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

1.荧光探针标记表达LTB_4受体的CHO细胞的最佳条件的确定：用4微摩尔的CMFDA或2微摩尔的CMTMR可以在荧光显微镜下充分标记CHO细胞，用该条件标记的表达LTB_4受体的CHO细胞在趋化小室中对LTB_4的趋化作用与未标记的细胞相同。评价不同药物气管内给药对小鼠肺内中性粒细胞聚集的影响及其重复性。作为中性粒细胞在小鼠肺内聚集的模型，我们在气管内给予LTB_4、博莱霉素和二氧化硅。在给药4小时后，只有大约20%的受试者在支气管肺泡灌洗液细胞中观察到中性粒细胞超过10%，而在给药24小时后，只有大约50%的受试者观察到中性粒细胞在…中聚集的重复性。这些方法增加肺活量是不够的。在加入二氧化硅的情况下，当从BALF中回收二氧化硅时，24小时后BALF细胞中有一致的显著中性粒细胞(40-80%)，但当二氧化硅未在BALF中回收时，未观察到中性粒细胞。这一发现表明，气管内给药的咯痰和无法接触到肺实质是手术重复性不佳的主要原因。BALF分析可以证实二氧化硅沉积到肺实质中，因此二氧化硅是一种合适的气管内给药药物。用CMFDA标记的LTB_4受体表达的CHO细胞对小鼠气管内染尘模型进行体内趋化试验。气管内给药20小时后，尾静脉注射CMFDA标记的LTB_4受体表达的CHO细胞和CMTMR标记的对照CHO细胞。而在4小时后获得的BALF中，CMTMR和CMFDA标记的细胞均无法恢复。由于表达LTB_4受体的CHO细胞可能不是自由存在于肺泡腔，而是附着在肺毛细血管内皮细胞或肺泡上皮细胞上，我们制作了肺组织标本，并对荧光探针标记的细胞进行了检测，但目前尚不能得到任何确定的结果。较少

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