Department of Obstetrics and Gynecology, Division of Maternal Fetal Medicine, Oregon Health and Science University, Portland, OR, USA.
Department of Obstetrics and Gynecology, Division of Maternal Fetal Medicine, Oregon Health and Science University, Portland, OR, USA; Center for Developmental Health, Oregon Health and Science University, Portland, OR, USA.
Placenta. 2017 Sep;57:137-143. doi: 10.1016/j.placenta.2017.06.344. Epub 2017 Jun 30.
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 min 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 min at 37 °C compared to 4.9 min 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.
动物模型研究表明，羊水在羊膜中的单向囊泡转运在调节羊水体积中起主要作用。我们的目的是使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、囊泡摄取和囊泡的细胞内分布。
剖宫产时获取胎盘羊膜，制备和培养羊膜细胞。在 20%-50%汇合度时，将细胞在 22°C 或 37°C 下用荧光标记的大分子孵育 1-30 分钟。荧光标记的大分子被选为受体介导的小窝和网格蛋白包被囊泡摄取以及非特异性内吞作用的标志物。荧光染料处理后，固定细胞，使用 Imaris 软件对细胞进行成像并计数囊泡。
囊泡摄取显示出一级饱和动力学，在 37°C 下的半饱和时间平均为 1.3 分钟，而在 22°C 下为 4.9 分钟，两种温度下的非特异性内吞作用都更快。摄取率在细胞间存在广泛的变异性。在超分辨率显微镜下，每种大分子的细胞内空间分布模式都不同。荧光标记大分子的共定位在囊泡尺寸下非常低。
在人胎盘羊膜细胞中，1）大分子的囊泡摄取非常迅速，这与囊泡跨羊膜转运在调节羊水体积中的作用的概念一致；2）摄取依赖于温度，并且在个体细胞之间存在差异；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。
