In vivo培養法を用いた気道粘液過分泌機構の解明

本研究主要进行了以下两方面的工作：1)建立以人呼吸道上皮细胞为载体的大鼠气管裸露移植物体内培养模型；2)探讨分泌型白蛋白水解酶抑制物(SLPI)在人肺各种非肿瘤状态下的作用。将传代细胞以-104个/只的密度接种于裸鼠气管移植物，并移植于裸鼠体内。3周后，将人中性粒细胞弹性蛋白酶(-30Omu/移植物)注入移植物腔内，以诱导粘液细胞增殖。注射后4周对移植物进行检查。在第一系列实验中，新建立的裸露气管上皮相当萎缩，后来显示出发育良好的粘液纤毛上皮；通过改变体外培养条件，结果得到了改善。到目前为止，弹性酶刺激还没有引起移植物上皮的形态变化。SLPI在各种非肿瘤性肺部疾病中的免疫组化研究表明，SLPI阳性细胞见于气管、支气管腺的浆液细胞、表面粘液细胞。也就是说，SLPI阳性粘液细胞的发生率与粘液细胞增生密切相关。有趣的是，SLPI阳性的细支气管Clara细胞在少数急性化脓性支气管炎中很少见到。SLPI具有抗弹性蛋白酶活性，弹性蛋白酶是诱导粘液细胞增殖和粘液高分泌的代表物质之一。因此，SLPI阳性细胞的异常出现可能是对浸润性中性粒细胞产生的增加的弹性蛋白酶的抑制作用。

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.