Ho S C, Huang L
J Histochem Cytochem. 1983 Mar;31(3):404-10. doi: 10.1177/31.3.6827078.
The endocytosis of large unilamellar liposomes composed of phosphatidylcholine by the cultured Chinese hamster V-79 cells is demonstrated with electron microscopy cytochemistry. A novel cytochemical marker, 5-Br,4-Cl,3-indolylphosphate (BCIP) is used. This marker is a soluble and colorless substrate for the lysosomal acid phosphatase and can be readily entrapped in liposomes. The product of the enzymatic reaction, 5-Br,4-Cl,3-hydroxy indole, rapidly self-condenses and becomes an insoluble derivative of indigo blue. In thin section transmission electron microscopy, the condensed product appears as electron-dense deposits in the lysosomes. Since the electron-dense deposit only appears when the endocytosed liposomes are delivered to the lysosomes as the result of phagosome-lysosome fusion, this marker provides a unique cytochemical means to reveal those liposomes that are lysosomotropic and are actually decomposed within the lysosomes. No electron-dense deposits are found in the liposome-treated cells in the presence of chloroquine, or a combination of NaN3 and deoxyglucose. As a comparison, we have also used horseradish peroxidase entrapped in liposomes to confirm the endocytic uptake of liposomes. Using a radioactive marker, 125I-labeled lysozyme, entrapped in liposomes, it is shown that about 20-30% of liposome uptake by V-79 cells is due to endocytosis.
利用电子显微镜细胞化学技术证实了培养的中国仓鼠V-79细胞对由磷脂酰胆碱组成的大单层脂质体的内吞作用。使用了一种新型细胞化学标记物5-溴-4-氯-3-吲哚磷酸酯（BCIP）。该标记物是溶酶体酸性磷酸酶的一种可溶性无色底物，可轻易包封在脂质体中。酶促反应产物5-溴-4-氯-3-羟基吲哚迅速自缩合，形成靛蓝的不溶性衍生物。在超薄切片透射电子显微镜下，缩合产物表现为溶酶体中的电子致密沉积物。由于仅当内吞的脂质体通过吞噬体-溶酶体融合被递送至溶酶体时才会出现电子致密沉积物，因此该标记物提供了一种独特的细胞化学方法来揭示那些具有溶酶体趋向性并实际在溶酶体内分解的脂质体。在存在氯喹或叠氮化钠与脱氧葡萄糖的组合的情况下，在脂质体处理的细胞中未发现电子致密沉积物。作为比较，我们还使用包封在脂质体中的辣根过氧化物酶来证实脂质体的内吞摄取。使用包封在脂质体中的放射性标记物125I标记的溶菌酶，结果表明V-79细胞摄取的脂质体中约20%-30%是由于内吞作用。
