PDF(Pubmed)
Abstract：
Therapeutic oligonucleotides have achieved great clinical interest since their approval as drug agents by regulatory agencies but their access and distribution in blood cells are not completely known. We evaluated by flow cytometry the ability of short fluorescent scramble oligonucleotides (ON*) to access human peripheral blood mononuclear cells (PBMC) after incubating with ON* during 1 h and 7 days of culture follow-up \'in vitro\'. Blood samples were treated with chemically modified oligonucleotides (phosphorothioate backbone and 2\' O-Me ends) to resist nuclease digestion under culture conditions. The ON* internalization was determined after discarding the membrane-associated fluorescence by trypan blue quenching. Whereas the oligonucleotide accessed neutrophils and monocytes rapidly, achieving their maximum in 1 h and 24 h, respectively, lymphocytes required 7 days to achieve the maximum (80% of cells) transfection. The ON*ability to access lymphocyte types (T, B, and NK) and T cell subtypes (CD4+, CD8+, and CD4-CD8-) were similar, with T cells being more accessible. Regulatory CD4+ and CD8+ T cells were classified in low and high Foxp3 expressers, whose expression proved not to alter the ON* internalization during the first hour, achieving 53% of CD4+Foxp3+ and 40% of CD8+Foxp3+ cells. Our results contribute to understanding and improving the management of therapeutic ONs.
摘要：
治疗性寡核苷酸已经获得了极大的临床兴趣,因为它们被管理机构批准作为药物试剂,但是它们在血细胞中的进入和分布并不完全已知。我们通过流式细胞术评估了短荧光乱序寡核苷酸（ON*）在体外培养随访1h和7天期间与ON*孵育后进入人外周血单核细胞（PBMC）的能力。用化学修饰的寡核苷酸(硫代磷酸酯主链和2'O-Me末端)处理血液样品以在培养条件下抵抗核酸酶消化。在通过台盼蓝猝灭丢弃膜相关荧光之后测定ON*内化。而寡核苷酸快速进入中性粒细胞和单核细胞，在1小时和24小时内达到最大值，分别,淋巴细胞需要7天才能达到最大（80％的细胞）转染。ON*访问淋巴细胞类型的能力(T，B,和NK）和T细胞亚型（CD4+，CD8+,和CD4-CD8-）相似，T细胞更容易获得。调节性CD4+和CD8+T细胞分为低和高Foxp3表达，其表达式被证明在第一个小时内不会改变ON*内化，获得53%的CD4+Foxp3+和40%的CD8+Foxp3+细胞。我们的结果有助于理解和改善治疗性ON的管理。
