Iversen P L, Crouse D, Zon G, Perry G
Department of Pharmacology, University of Nebraska Medical Center, Omaha.
Antisense Res Dev. 1992 Fall;2(3):223-33. doi: 10.1089/ard.1992.2.223.
A phosphorothioate oligonucleotide that has been employed to inhibit HIV-1 viral expression in chronically infected H9 cells was examined for its ability to associate with murine lymphoid cells. The relationship between cellular oligonucleotide concentration and the lymphoid target tissues is important to the selection of an animal model, evaluation of potential side effects, and understanding the actions of a therapeutically useful antisense oligonucleotide. Lymphoid cells were harvested from murine peripheral blood, bone marrow, thymus, lymph node, and spleen. Cell subpopulations that bind the oligonucleotide were distinguished by two-color flow cytometry employing a fluorescein-labeled anti-rev oligonucleotide and phycoerythrin-labeled antibodies to selected cell surface molecules associated with unique subpopulations of cells. Very little oligonucleotide binding was observed in peripheral blood mononuclear cells or thymic T cells, but substantial numbers of cells, primarily B cells from bone marrow and spleen, accumulated the oligonucleotide. The cell-associated oligonucleotide was increased significantly in lymphoid populations when the cells were mitogen pretreated with either concanavalin-A (ConA), a T cell mitogen, or lipopolysaccharide (LPS), a B cell mitogen. These data clearly demonstrate the ability of fluorescein-conjugated oligonucleotides to bind to unique cell populations in suspension, allowing simultaneous two-color phenotypic analysis, suggesting that fluorescein-conjugated oligonucleotides may be a useful bridge between in vitro molecular biology techniques and in vivo cell biology. In addition, these data provide optimism concerning the in vivo treatment of chronically infected HIV patients using antisense oligonucleotides.
一种已被用于抑制慢性感染的H9细胞中HIV-1病毒表达的硫代磷酸酯寡核苷酸，被检测其与鼠类淋巴细胞结合的能力。细胞内寡核苷酸浓度与淋巴靶组织之间的关系，对于动物模型的选择、潜在副作用的评估以及理解治疗用反义寡核苷酸的作用非常重要。从鼠类外周血、骨髓、胸腺、淋巴结和脾脏中采集淋巴细胞。通过双色流式细胞术区分结合寡核苷酸的细胞亚群，该技术使用荧光素标记的抗rev寡核苷酸和藻红蛋白标记的抗体来识别与特定细胞亚群相关的选定细胞表面分子。在外周血单核细胞或胸腺T细胞中观察到极少的寡核苷酸结合，但大量细胞，主要是来自骨髓和脾脏的B细胞，积累了寡核苷酸。当细胞用T细胞促有丝分裂原刀豆球蛋白A（ConA）或B细胞促有丝分裂原脂多糖（LPS）进行促有丝分裂预处理时，淋巴群体中与细胞相关的寡核苷酸显著增加。这些数据清楚地证明了荧光素偶联的寡核苷酸与悬浮中的特定细胞群体结合的能力，允许同时进行双色表型分析，这表明荧光素偶联的寡核苷酸可能是体外分子生物学技术和体内细胞生物学之间的有用桥梁。此外，这些数据为使用反义寡核苷酸对慢性感染的HIV患者进行体内治疗带来了希望。
