Use of fluorescent ANTS to examine the BBB-permeability of polysaccharide.
Use of fluorescent ANTS to examine the BBB-permeability of polysaccharide.
复制标题
作者：
Christopher K;Makani V;Judy W;Lee E;Chiaia N;Kim DS;Park J
最近，一些多糖显示出用于治疗神经退行性疾病的治疗潜力，而最重要的性质，其对包裹大脑和脊髓的血脑屏障（BBB）的渗透性尚未确定。由于在动物体内内源性多糖中难以追踪目标多糖，导致测定延迟。我们开发了一种简单的方法来检查BBB渗透性，并可能，在动物中的目标多糖的组织分布。我们用荧光8-氨基萘-1，3，6-三磺酸二钠盐（ANTS）标记多糖用于追踪。我们还开发了一种简单的方法，使用75%乙醇从未结合的游离ANTS中分离ANTS标记的多糖。动物鼻内注射ANTS-多糖后，我们可以通过荧光细胞计量法定量测定脑和血清中ANTS-多糖的含量。用三氯乙酸（TCA）和75%乙醇提取血清蛋白中的游离ANTS-多糖。该方法将有助于在动物体内追踪多糖。ANTS标记不像放射性标记那么繁琐，但与放射性标记一样强大，用于追踪动物中的靶多糖。我们简单的方法可以从动物血清和组织中分离出结构完整的ANTS-多糖。该方法适用于组织中ANTS结合大分子的荧光检测。
Recently, some polysaccharides showed therapeutic potentials for the treatment of neurodegenerative diseases while the most important property, their permeability to the blood brain barrier (BBB) that sheathes the brain and spinal cord, is not yet determined. The determination has been delayed by the difficulty in tracking a target polysaccharide among endogenous polysaccharides in animal. We developed an easy way to examine the BBB-permeability and, possibly, tissue distribution of a target polysaccharide in animal. We tagged a polysaccharide with fluorescent 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt (ANTS) for tracking. We also developed a simple method to separate ANTS-tagged polysaccharide from unconjugated free ANTS using 75% ethanol. After ANTS-polysaccharide was intra-nasally administered into animals, we could quantify the amounts of ANTS-polysaccharide in the brain and the serum by fluorocytometry. We could also separate free ANTS-polysaccharide from serum proteins using trichloroacetic acid (TCA) and 75% ethanol. Our method will help to track a polysaccharide in animal easily. ANTS-labeling is less tedious than but as powerful as radiolabeling for tracking a target polysaccharide in animal. Our simple method can separate structurally intact ANTS-polysaccharide from animal serum and tissues. This method is good for the fluorometry-based measurement of ANTS-conjugated macromolecules in tissues.