Analysis of mechanisms of neutrophil accumulation in the lung ; research utilizing LTB_4 receptor-expressed CHO cells.

中性粒细胞在肺部积聚的机制分析；

• 批准号: 11670569
• 负责人: OHISHI Nobuya
• 金额: $ 1.66万
• 依托单位: University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670569/
• 关键词: Neutrophil; Leukotriene B_4 receptor; CHO cell; Chemotaxis; Acute lung injury; ロイコトリエンB_4受容体; LTB4; LTB4受容体; シリカ

1. Determination of optimal condition of labeling LTB_4 receptor-expressed CHO cells with fluorescent probes.CHO cells could be adequately labeled for visualization with fluorescent microscopy with 4 micro molar CMFDA or 2 micro molar CMTMR.LTB_4 receptor-expressed CHO cells labeled with this conditions showed the same chemotaxis against LTB_4 as unlabeled cells in chemotaxis assay using chemotaxis chamber.2. Evaluation of neutrophil accumulation in mice lung with intratracheal administration of various agents and its reproducibility.As models of neutrophil accumulation in mice lung, we administered LTB_4, bleomycin, and silica intratracheally. After 4 hours of LTB_4 administration, neutrophilia over 10% in bronchoalveolar lavage fluid (BALF) cells were observed in only about 20% of examined mice, and after 24 hours of bleomycin administration, neutrophilia over 50% were observed in only about 50% of examined mice, suggesting that the reproducibility of neutrophil accumulation in mice … More lung by these methods was not adequate. In case of administration of silica, when silica was recovered in BALF, there were consistent marked neutrophilia (40-80%) in BALF cells 24 hours after silica administration, but, when silica was not recovered in BALF, neutrophilia were not observed. This findings indicated that expectoration and inaccessibility to lung parenchyme of intratracheally administered agents were the main cause of inadequate reproducibility of the procedures. Silica was a suitable agent for intratracheal administration in that its deposition into lung parenchyme could be verified by BALF analysis.3. in vivo Chemotaxis assay in mice model of intratracheal administration of silica using CMFDA-labeled LTB_4 receptor-expressed CHO cells.CMFDA-labeled LTB_4 receptor-expressed CHO cells and CMTMR-labeled control CHO cells were injected to mice intravenously via tail veins after 20 hours of intratracheal administration of silica. However, in BALF obtained 4 hours later, neither CMTMR nor CMFDA-labeled cells could not be recovered. Because there was a possibility that LTB_4 receptor-expressed CHO cells was not present freely in alveolar space but attached to pulmonary capillary endothelium or alveolar epithelium, we made histological specimens of lung tissues and examined fluorescent probe-labeled cells, but, at present, we could not obtain any definite results. Less

1。用荧光问题标记LTB_4受体表达的CHO细胞的最佳条件。可以充分地标记Cho细胞用荧光显微镜使用4微摩尔CMFDA或2个微摩尔CMTMR的荧光显微镜进行可视化，以与该条件相同的Cho the chos sized，与同一化学细胞相同的Cho sys sississ sissips sississ sissips sissips sissips sissips。使用趋化室2。通过气管内给药各种药物及其可重复性的肺中性粒细胞积累的评估。作为嗜中性粒细胞在肺中的中性粒细胞积累的模型，我们施用了LTB_4，博霉素和二氧化硅肠内。在使用LTB_4 4小时后，仅在大约20％的检查小鼠中观察到了支气管肺泡灌洗液（BALF）细胞超过10％的中性粒细胞，并且在给药24小时后，只有大约50％的小鼠中性粒细胞的嗜中性粒细胞嗜性粒细胞，这表明是由中性群的50％。如果使用二氧化硅，当二氧化硅在BALF中回收时，在二氧化硅施用后24小时在BALF细胞中有一致的嗜中性粒细胞（40-80％），但是当未在BALF中回收二氧化硅时，未观察到嗜中性粒细胞。这一发现表明，气管内施用药物的肺实质的期望和无法访问性是该手术可重复性不足的主要原因。二氧化硅是气管内给药的合适药物，因为它可以通过BALF分析来验证其沉积到肺实质中。3。 in vivo Chemotaxis assay in mice model of intratracheal administration of silica using CMFDA-labeled LTB_4 receptor-expressed CHO cells.CMFDA-labeled LTB_4 receptor-expressed CHO cells and CMTMR-labeled control CHO cells were injected to mice intravenously via tail veins after 20 hours of intratracheal administration of silica.但是，在4小时后获得的BALF中，CMTMR和CMFDA标记的细胞都无法恢复。因为有可能在肺泡空间中自由地存在LTB_4受体表达的CHO细胞，而是附着在肺毛细血管山上或肺泡上皮上的，所以我们制造了肺组织的组织学物种并检查了荧光探针标记的细胞，但是目前，我们无法获得任何定义的结果。较少的

项目成果

Ishizaka,N.,Nakao,A.,Ohishi,N. et al.: "Increased leukctaere A4 hydrolase expression in the heart of angiotousin II-induced hypertousive rat."FEBS Lett.. 463. 155-159 (1999)

石坂，N.，中尾，A.，大石，N.

Ishizaka, N., Nakao, A., Ohishi, N., Suzuki, M., Aizawa, T., Taguchi, J., Nagai, R., Shimizu, T.and Ohno, M.: "Increased leukotriene A_4 hydrolase expression in the heart of angiotensin II-induced hypertensive rat."FEBS Lett.. 463. 155-159 (1999)

Ishizaka, N.、Nakao, A.、Ohishi, N.、Suzuki, M.、Aizawa, T.、Taguchi, J.、Nagai, R.、Shimizu, T. 和 Ohno, M.：“增加白三烯 A_4 水解酶

Ishizaka,N.,Nakao,A.,Ohishi,N. et al.: "Increased leukotriene A_4 hydrolase expression in the heart of angiotensin II-induced hypertensive rat."FEBS Lett.. 463. 155-159 (1999)

石坂，N.，中尾，A.，大石，N.