LOW TEMPERATURE SEM AND X RAY MICROANALYSIS OF ASL

该项目有两个目标。首先，在正常组织中， 表征上皮过程（腺体分泌、表面上皮 离子转运、杯状细胞放电）影响气道表面的深度 液体然后我们将确定ASL深度的调节是如何改变的 囊性纤维化（CF）其次，我们将确定离子和粘蛋白 CF中ASL和腺体分泌物的浓度改变。的深度 ASL将通过低温扫描电子显微镜测量 （LTSEM）的冷冻水合组织垂直断裂的平面， 表面上皮。离子含量将通过X射线测量 在LTSEM中对样品进行微观分析。粘蛋白浓度将是 根据X射线微量分析光谱中的硫峰估计。作为 替代X射线微量分析，我们将估计Na和Cl水平， 通过添加氚化水（或 甘露醇）、136 Cl和122 Na至细胞培养物的基底外侧。Na 和Cl浓度，然后估计从平衡的比例 从22/Na或36/C1的吸收到3/H/2/0（或/3 H-）的平衡吸收， 甘露醇）。测定腺体中Na、Cl和粘蛋白的含量 分泌物、冷冻水合部分将在平行平面上断裂 在表皮下。这将产生以下配置文件： 大约50 μ m直径的腺管，每mm/2 1个，在其上 电子束将居中。最后，我们将估计力 表面张力将纤毛周围的溶胶固定在原位。当溶质 加入到基底外侧介质中，应该有一个点， 渗透梯度超过了表面张力。在这一点上， 溶胶应该消失，纤毛塌陷到上皮上。的 本项目所获得的有关ASL深度和组成的信息将 作为其他项目的指导方针。

This project has two objectives. First, in normal tissues we will characterize how epithelial processes (gland secretion, surface epithelial ion transport, goblet cell discharge) affect the depth of airway surface liquid. We will then determine how the regulation of ASL depth is altered in cystic fibrosis (CF). Second, we will determine how the ion and mucin concentrations of ASL and gland secretions are altered in CF. The depth of ASL will be measured by low temperature scanning electron microscopy (LTSEM) of frozen hydrated tissues fractured perpendicular to the plane of the surface epithelium. Ion contents will be measured by X-ray microanalysis of specimens in the LTSEM. Mucin concentrations will be estimated from the sulfur peak in the X-ray microanalysis spectrum. As an alternative to X-ray microanalysis we will estimate Na and Cl levels in ASL of surface epithelial cultures by adding tritiated water (or mannitol), /36 Cl and /22 Na to the basolateral side of cell cultures. Na and Cl concentrations are then estimated from the ration of equilibrium uptake for 22/Na or 36/C1 to the equilibrium uptakes of 3/H/2/0 (or /3 H- mannitol). To determine concentrations of Na, Cl and mucin in gland secretions, frozen hydrated sections will be fractured in a plane parallel to, and just below the surface epithelium. This will result in profiles of gland ducts of approximately 50 mum diameter and 1 per mm/2, over which the electron beam will be centered. Finally, we will estimate the forces of surface tension holding the periciliary sol in place. When solute is added to the basolateral medium, there should come a point at which the osmotic grandient exceeds the forces of surface tension. At this point, the sol should disappear and the cilia collapse onto the epithelium. The information on depth and composition of ASL obtained in this project will serve as guidelines for the other projects.