Isolation and culture of human alveolar type II epithelial cells. Characterization of their phospholipid secretion.
Isolation and culture of human alveolar type II epithelial cells. Characterization of their phospholipid secretion.
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人肺泡II型上皮细胞的分离和培养。
DOI：
10.1164/arrd.1984.130.6.1156
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发表时间：
1984
期刊：
影响因子：
--
通讯作者：
Mason,RJ
中科院分区：
文献类型：
--
作者：
Robinson,PC;Voelker,DR;Mason,RJ
肺泡II型上皮细胞以前从啮齿类动物身上分离出来，但从人类身上没有分离出来。本研究的目的是分离培养人肺泡II型细胞，并对其磷脂分泌进行研究。肺组织取自13例肺叶或全肺切除术后患者。用猪胰腺弹性蛋白酶和不连续的甲硝唑胺密度梯度分离大鼠II型细胞的方法进行了改进。差速贴壁法进一步纯化细胞，然后在牛角膜内皮细胞制备的细胞外基质上进行培养。每克组织可获得1.3~4.8×106个细胞，细胞存活率为96±1%(平均±SE，n=10)。13个实验中有9个实验的II型细胞纯度为84±4%(平均值±SE)。在其余4次实验中，纯度均低于50%。电子显微镜显示超微结构保存完好。~(32)PI标记的细胞分泌肺表面活性物质特有的磷脂，主要是磷脂酰胆碱(68%)和磷脂酰甘油(12%)。磷脂分泌可被乙酸十四酰佛波酯(4.5倍基线)和β肾上腺素能激动剂特布他林(2.0倍基线)刺激，但不能被胆碱能激动剂氨甲胆碱所刺激。综上所述，人肺泡II型上皮细胞可以从切除的肺中分离出来，并在培养中保持，它们分泌具有肺表面活性物质特征的磷脂。
Alveolar type II epithelial cells have been isolated previously from rodents but not from humans. The aims of this study were to isolate and culture alveolar type II cells from human lungs, and to study their phospholipid secretion. Lung tissue was obtained from 13 patients after lobectomy or pneumonectomy. Type II cells were isolated by a modification of our method for isolating rat type II cells with porcine pancreatic elastase and discontinuous metrizamide density gradients. Cells were purified further by differential adherence and they were then cultured on an extracellular matrix prepared from bovine corneal endothelial cells. We obtained 1.3 to 4.8 × 106cells per gram of tissue with cell viability on the day of isolation of 96 ± 1% (mean ± SE, n = 10). Type II cell purity, assessed by Papanicolaou stain on Day 1 in culture, was 84 ± 4% (mean ± SE) in 9 of the 13 experiments. In the remaining 4 experiments, the purity was less than 50%. Electron microscopy showed well-preserved ultrastructure. The cells, radiolabeled with32Pi, secreted the phospholipids characteristic of pulmonary surfactant, primarily phosphatidylcholine (68%) and phosphatidylglycerol (12%). Phospholipid secretion was stimulated by tetradecanoyl phorbol acetate (4.5 times baseline) and by the beta adrenergic agonist terbutaline (2.0 times baseline), but not by the cholinergic agonist carbamylcholine chloride. In summary, human alveolar type II epithelial cells can be isolated from resected lung and maintained in culture, and they secrete the phospholipids characteristic of pulmonary surface active material.