In vivo培養法を用いた気道粘液過分泌機構の解明

利用体内培养方法阐明气道粘液分泌过多的机制

• 批准号: 08670673
• 负责人: INAYAMA Yoshiaki
• 金额: $ 1.47万
• 依托单位: Yokohama City University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08670673/
• 关键词: Airway epithelial cells; Mucus cell hyperplasia; Mucus hypersecretion; SLPI; elastase; culture model; MUC; MUC

This research project primarily has been conducted, 1) to establish an in vivo culture model using denuded rat tracheal grafts repopulated with human airway epithelial cells, and 2) to clarify role of secretory leukoprotease inhibitor (SLPI) in various nonneoplastic conditions of human lungs.Human airway bronchial cells were purchased from Clonetics Corp. and cultured several weeks in vitro in order to obtain plenty of cells. Subcultured cells were inoculated into denuded rat tracheal grafts at density of - 10^4 cells/graft, and the grafts were implanted into nude mice. Three weeks later, human neutrophil elastase ( - 3OOmu/graft) was injected into the graft lumen in order to induce mucus cell hyperplasia. The grafts were examined 4 weeks after the injection. The newly established epithelium in denuded tracheas was rather atrophic in the first series of the experiments, and later showed well-developed mucociliary epithelium ; the results were improved by changing the in vitro culture condition. Elastase stimuli have evoked no morphological change in the graft epithelium so far. Further investigation will be necessary for establishment of this culture model.Immunonistochemical study of SLPI in various non-neoplastic pulmonary diseases indicated that SLPI-positive cells were seen in serous cells of tracheobronchial glands, surface mucous cells. Namely, incidence of SLPI-positive mucous cells was closely related to mucous cell hyperplasia. Interestingly, SLPI-positive bronchiolar Clara cells were rarely observed in a few case of acute, purulent bronchopneumonia. SLPI is well known to have anti-elastase activity, and elastase is one of representative substance to induce mucous cell hyperplasia and mucus hypersecretion. Therefore, unusual occurrence of SLPI-positive cells may act as inhibitors against increased-elastase derived from infiltrating neutrophils.

该研究项目的主要目的是：1）利用裸露的大鼠气管移植物重建人气道上皮细胞建立体内培养模型，2）阐明分泌性白细胞蛋白酶抑制剂（SLPI）在人肺各种非肿瘤条件下的作用。人气道支气管细胞购自Clonetics Corp.，并在体外培养数周以获得大量细胞。将传代培养的细胞以-10^4细胞/移植物的密度接种到裸露的大鼠气管移植物中，并将移植物植入裸鼠体内。三周后，将人中性粒细胞弹性蛋白酶（～300μ/移植物）注射到移植物管腔中以诱导粘液细胞增生。注射后4周检查移植物。在第一个系列的实验中，裸露气管中新形成的上皮相当萎缩，后来显示出发育良好的粘膜纤毛上皮；通过改变体外培养条件，结果得到改善。到目前为止，弹性蛋白酶刺激尚未引起移植上皮的形态变化。 SLPI在多种非肿瘤性肺部疾病中的免疫化学研究表明，SLPI阳性细胞见于气管支气管腺浆液细胞、表面粘液细胞。即SLPI阳性粘液细胞的发生与粘液细胞增生密切相关。有趣的是，在少数急性化脓性支气管肺炎病例中很少观察到SLPI阳性细支气管Clara细胞。众所周知，SLPI具有抗弹性蛋白酶活性，而弹性蛋白酶是诱导粘液细胞增生和粘液分泌过多的代表性物质之一。因此，SLPI 阳性细胞的异常出现可能会抑制源自浸润性中性粒细胞的弹性蛋白酶增加。

项目成果

蟹沢成好: "肺癌の発生機構:実験病理の新しい展開" 病理と臨床. 14. 22-33 (1996)

Nariyoshi Kanizawa：“肺癌发生机制：实验病理学的新进展”《病理学与临床研究》14. 22-33 (1996)。

Yoshiaki Inayama: "In vivo growth and differentiation potential of tracheal basal cells of rabbits in vitamin A deficiency" Int J Exp Path. 77. 89-97 (1996)

Yoshiaki Inayama：“维生素 A 缺乏时兔子气管基底细胞的体内生长和分化潜力”Int J Exp Path。

Inayama Y, Kitamura H, Shibagaki T, Usuda Y, Ito Y, Nakatani Y, and Kanisawa M: "In vivo growth and differentiation potential of tracheal basal cells of rabbits in vitamin A deficiency" Int J Exp Path. 77. 89-97 (1996)

Inayama Y、Kitamura H、Shibagaki T、Usuda Y、Ito Y、Nakatani Y 和 Kanisawa M：“维生素 A 缺乏时兔子气管基底细胞的体内生长和分化潜力”Int J Exp Path。