Viani P, Marchesini S, Cestaro B, Gatt S
Department of Membrane Biochemistry and Neurochemistry, Hebrew University, Hadassah Medical School, Jerusalem, Israel.
Biochim Biophys Acta. 1989 Mar 14;1002(1):20-7. doi: 10.1016/0005-2760(89)90059-3.
This study aimed at increasing the efficiency and shortening the time required for administering cerebroside sulfate to cultured cells. For this purpose several modes of dispersion of a fluorescent derivative of cerebroside sulfate (sulfatide), in which the natural fatty acid has been replaced by pyrenedodecanoic acid (P12), were incubated with the cells. This fluorescent derivative of cerebroside sulfate (P12-CS) was introduced into the growth medium of the cells using the three following modes of dispersion: (1) P12-CS was dissolved in dimethylsulfoxide and added to the medium, (2) it was precomplexed with serum albumin or (3) incorporated into small, unilamellar vesicles (SUV) of phosphatidylcholine. With each of these respective modes of dispersion, the P12-CS was incubated for periods up to 48 h with cultured lymphoblasts or fibroblasts. Uptake by the cells could be determined by recording directly the cell-associated fluorescence, using a suspension of washed intact cells. The cell lipids were subsequently extracted with mixtures of chloroform/methanol and their fluorescence recorded. When related to the incubation time, uptake of P12-CS by the cells increased continuously using each of the above dispersions. The appearance of fluorescence at 475 nm ('excimer') and the ratio of this to the monomolecular fluorescence at 378 nm ('E/M') could be used as a measure for the presence of the internalized P12-CS in aggregated or fully dispersed states. These values (i.e., E/M), recorded on the suspensions of intact cells were rather high using the aqueous dispersions, intermediate values were observed using the SUV and rather low E/M values (0.5 or less) were observed using the preformed albumin-(P12-CS) complexes. Increasing the mole ratio of albumin to P12-CS (i.e., from 1:2 to 2:1 m/m), decreased the quantity of sulfatide which was taken up by the cells but also further decreased the E/M ratio, suggesting a fully dispersed state of the pyrene lipid within the cell. This indicated that, using an optimal albumin to P12-CS ratio of 1-2 (or its equivalent values in fetal calf serum) permitted an influx of single molecules of P12-CS into the cells. After 48 h, about 50% of the fluorescence of skin fibroblasts was found in metabolic degradation products of P12-CS. The parallel value for fibroblasts derived from a patient with metachromatic leukodystrophy was only about 5%. Appearance of the excimeric emission of a dispersion of P12-CS in water permitted estimation of its critical micellar concentration as being 7.5 x 10(-7) M.
本研究旨在提高向培养细胞施用硫酸脑苷脂的效率并缩短所需时间。为此，将硫酸脑苷脂（硫脂）的荧光衍生物（其中天然脂肪酸已被芘十二烷酸（P12）取代）的几种分散模式与细胞一起孵育。硫酸脑苷脂的这种荧光衍生物（P12-CS）通过以下三种分散模式引入细胞的生长培养基中：（1）将P12-CS溶解在二甲基亚砜中并添加到培养基中，（2）将其与血清白蛋白预先复合，或（3）掺入磷脂酰胆碱的小单层囊泡（SUV）中。对于这些各自的分散模式中的每一种，将P12-CS与培养的成淋巴细胞或成纤维细胞孵育长达48小时。通过使用洗涤过的完整细胞悬液直接记录细胞相关荧光，可以确定细胞的摄取情况。随后用氯仿/甲醇混合物提取细胞脂质并记录其荧光。与孵育时间相关，使用上述每种分散方式，细胞对P12-CS的摄取持续增加。在475nm处荧光的出现（“准分子”）及其与378nm处单分子荧光的比率（“E/M”）可用于衡量内化的P12-CS处于聚集或完全分散状态的情况。使用水性分散体时，在完整细胞悬液上记录的这些值（即E/M）相当高，使用SUV时观察到中间值，使用预先形成的白蛋白 - （P12-CS）复合物时观察到相当低的E/M值（0.5或更低）。增加白蛋白与P12-CS的摩尔比（即从1:2增加到2:1 m/m），会减少细胞摄取硫脂的量，但也会进一步降低E/M比率，这表明芘脂质在细胞内处于完全分散状态。这表明，使用1-2的最佳白蛋白与P12-CS比率（或其在胎牛血清中的等效值）可使P12-CS的单分子流入细胞。48小时后，在皮肤成纤维细胞中约50%的荧光存在于P12-CS的代谢降解产物中。来自异染性脑白质营养不良患者的成纤维细胞的相应值仅约为5%。P12-CS在水中分散体的准分子发射的出现使得能够估计其临界胶束浓度为7.5×10^(-7) M。
