RECONSTITUTED LOW-DENSITY LIPOPROTEIN - VEHICLE FOR THE DELIVERY OF HYDROPHOBIC FLUORESCENT-PROBES TO CELLS
RECONSTITUTED LOW-DENSITY LIPOPROTEIN - VEHICLE FOR THE DELIVERY OF HYDROPHOBIC FLUORESCENT-PROBES TO CELLS
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DOI：
10.1002/jss.400100409
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发表时间：
1979-01-01
期刊：
影响因子：
--
通讯作者：
BROWN, MS
中科院分区：
文献类型：
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作者：
KRIEGER, M;SMITH, LC;BROWN, MS
以往的研究表明，血浆低密度脂蛋白（LDL）的胆固醇酯核心可以用庚烷提取，并被各种疏水分子取代。该重建技术用于掺入2种荧光探针，3-芘甲基-23，24-二去甲-5-胆烯-22-酸酯-3 β-油酸酯（PMCA油酸酯）和二油基荧光素，转化为庚烷提取的LDL。这两种荧光脂蛋白制剂是有用的探针可视化受体介导的内吞LDL在培养的人成纤维细胞。当正常成纤维细胞在37 ℃孵育时，C与任何一种荧光LDL制剂相比，荧光颗粒积聚在细胞的核周区域。来自缺乏功能性LDL受体的家族性高胆固醇血症（FH）纯合子患者的成纤维细胞在与荧光重建LDL孵育时没有积累可见的荧光颗粒。使用荧光激活的细胞分选仪来定量已经与用二油基荧光素重构的LDL孵育的单个细胞的荧光强度。利用该技术，可以将正常成纤维细胞群体与FH成纤维细胞群体区分开。显然，使用荧光重建LDL与细胞分选仪结合来分离缺乏功能性LDL受体的突变细胞是可行的。
Previous studies showed that the cholesteryl ester core of plasma low density lipoprotein (LDL) can be extracted with heptane and replaced with a variety of hydrophobic molecules. This reconstitution technique was used to incorporate 2 fluorescent probes, 3-pyrenemethyl-23,24-dinor-5-cholen-22-oate-3.beta.-yl oleate (PMCA oleate) and dioleyl fluorescein, into heptane-extracted LDL. Both fluorescent lipoprotein preparations were useful probes for visualizing the receptor-mediated endocytosis of LDL in cultured human fibroblasts. When normal fibroblasts were incubated at 37.degree. C with either of the fluorescent LDL preparations, fluorescent granules accumulated in the perinuclear region of the cell. Fibroblasts from patients with the homozygous form of familial hypercholesterolemia (FH) that lack functional LDL receptors did not accumulate visible fluorescent granules when incubated with the fluorescent reconstituted LDL. A fluorescence-activated cell sorter was used to quantify the fluorescence intensity of individual cells that had been incubated with LDL reconstituted with dioleyl fluorescein. With this technique a population of normal fibroblasts could be distinguished from a population of FH fibroblasts. It is apparently feasible to use fluorescent reconstituted LDL in conjunction with the cell sorter to isolate mutant cells lacking functional LDL receptors.