Levi-Schaffer F, Austen K F, Caulfield J P, Hein A, Gravallese P M, Stevens R L
J Immunol. 1987 Jul 15;139(2):494-500.
Human mast cells, dispersed from lung tissue by proteolytic treatment and enriched to a purity of 23 to 68% by density-gradient centrifugation, were maintained ex vivo for up to 13 days when co-cultured with mouse skin-derived 3T3 fibroblasts in RPMI 1640 containing 10% fetal calf serum. The human mast cells were adherent to the fibroblast cultures within 2 to 4 hr after seeding, and after 7 days of co-culture were localized between the layers of fibroblasts. The cell surfaces of the mast cells and the fibroblasts did not form tight junctions, but rather approached within 20 nm of each other. The co-cultured mast cells did not divide; they maintained their cellular content of histamine and TAMe esterase and resembled in vivo mast cells in that their secretory granules exhibited scroll patterns and their nuclei were oval. Both the freshly isolated and the co-cultured mast cells responded to activation with anti-human IgE by exocytosing histamine and generating and releasing arachidonic acid metabolites. When freshly isolated mast cells were activated immunologically, they exocytosed 38 +/- 8% of their total histamine content and released 28 +/- 1.9 ng (mean +/- range, n = 2) of immunoreactive equivalents of prostaglandin D2 (PGD2) per microgram of total cellular histamine, but did not generate significant amounts of either leukotriene C4 (LTC4) or leukotriene B4 (LTB4). The 1-wk co-cultured mast cells, on the other hand, exocytosed 43 +/- 2.4% of their total histamine content, and released 86 +/- 10, 43 +/- 20, and 5.2 +/- 5.2 ng (mean +/- SD, n = 4) of immunoreactive equivalents of PGD2, LTC4, and LTB4, respectively, per microgram of histamine. Thus, human lung-derived mast cells can be maintained ex vivo when co-cultured with fibroblasts, and, when treated with anti-IgE, they metabolize arachidonic acid via both the cyclooxygenase and the 5-lipoxygenase pathways.
通过蛋白水解处理从肺组织中分离并经密度梯度离心富集至纯度为23%至68%的人肥大细胞，在含有10%胎牛血清的RPMI 1640培养基中与小鼠皮肤来源的3T3成纤维细胞共培养时，可在体外维持长达13天。人肥大细胞在接种后2至4小时内附着于成纤维细胞培养物上，共培养7天后位于成纤维细胞层之间。肥大细胞和成纤维细胞的细胞表面未形成紧密连接，而是彼此靠近至20纳米以内。共培养的肥大细胞不分裂；它们维持其组胺和TAMe酯酶的细胞内含量，并且在体内肥大细胞方面表现相似，即其分泌颗粒呈现涡旋模式且细胞核为椭圆形。新鲜分离的和共培养的肥大细胞在受到抗人IgE激活时，都会通过组胺胞吐作用以及生成和释放花生四烯酸代谢物做出反应。当新鲜分离的肥大细胞受到免疫激活时，它们会胞吐出其总组胺含量的38±8%，并每微克总细胞组胺释放28±1.9纳克（平均值±范围，n = 2）的前列腺素D2（PGD2）免疫反应当量，但不会产生大量的白三烯C4（LTC4）或白三烯B4（LTB4）。另一方面，共培养1周的肥大细胞胞吐出其总组胺含量的43±2.4%，并且每微克组胺分别释放86±10、43±20和5.2±5.2纳克（平均值±标准差，n = 4）的PGD2、LTC4和LTB4免疫反应当量。因此，与人肺来源的肥大细胞与成纤维细胞共培养时可在体外维持，并且在受到抗IgE处理时，它们会通过环氧化酶和5-脂氧合酶途径代谢花生四烯酸。
