Sokol J, Blanchette-Mackie J, Kruth H S, Dwyer N K, Amende L M, Butler J D, Robinson E, Patel S, Brady R O, Comly M E
Developmental and Metabolic Neurology Branch, National Institute of Neurological and Communicative Disorders and Stroke, Bethesda, Maryland 20892.
J Biol Chem. 1988 Mar 5;263(7):3411-7.
The intracellular accumulation of unesterified cholesterol was examined during 24 h of low density lipoprotein (LDL) uptake in normal and Niemann-Pick C fibroblasts by fluorescence microscopy with filipin staining and immunocytochemistry. Perinuclear fluorescence derived from filipin-sterol complexes was observed in both normal and mutant cells by 2 h. This perinuclear cholesterol staining reached its peak in normal cells at 6 h. Subsequent development of fluorescence during the remaining 18 h of LDL incubation was primarily limited to the plasma membrane region of normal cells. In contrast, mutant cells developed a much more intense perinuclear fluorescence throughout the entire 24 h of LDL uptake with little enhancement of cholesterol fluorescence staining in the plasma membranes. Direct mass measurements confirmed that internalized LDL cholesterol more readily replenishes the plasma membrane cholesterol of normal than of mutant fibroblasts. Perinuclear filipin-cholesterol fluorescence of both normal and mutant cells was colocalized with lysosomes by indirect immunocytochemical staining of lysosomal membrane protein. Abnormal sequestration of LDL cholesterol in mutant cells within a metabolically latent pool is supported by the finding that in vitro esterification of cellular cholesterol could be stimulated in mutant but not in normal cell homogenates by extensive disruption of the intracellular membranous structures of cells previously cultured with LDL. Deficient translocation of exogenously derived cholesterol from lysosomes to other intracellular membrane sites may be responsible for the delayed homeostatic responses associated with LDL uptake by mutant Niemann-Pick Type C fibroblasts.
通过用制霉菌素染色的荧光显微镜检查和免疫细胞化学方法，研究了正常和尼曼-匹克C型成纤维细胞在摄取低密度脂蛋白（LDL）24小时期间未酯化胆固醇的细胞内积累情况。2小时时，在正常细胞和突变细胞中均观察到源自制霉菌素-固醇复合物的核周荧光。这种核周胆固醇染色在正常细胞中6小时达到峰值。在LDL孵育的剩余18小时内，荧光的后续发展主要局限于正常细胞的质膜区域。相比之下，在整个24小时的LDL摄取过程中，突变细胞产生了更强烈的核周荧光，而质膜中的胆固醇荧光染色几乎没有增强。直接质量测量证实，内化的LDL胆固醇比突变成纤维细胞更容易补充正常细胞的质膜胆固醇。通过溶酶体膜蛋白的间接免疫细胞化学染色，正常细胞和突变细胞的核周制霉菌素-胆固醇荧光都与溶酶体共定位。发现先前用LDL培养的细胞的细胞内膜结构被广泛破坏后，突变细胞匀浆中的细胞胆固醇体外酯化可被刺激，而正常细胞匀浆中则不能，这支持了突变细胞中LDL胆固醇在代谢潜伏池中异常隔离的观点。外源性胆固醇从溶酶体向其他细胞内膜位点的转运不足可能是导致突变的尼曼-匹克C型成纤维细胞摄取LDL时稳态反应延迟的原因。
