Vesicular uptake of macromolecules by human placental amniotic epithelial cells.
Vesicular uptake of macromolecules by human placental amniotic epithelial cells.
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DOI：
10.1016/j.placenta.2017.06.344
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发表时间：
2017-09
期刊：
影响因子：
3.8
通讯作者：
Cheung CY
中科院分区：
文献类型：
--
作者：
Sharshiner R;Brace RA;Cheung CY
动物模型研究表明，羊水在羊膜上的单向囊性运输在调节羊水体积中起主要作用。我们的目的是利用高分辨率和超分辨率荧光显微镜探索人羊膜细胞中的囊泡类型、囊泡摄取和囊泡在细胞内的分布。剖宫产获得胎盘羊膜，制备并培养羊膜细胞。在20%-50%的合度下，将细胞与荧光基团结合的大分子在22℃或37℃下孵育1-30分钟。选择荧光基团标记的大分子作为受体介导的囊泡和网格蛋白包被的囊泡摄取以及非特异性内吞的标记物。荧光团处理后，将细胞固定、成像并使用Imaris®软件对囊泡进行计数。泡状摄取表现出一级饱和动力学，在37°C时半饱和时间平均为1.3分钟，而在22°C时为4.9分钟，在两种温度下非特异性内吞摄取更快。细胞间摄取速率存在广泛的差异性。在超分辨显微镜下，每个大分子的胞内空间分布模式是不同的。荧光标记的大分子在囊泡尺寸上的共定位非常低。在人胎盘羊膜细胞中，1)水疱性摄取大分子是迅速的，这与水疱性跨羊膜胞吞作用在羊水体积调节中起作用的概念一致；2)摄取依赖于温度，并且在单个细胞中是可变的；3)独特的细胞内分布表明每种囊泡类型具有不同的功能；4)非受体介导的囊泡摄取可能是主要的囊泡摄取机制。
Studies in animal models have shown that unidirectional vesicular transport of amniotic fluid across the amnion plays a primary role in regulating amniotic fluid volume. Our objective was to explore vesicle type, vesicular uptake and intracellular distribution of vesicles in human amnion cells using high- and super-resolution fluorescence microscopy. Placental amnion was obtained at cesarean section and amnion cells were prepared and cultured. At 20%–50% confluence, the cells were incubated with fluorophore conjugated macromolecules for 1–30 minutes at 22°C or 37°C. Fluorophore labeled macromolecules were selected as markers of receptor-mediated caveolar and clathrin-coated vesicular uptake as well as non-specific endocytosis. After fluorophore treatment, the cells were fixed, imaged and vesicles counted using Imaris® software. Vesicular uptake displayed first order saturation kinetics with half saturation times averaging 1.3 minutes at 37°C compared to 4.9 minutes at 22°C, with non-specific endocytotic uptake being more rapid at both temperatures. There was extensive cell-to-cell variability in uptake rate. Under super-resolution microscopy, the pattern of intracellular spatial distribution was distinct for each macromolecule. Co-localization of fluorescently labeled macromolecules was very low at vesicular dimensions. In human placental amnion cells, 1) vesicular uptake of macromolecules is rapid, consistent with the concept that vesicular transcytosis across the amnion plays a role in the regulation of amniotic fluid volume; 2) uptake is temperature dependent and variable among individual cells; 3) the unique intracellular distributions suggest distinct functions for each vesicle type; 4) non-receptor mediated vesicular uptake may be a primary vesicular uptake mechanism.
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DOI：
10.1152/ajprenal.00532.2013
发表时间：
2014-05-01
影响因子：
4.2
作者：
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发表时间：
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影响因子：
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通讯作者：
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作者：
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