Laporte J, Hallee A, Maghni K, Robidoux C, Borgeat P, Sirois P
Department of Pharmacologie, Faculté de Médecine, Université de Sherbrooke, P.Q., Canada.
Prostaglandins. 1991 Mar;41(3):263-81. doi: 10.1016/0090-6980(91)90046-i.
A method for the isolation of non-ciliated bronchiolar epithelial (Clara) cells from the guinea pig is described. Following digestion of the lung tissue with Type XXIV protease, the isolated lung cells showed a viability greater than 90% and contained 3% of Clara cells. Several cell populations were then separated on the basis of size using 2 centrifugal elutriations. The macrophages and endothelial cells were removed from the Clara cells enriched fractions by differential adherence on Petri dishes. The Clara cell-rich suspension was then further purified by centrifugation on Percoll non-continuous density gradients consisting of 48-52-55% Percoll solution. The lower interface and the pellet of the non-continuous gradient consisted of approximately 80% Clara cells. Identification of isolated Clara cells was confirmed by light microscopic observations after nitroblue tetrazolium staining and by ultrastructural characteristic features as observed by electron microscopy. The metabolism of arachidonic acid into prostaglandins and TxB2 by purified Clara cells was examined by enzyme immunoassay (EIA) and leukotriene formation was investigated by reverse phase high performance liquid chromatography (RP-HPLC). Enriched guinea pig Clara cells incubated with arachidonic acid released TxB2, PGE2 and 6-keto PGF1 alpha, but did not produce leukotrienes. These cells could however transform exogenous leukotriene A4 into leukotriene B4. These results suggest that guinea pig Clara cells possess the enzymes of the cyclooxygenase pathway required for TxB2, PGE2 and 6-keto-PGF1 alpha synthesis. Clara cells do not possess the 5-lipoxygenase enzyme but show some leukotriene A4 hydrolase activity since they can produce leukotriene B4 upon incubation with leukotriene A4.
本文描述了一种从豚鼠中分离非纤毛细支气管上皮（克拉拉）细胞的方法。用XXIV型蛋白酶消化肺组织后，分离出的肺细胞活力大于90%，其中含3%的克拉拉细胞。然后利用两次离心淘析根据细胞大小分离出几个细胞群体。通过在培养皿上的差异贴壁从富含克拉拉细胞的组分中去除巨噬细胞和内皮细胞。接着，将富含克拉拉细胞的悬浮液在由48%-52%-55% Percoll溶液组成的不连续密度梯度上离心进一步纯化。不连续梯度的下层界面和沉淀中约含80%的克拉拉细胞。通过硝基蓝四氮唑染色后的光学显微镜观察以及电子显微镜观察到的超微结构特征，证实了分离出的克拉拉细胞的身份。通过酶免疫测定（EIA）检测纯化的克拉拉细胞将花生四烯酸代谢为前列腺素和TXB2的情况，并通过反相高效液相色谱（RP-HPLC）研究白三烯的形成。用花生四烯酸孵育富含克拉拉细胞的豚鼠细胞后，释放出TXB2、PGE2和6-酮-PGF1α，但未产生白三烯。然而，这些细胞能够将外源性白三烯A4转化为白三烯B4。这些结果表明，豚鼠克拉拉细胞具有合成TXB2、PGE2和6-酮-PGF1α所需的环氧化酶途径的酶。克拉拉细胞不具有5-脂氧合酶，但具有一定的白三烯A4水解酶活性，因为它们在与白三烯A4孵育时能够产生白三烯B4。
