Development of protein drug delivery system from the lung based on ligand recognition mechanisms of the receptors

本研究的目的是为肺靶向蛋白质给药系统的开发提供依据。本研究使用培养的RLE-6 TN细胞以及分离自大鼠的原代培养的肺泡II型上皮细胞检测了肺泡II型上皮细胞对FITC-1标记的白蛋白和胰岛素的摄取。RLE-6 TN细胞对FITC-白蛋白的摄取具有温度、浓度和能量依赖性。高亲和力系统和低亲和力系统介导了细胞的摄取，其中高亲和力系统为网格蛋白介导的内吞. RLE-6 TN细胞对FITC-胰岛素的摄取部分由网格蛋白介导的内吞作用介导，但也可能涉及其他系统.检查RLE-6 TN细胞摄取的FITC-白蛋白和胰岛素的命运。这些蛋白质部分定位于溶酶体中，并随着时间的推移逐渐降解.配体印迹分析表明，RLE-6 TN细胞质膜上存在一些可能的白蛋白受体蛋白.原代培养的大鼠肺泡II型细胞对FITC-白蛋白的摄取与RLE-6 TN细胞的摄取一样，是通过网格蛋白介导的内吞作用进行的，进一步研究肺泡II型和I型细胞对蛋白药物的摄取活性、相关受体、受体识别配体的机制，将有助于开发新的肺部蛋白药物释放系统。

The purpose of this study is to obtain the information for the development of protein drug delivery system from the lung. I examined the uptake of FITC-1abeled albumin and insulin by alveolar type II epithelial cells using cultured RLE-6TN cells as well as primary cultured alveolar type II cells isolated from rats.1. FITC-Albumin uptake by RLE-6TN cells was temperature-, concentration-, and energy-dependent. The uptake was mediated by high-affinity and low-affinity systems, and the former system was found to be the clathrin-mediated endocytosis.2. The uptake of FITC-insulin by RLE-6TN cells was partly mediated by the clathrin-mediated endocytosis, but other systems would also be involved.3. The fate of FITC-albumin and insulin taken up by RLE-6TN cells was examined. These proteins were partly localized in lysosomes, and gradually degraded over time.4. Ligand blot analysis showed that there were some possible receptor proteins for albumin in the plasma membranes of RLE-6TN cells.5. FITC-Albumin uptake by primary cultured alveolar type II cells isolated from rats was shown to be mediated by the clathrin-mediated endocytosis, like the uptake in RLE-6TN cells.Further studies concerning the activities of protein drug uptake by alveolar type II and I cells, related receptors, the mechanisms of ligand recognition by the receptors would help to develop new strategies for protein drug delivery system from the lung.