Reconstituted low density lipoprotein: A vehicle for the delivery of hydrophobic fluorescent probes to cells

Wiley - Tập 10 Số 4 - Trang 467-478 - 1979
Monty Krieger1, Richard G.W. Anderson1, Joseph L. Goldstein1, Michael S. Brown1, Louis C. Smith2, Yin J. Kao2, Henry J. Pownall2, Antonio M. Gotto2
1Departments of Molecular Genetics and Cell Biology, University of Texas Health Science Center at Dallas, Dallas, Texas 75235
2Department of Medicine, Baylor College of Medicine, Houston, Texas 77030.

Tóm tắt

AbstractPrevious studies have shown that the cholesteryl ester core of plasma low density lipoprotein (LDL) can be extracted with heptane and replaced with a variety of hydrophobic molecules. In the present report we use this reconstitution technique to incorporate two fluorescent probes, 3‐pyrenemethyl‐23, 24‐dinor‐5‐cholen‐22‐oate‐3β‐yl oleate (PMCA oleate) and dioleyl fluorescein, into heptane‐extracted LDL. Both fluorescent lipoprotein preparations were shown to be useful probes for visualizing the receptor‐mediated endocytosis of LDL in cultured human fibroblasts. When normal fibroblasts were incubated at 37°C with either of the fluorescent LDL preparations, fluorescent granules accumulated in the perinuclear region of the cell. In contrast, 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. The current studies demonstrate the feasibility of using fluorescent reconstituted LDL in conjunction with the cell sorter to isolate mutant cells lacking functional LDL receptors.

Từ khóa


Tài liệu tham khảo

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Wiley

Tập 10 Số 4

467-478

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