁珠从MNC分离MPNCD34+和CD34耗尽的群体。CD34+/耗竭群体的单独细胞培养物在不同的氧气浓度下进行管理，包括缺氧（~0%），缺氧(~3%)，和24小时的常氧(~20%)条件。用实时RT-PCR检测每个群体中与JAK2V617F状态相关的HIF-1α和NOS3基因表达变化。
结果：显示所有样品中的相对HIF-1α和NOS3表达均响应于氧浓度的降低而显着增加。与携带野生型JAK2的MPN患者相比，发现HIF-1α和NOS3的相对表达在携带JAK2V617F突变的CD34和CD34缺失人群中尤其更高。
结论：JAK2V617F可能在HIF-1α和NOS3调节Ph-MPN低氧浓度方面具有特定作用。进一步的评估可能揭示JAK2V617F对骨髓微环境中Ph-MPN发病机制的影响。
方法：收集12例Ph-MPN患者的外周血单个核细胞（MNC）。用等位基因特异性巢式PCR分析确定JAK2V617F等位基因状态的存在。通过磁珠从MNC分离MPNCD34+和CD34耗尽的群体。CD34+/耗竭群体的单独细胞培养物在不同的氧气浓度下进行管理，包括缺氧（~0%），缺氧(~3%)，和24小时的常氧(~20%)条件。用实时RT-PCR检测每个群体中与JAK2V617F状态相关的HIF-1α和NOS3基因表达变化。
结果：显示所有样品中的相对HIF-1α和NOS3表达均响应于氧浓度的降低而显着增加。与携带野生型JAK2的MPN患者相比，发现HIF-1α和NOS3的相对表达在携带JAK2V617F突变的CD34和CD34缺失人群中尤其更高。
结论：JAK2V617F可能在HIF-1α和NOS3调节Ph-MPN低氧浓度方面具有特定作用。进一步的评估可能揭示JAK2V617F对骨髓微环境中Ph-MPN发病机制的影响。
