Molecular Genomics and Therapeutics Section, Molecular Medicine Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.
PLoS One. 2013 Jul 8;8(7):e68307. doi: 10.1371/journal.pone.0068307. Print 2013.
Based upon the lack of clinical samples available for research in many laboratories worldwide, a significant gap exists between basic and clinical studies of beta-thalassemia major. To bridge this gap, we developed an artificially engineered model for human beta thalassemia by knocking down beta-globin gene and protein expression in cultured CD34+ cells obtained from healthy adults. Lentiviral-mediated transduction of beta-globin shRNA (beta-KD) caused imbalanced globin chain production. Beta-globin mRNA was reduced by 90% compared to controls, while alpha-globin mRNA levels were maintained. HPLC analyses revealed a 96% reduction in HbA with only a minor increase in HbF. During the terminal phases of differentiation (culture days 14-21), beta-KD cells demonstrated increased levels of insoluble alpha-globin, as well as activated caspase-3. The majority of the beta-KD cells underwent apoptosis around the polychromatophilic stage of maturation. GDF15, a marker of ineffective erythropoiesis in humans with thalassemia, was significantly increased in the culture supernatants from the beta-KD cells. Knockdown of beta-globin expression in cultured primary human erythroblasts provides a robust ex vivo model for beta-thalassemia.
由于全球许多实验室缺乏用于研究的临床样本，因此β-地中海贫血的基础研究与临床研究之间存在很大差距。为了弥补这一差距，我们通过敲低来自健康成年人的培养 CD34+细胞中的β-珠蛋白基因和蛋白表达，开发了一种用于人类β-地中海贫血的人工工程模型。慢病毒介导的β-珠蛋白 shRNA（β-KD）转导导致珠蛋白链产生失衡。与对照组相比，β-珠蛋白 mRNA 减少了 90%，而α-珠蛋白 mRNA 水平保持不变。HPLC 分析显示，HbA 减少了 96%，而 HbF 仅略有增加。在分化的终末阶段（培养第 14-21 天），β-KD 细胞表现出不溶性α-珠蛋白水平升高，以及 caspase-3 激活。大多数β-KD 细胞在成熟的多色性阶段周围发生凋亡。GDF15 是地中海贫血患者无效红细胞生成的标志物，β-KD 细胞培养上清液中的 GDF15 显著增加。在培养的原代人类红细胞中敲低β-珠蛋白表达提供了一种用于β-地中海贫血的强大的体外模型。
