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

1. 荧光探针标记LTB_4受体表达CHO细胞的最佳条件的确定。用4微摩尔CMFDA或2微摩尔CMTMR荧光显微镜可以充分标记CHO细胞。在趋化室进行趋化实验时，经此条件标记的LTB_4受体表达的CHO细胞对LTB_4的趋化性与未标记的细胞相同。气管内给药对小鼠肺中性粒细胞积累的影响及其可重复性。作为小鼠肺中性粒细胞积累模型，我们气管内给药LTB_4、博来霉素和二氧化硅。LTB_4给药4小时后，只有约20%的小鼠支气管肺泡灌洗液（BALF）细胞中性粒细胞超过10%，博来霉素给药24小时后，只有约50%的小鼠中性粒细胞超过50%，表明中性粒细胞在小鼠体内积累的可重复性不够。在给予二氧化硅的情况下，当二氧化硅在BALF中恢复时，在给予二氧化硅24小时后，BALF细胞中存在一致的显著中性粒细胞（40-80%），但当二氧化硅在BALF中未恢复时，未观察到中性粒细胞。这一发现表明，气管内给药的咳痰和肺实质难以接近是导致该过程重复性不足的主要原因。二氧化硅是一种适合气管内给药的药物，因为它在肺实质的沉积可以通过BALF分析得到证实。用cmfda标记的LTB_4受体表达的CHO细胞对气管内注射二氧化硅小鼠模型进行体内趋化实验。将cmfda标记的LTB_4受体表达的CHO细胞和cmtmr标记的对照CHO细胞在气管内给药20小时后经尾静脉注射到小鼠体内。然而，在4小时后获得的BALF中，CMTMR和cmfda标记的细胞都无法恢复。由于LTB_4受体表达的CHO细胞有可能不是在肺泡腔内自由存在，而是附着在肺毛细血管内皮或肺泡上皮上，我们制作了肺组织组织学标本，并检测了荧光探针标记的细胞，但目前还不能得到明确的结果。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