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. 确定用荧光探针标记 LTB_4 受体表达的 CHO 细胞的最佳条件。使用 4 微摩尔 CMFDA 或 2 微摩尔 CMTMR 可以充分标记 CHO 细胞，以便用荧光显微镜观察。用此条件标记 LTB_4 受体表达的 CHO 细胞在使用趋化室的趋化测定中显示出与未标记细胞相同的针对LTB_4的趋化性。2．气管内施用各种药物及其重现性。作为小鼠肺部中性粒细胞积聚的模型，我们气管内施用LTB_4、博来霉素和二氧化硅。LTB_4施用4小时后，观察到支气管肺泡灌洗液(BALF)细胞中中性粒细胞增多超过10%。只有大约 20% 的受检小鼠在服用博来霉素 24 小时后，中性粒细胞增多仅在约 50% 的受检小鼠中观察到 50%，这表明通过这些方法在小鼠肺中的中性粒细胞积累的再现性不充分。在施用二氧化硅的情况下，当在 BALF 中回收二氧化硅时，存在一致的标记。施用二氧化硅后24小时，BALF细胞中出现中性粒细胞增多（40-80%），但是，当BALF中没有回收二氧化硅时，没有观察到中性粒细胞增多。气管内给药药物的预期和难以进入肺实质是该过程重复性不足的主要原因，因为二氧化硅在肺实质中的沉积可以通过体内BALF分析来验证。使用 CMFDA 标记的 LTB_4 受体表达的 CHO 细胞气管内给予二氧化硅的小鼠模型。CMFDA 标记的 LTB_4 受体表达气管内给予二氧化硅20小时后，将CHO细胞和CMTMR标记的对照CHO细胞通过尾静脉静脉注射至小鼠，但在4小时后获得的BALF中，均未回收CMTMR和CMFDA标记的细胞。 LTB_4 受体表达的 CHO 细胞可能不是自由存在于肺泡腔中，而是附着在肺毛细血管内皮或肺泡上皮上，我们制作了肺组织的组织学标本并检查了荧光探针标记的细胞，但目前我们无法获得任何明确的结果。

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.