LOW TEMPERATURE SEM AND X RAY MICROANALYSIS OF ASL

这个项目有两个目标。首先，在正常组织中，我们会 描述上皮细胞如何处理(腺体分泌、表面上皮细胞 离子传输、杯状细胞放电)影响呼吸道表面的深度 液体。然后我们将确定如何改变ASL深度的规则 囊性纤维化(CF)。第二，我们将确定离子和粘蛋白是如何 在CF中，ASL和腺体分泌物的浓度发生变化。地球的深度 ASL将通过低温扫描电子显微镜进行测量 (LTSEM)垂直于关节平面断裂的冷冻水化组织 表面上皮细胞。离子含量将用X射线测量 样品在低温扫描电子显微镜下的显微分析。粘蛋白浓度将是 根据X射线微区分析光谱中的硫峰进行估算。作为一种 作为X射线微量分析的替代方法，我们将估计 表面上皮细胞培养中加入氚水(或 甘露醇)、/36Cl和/22Na到细胞培养基侧。北美 然后从平衡比估计氯的浓度 对22/Na或36/C1的吸收达到3/H/2/0(或/3 H- 甘露醇)。测定腺体中钠、氯和粘蛋白的浓度 分泌物、冰冻水化切片会平行平面断裂 到表面上皮，并恰好在表面上皮下。这将产生以下配置文件 直径约50微米，每毫米/2 1个的腺体导管，其上 电子束将居中。最后，我们将估计部队 表面张力将睫毛周围的溶胶固定在适当的位置。当溶质为 加上基底侧向介质，应该会出现一个点，在那里 渗透压强超过表面张力。在这点上， 溶胶体应该消失，纤毛塌陷到上皮上。这个 在这个项目中获得的关于ASL的深度和组成的信息将 对其他项目起到指导作用。

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.