Dobbs L G, Geppert E F, Williams M C, Greenleaf R D, Mason R J
Biochim Biophys Acta. 1980 Jun 23;618(3):510-23. doi: 10.1016/0005-2760(80)90270-2.
We used porcine pancreatic elastase to isolate type II cells from the lungs of rats; the yield and purity of the type II cells was better than that obtained by methods using trypsin. In 102 experiments we obtained 82 +/- 23 . 10(6) cells/rat, 68 +/- 11% (mean +/- S.D.) of which type II cells. This preparation of cells, when centrifuged over a discontinuous density gradient, yielded 25 +/- 10 . 10(6) cells/rat, 80 +/- 13% of which were type II cells (n = 102). The cells, after density gradient centrifugation, could be futher purified by centrifugal elutriation (94 +/- 3% type II cells, n = 22) or adherence in primary culture (94 +/- 2% type II cells, n = 34). Type II cells isolated with elastase are similar morphologically and biochemically to type II cells isolated from rats with trypsin. The preparations of cells appeared healthy by several different criteria: ultrastructure, exclusion of vital dye, lack of stimulation of oxygen consumption by exogenous sodium succinate, and linear rates of oxidation of [1-14C]palmitic acid and of incorporation of [1-14C]acetate into fatty acids. Type II cells consumed 75 +/- 20 nmol O2/10(6) cell per h, oxidized [1-14C]palmitic acid at a rate of 0.4 nmol/10(6) cells per h, and incorporated [1-14C]acetate into fatty acids at a rate of 7.5 nmol/10(6) cells per h.
我们使用猪胰弹性蛋白酶从大鼠肺中分离II型细胞；与使用胰蛋白酶的方法相比，II型细胞的产量和纯度更高。在102次实验中，我们每只大鼠获得82±23×10⁶个细胞，其中II型细胞占68±11%（平均值±标准差）。这种细胞制剂在不连续密度梯度上离心后，每只大鼠可获得25±10×10⁶个细胞，其中80±13%为II型细胞（n = 102）。密度梯度离心后的细胞可通过离心淘析进一步纯化（II型细胞占94±3%，n = 22）或在原代培养中贴壁纯化（II型细胞占94±2%，n = 34）。用弹性蛋白酶分离的II型细胞在形态和生化方面与用胰蛋白酶从大鼠中分离的II型细胞相似。通过几种不同标准判断，这些细胞制剂看起来是健康的：超微结构、对活性染料的排斥、外源琥珀酸钠不刺激耗氧量以及[1-¹⁴C]棕榈酸氧化和[1-¹⁴C]乙酸掺入脂肪酸的线性速率。II型细胞每小时每10⁶个细胞消耗75±20 nmol氧气，以每小时每10⁶个细胞0.4 nmol的速率氧化[1-¹⁴C]棕榈酸，并以每小时每10⁶个细胞7.5 nmol的速率将[1-¹⁴C]乙酸掺入脂肪酸。
