Butler M, Stecker K, Bennett C F
Isis Pharmaceuticals, Carlsbad, California 92008, USA.
Lab Invest. 1997 Oct;77(4):379-88.
The distribution of intravenously injected phosphorothioate oligodeoxynucleotides (P = S ODN) was studied in vivo in rodent tissues using three histologic methods: immunohistochemistry with a monoclonal antibody that recognizes P = S ODN ISIS 2105; direct fluorescence microscopy of P = S ODN ISIS 2105 conjugated to rhodamine; and autoradiography of 14C-labeled P = S ODN ISIS 2302. All three methods gave the same pattern of oligonucleotide distribution, and the intensity of the histologic signal agreed with previously published pharmacokinetic data on the relative concentration of P = S ODN in different organs. Proximal tubule cells in the kidney and Kupffer and endothelial cells in the liver were among the most heavily labeled with P = S ODN at all doses and time-points. Connective tissues proper, such as the lamina propria and submucosa of the intestine and the dermis and subcutaneous layer of the skin, were also labeled, whereas the P = S ODN signal was weak or negative in epithelial and muscle cells in the skin and intestine. At 2 hours postinjection, P = S ODN were clearly detectable in the extracellular matrix in loose and dense connective tissues, although by 24 hours, the label was predominantly intracellular. Large, nucleated cells in red marrow, and the connective tissues around bone and skeletal muscle cells and lining the knee joint, were positive for oligonucleotide, whereas P = S ODN were not detected in erythrocytes, cartilage, compact bone, and skeletal muscle. In spleen, white pulp was negative for P = S ODN, whereas cells surrounding the sinusoids and nucleated cells in the red pulp were strongly positive for P = S ODN. Our results provide specific information on the tissue and cellular localization of P = S ODN within organs in vivo. The data presented will be used as a reference for studies of P = S ODN distribution in diseased tissues and the distribution of modified oligonucleotides. Furthermore, because our results indicate which cell types are likely to be affected by antisense oligonucleotides, they can be used to guide future in vivo applications of the technology.
采用三种组织学方法在啮齿动物体内研究静脉注射硫代磷酸酯寡脱氧核苷酸（P = S ODN）的分布情况：使用识别P = S ODN ISIS 2105的单克隆抗体进行免疫组织化学；对与罗丹明偶联的P = S ODN ISIS 2105进行直接荧光显微镜检查；以及对14C标记的P = S ODN ISIS 2302进行放射自显影。这三种方法得到的寡核苷酸分布模式相同，组织学信号强度与先前发表的关于不同器官中P = S ODN相对浓度的药代动力学数据一致。在所有剂量和时间点，肾脏近端小管细胞以及肝脏中的库普弗细胞和内皮细胞被P = S ODN标记的程度最高。固有结缔组织，如肠黏膜固有层和黏膜下层以及皮肤真皮层和皮下层，也有标记，而皮肤和肠道的上皮细胞和肌肉细胞中的P = S ODN信号较弱或为阴性。注射后2小时，在疏松和致密结缔组织的细胞外基质中可明显检测到P = S ODN，不过到24小时时，标记主要位于细胞内。红骨髓中的大核细胞、骨骼周围的结缔组织、骨骼肌细胞以及膝关节内衬组织对寡核苷酸呈阳性反应，而在红细胞、软骨、致密骨和骨骼肌中未检测到P = S ODN。在脾脏中，白髓对P = S ODN呈阴性，而窦周细胞和红髓中的有核细胞对P = S ODN呈强阳性。我们的结果提供了关于体内器官中P = S ODN的组织和细胞定位的具体信息。所呈现的数据将作为研究P = S ODN在患病组织中的分布以及修饰寡核苷酸分布的参考。此外，由于我们的结果表明哪些细胞类型可能受反义寡核苷酸影响，它们可用于指导该技术未来的体内应用。
