In vivo培養法を用いた気道粘液過分泌機構の解明

本研究项目主要进行：1)利用去皮大鼠气管移植物填充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）在人肺各种非肿瘤性疾病中的作用。从克隆公司购买人气道支气管细胞，体外培养数周以获得大量细胞。将传代培养的细胞以- 10^4个细胞/片的密度接种于去皮大鼠气管移植物中，并将移植物植入裸鼠体内。3周后，将人中性粒细胞弹性蛋白酶（- 300omu /移植物）注入移植物腔内，诱导粘液细胞增生。注射后4周检查移植物。剥脱气管新生上皮在第一次实验中相当萎缩，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，提高了结果。到目前为止，弹性蛋白酶刺激未引起移植物上皮的形态学改变。这一文化模式的建立需要进一步的研究。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浆液细胞、表面黏液细胞。即slpi阳性黏液细胞的发生率与黏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阳性的细支气管Clara细胞。SLPI具有抗弹性酶活性，弹性酶是诱导黏液细胞增生和黏液高分泌的代表性物质之一。因此，异常出现的slpi阳性细胞可能作为抑制来自浸润中性粒细胞的增加弹性酶的抑制剂。

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.