| Summary: | 碩士 === 東海大學 === 生物學系 === 87 === High density lipoproteins (HDL) were labeled with fluorescein 1, 1-dioctadecyl-3, 3, 3’, 3’-tetramethylindocarbocyanine perchlorate (DiI) or colloidal gold for investigation of binding and internalization of HDL by cultured endothelial cells and smooth muscle cells from the rat aorta.
The confluent monolayers of endothelial and smooth muscle cells were first incubated in the serum free medium containing cholesterol for 48 hr at 37℃. For binding experiment, cells were incubated for 2 hr at 4℃ with serum free medium containing HDL-DiI. Then the cells were incubated for 0, 5, 15, and 30 min at 37℃for fluorescence microscopy. The concentrations of cholesterol in the medium were measured by cholesterol enzymatic reagent. For transmission electromicroscopy, the cholesterol fed cells were first incubated for 2 hr at 4℃ in the serum free medium containing HDL-colloidal gold. After medium was washed out with PBS, cells were then incubated in the serum free medium without HDL at 37℃ for 0, 5, 15, and 30 min for internalization experiment.
The results indicated that when cells were incubated with HDL-DiI or HDL-gold complexes for 2 hr at 4℃, the complexes were found only on the cell surface. When the cells were incubated at 37℃ for 5-30 min, the HDL-DiI was observed in the cytoplasmic vesicles. The cholesterol concentrations in the culture medium were increased with incubation time. The HDL-colloidal gold was revealed in the plasmalemmal vesicles close to the cytoplasmic membrane for 5 min at 37℃. At 15 min, the complexes were appeared in the large multivesicular bodies. At 30 min , most of the complexes were reappeared on the cell surface. Whether HDLs are acceptors for excess intracellular cholesterol needs further investigation.
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