Functional and morphological analysis of the main excretory duct epithelium of rat submandibular gland. -VII Molecular biological study of the main excretory duct-

我们进行了这项研究，以阐明大鼠(9周龄雄性Wistar大鼠)颌下腺主要排泄管的功能。1.应用免疫细胞化学(光镜和电子显微镜)、RT-PCR法和原位杂交技术，对参与味觉信号转导和离子通道的Gα蛋白和GαO亚基在大鼠颌下腺主要排泄管上皮细胞中的定位进行了研究，并在电子显微镜下观察到GαGustducin在绒毛细胞和II型光细胞中的免疫反应。在簇状细胞中，免疫金颗粒位于微绒毛和顶端小泡附近。在II型光细胞中，管腔表面和管腔下的囊泡与免疫金颗粒反应。从MED中提取的总RNA的RT-PCR示出与成熟的(剪接的)αGustducin mRNA相对应的阳性条带。Spin…中存在信号用地高辛标记的反义核糖核苷酸探针进行原位杂交，可观察到较多的梭形细胞(簇状细胞)和顶端膨大的细胞(轻细胞II型)。GαO免疫反应阳性的细胞在所有类型的细胞中均可观察到，而不包括构成主排泄管的基底细胞。尤其是簇状细胞和II型光细胞具有较强的免疫反应。除了GαGustducin免疫反应外，免疫金颗粒还存在于绒毛细胞的微绒毛和顶端小泡附近，以及II型光细胞的管腔表面和腔下的小泡上。用地高辛标记的反义核糖核苷酸探针进行原位杂交，所有类型的无基底细胞均有信号。2.用免疫细胞化学方法(光镜和电子显微镜)检测慢性酸中毒和慢性碱中毒时H+/K+ATPase的变化。对照组H^+/K^+ATPase免疫反应阳性分布于细胞内。慢性代谢性酸中毒使H^+/K^+ATPase从弥漫性分布转向尖端定位，提示H^+/K^+ATPase可能参与了酸碱平衡的调节。慢性代谢性碱中毒与H+/K+ATPase呈弱免疫反应。较少

We performed this research to elucidate the function of the main excretory duct of the submandibular gland of the rat (9-week-old male Wistar rat). Following results were obtained during 2003-2005.1.The localization of G α gustducin and G α O, the GTP-binding protein a subunits implicated in taste signal transduction and ion channel were investigated in the main excretory duct epithelium of the rat submandibular gland using immunocytochemistry (light microscopy and electron microscopy), RT-PCR and in situ hybridization.G α gustducin immunoreactivity was observed in the tuft cell and light cell type II under electron microscopy. In the tuft cell, immuno-gold particles were present on the microvilli and near the apical vesicles. In light cell type II, luminal surface and vesicles just beneath the lumen were reacted with immuno-gold particles. RT-PCR of total RNA extracted from the MED showed positive bands corresponding to the mature (spliced) α gustducin mRNA. Signal was present in spin … More dle-shaped cells (tuft cells) and cells with a bulging apex (light cells type II) with in situ,hybridization using DIG-labeled antisense ribonucleotide probesG α O immunoreactivity was observed in the all cell types without the basal cell composing the main excretory duct. Especially, tuft cells and light cells type II had the strong immunoreactivity. As well as G α gustducin immunoreactivity, immuno-gold particles were present on the microvilli and near the apical vesicles in the tuft cells, and luminal surface and vesicles just beneath the lumen in the light cells type II. RT-PCR of total RNA extracted from the MED showed positive bands corresponding to the mature (spliced) α O mRNA. Signal was present in all cell types without basal cells with in situ hybridization using DIG-labeled antisense ribonucleotide probes.2.We examined the change of H^+/K^+ATPase in chronic acidosis and chronic alkalosis by immunocytochemistry (light microscopy and electron microscopy). H^+/K^+ATPase immunoreactivity was distributed intracellularly in control. Chronic metabolic acidosis caused a shift in H^+/K^+ATPase away from diffuse distribution towards apical localization, suggesting that H^+/K^+ATPase might be involved in the regulation of acidbase homeostasis. The week immunoreactivity with H^+/K^+ATPase was present in Chronic metabolic alkalosis. Less