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Studies on regulation mechanisms of respiratory epithelial cell differentiation using human nasal epithelial cell culture.

利用人鼻上皮细胞培养研究呼吸道上皮细胞分化的调控机制。

基本信息

批准号：
05671436
负责人：
HANAMURE Yutaka
金额：
$ 1.41万
依托单位：
Kagoshima University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1995
项目状态：
已结题

项目摘要

(1) We demonstrated a floating culture model which allowed effective induction of cell differentiation of human respiratory epithelial cells in vitro. Human nasal epithelial cells plated on type I collagen gel rapidly proliferated to reach confluence and showed flat squamous-like appearance, and after detached and floated gradually differentiate to secretory cells and ciliated cells and then displayd pseudostratified mucociliary epithelium after 14 days in floating culture. Ultrastructural and morphometric studies demonstrated fibrous granules, deuterosomes, and centrioles, the intracellular characteristic of ciliogenesis during floating and final high density of ciliated cells.(2) Surface of matured ciliated cells are covered with glycocaryx that contains sialic acid as terminal carbohydrate of glycoconjugates. We used lectins, Macckia amurensis agglutinin and Sambucus nigra agglutinin which recognize sialic acid residues to observe expression of sialic acid residue during respiratory cell differentiation. Sialic acid was not present before start of ciliogenesis, gradually appeared during ciliogenesis and finally covered the full surface of matured ciliated cells. This result indicates that sialic acid is one of markers of respiratory cell differentiation.(3) A novel monoclonal antibody, MAb-RPCS was obtained after immunization of mice with whole cells of 3 days floating eulture. MAb-RPCS recognized the secretory cells appeared at preciliated state. Western blotting with periodate oxidation revealed that MAb-RPCS recognized carbohydrate moieties.(4) Interleukin-1beta (IL-1beta) is one of inflammatory cytokines that modulates inflammatory process. We observed the effects of IL-1beta during the differentiation of respiratory epithelial cells. The results suggest that IL-1beta accelerates the appearance of secretory cells and suppresses the onset of ciliogenesis and the appearance of matured ciliated cells.

(1)建立了一种在体外有效诱导人呼吸道上皮细胞分化的漂浮培养模型。人鼻上皮细胞经I型胶原凝胶处理后迅速增殖达到合流，呈扁平鳞状外观，分离漂浮后逐渐分化为分泌细胞和纤毛细胞，漂浮培养14天后形成假层状纤毛黏膜上皮。超微结构和形态计量学研究表明，纤毛细胞在漂浮过程中具有纤维颗粒、后粒和中心粒等细胞内特征，并最终形成高密度纤毛细胞。(2)成熟纤毛细胞表面覆盖糖核，糖核中含有唾液酸作为糖缀合物的末端碳水化合物。本研究利用识别唾液酸残基的凝集素、猕猴桃凝集素和黑树苗凝集素，观察呼吸细胞分化过程中唾液酸残基的表达。唾液酸在纤毛发生前不存在，在纤毛发生过程中逐渐出现，最终覆盖成熟纤毛细胞的整个表面。提示唾液酸是呼吸细胞分化的标志物之一。(3)用漂浮培养3 d的全细胞免疫小鼠，获得一种新的单克隆抗体MAb-RPCS。单克隆抗体- rpcs识别出处于脆弱状态的分泌细胞。高酸盐氧化Western blotting显示，单克隆抗体- rpcs识别碳水化合物部分。(4)白细胞介素-1 β (il -1 β)是调节炎症过程的炎症细胞因子之一。我们观察了il -1 β在呼吸道上皮细胞分化过程中的作用。结果表明，il -1 β加速分泌细胞的出现，抑制纤毛发生的发生和成熟纤毛细胞的出现。