Role of Alveolar Type II Epithelial Cells in the Repairing Process after Lung Injury; Stimulating Factors for Cell Proliferation and Ion Transport

1. 从切除的人肺中分离和培养人肺泡II型细胞：标本来自所有接受肺叶切除术或全肺切除术的原发性肿瘤患者。采用弹性蛋白酶和密度梯度对大鼠ⅱ型细胞分离方法进行改进，分离肺泡ⅱ型细胞。II型细胞的产量为2.7 x10^6个细胞/g组织重。培养1 d后，8个分离株中ⅱ型细胞的百分率为71.4%。这些人类细胞比分离的大鼠II型细胞有更多更小的颗粒。细胞电镜显示特征性的表面微绒毛和胞浆内板层体，部分为多中心。通过亲和素-生物素染色检查细胞骨架结构，它们具有细小的中间纤维，如肌动蛋白和角蛋白。我们测量了人II型细胞在培养1天后3-6小时肺表面活性剂载脂蛋白的分泌。Apoprotein在3 h内分泌33.1 ng/10^6个细胞，在6 h内分泌35.3 ng/10^6个细胞。ADP 10^<-3>M.2对这种分泌的刺激几乎是2倍。肺泡II型细胞的功能：(1)二氧化硅的体外作用；肺泡II型细胞在二氧化硅处理的肺中出现增生性和增生性，我们通过测量氚化胸腺嘧啶掺入来研究II型细胞的增殖。II型细胞与经二氧化硅刺激的肺泡巨噬细胞共培养，使用transwell膜，胸苷的掺入被抑制为对照组的46.9%。(2)体内培养；由于体外分离的过程和条件，细胞必然会受到一定程度的损伤和失去表面受体，我们开发了一种新的体内培养系统，将分离的细胞利用扩散室再次植入另一只大鼠的腹膜或皮下区域。体外培养的细胞增殖更大，离子转运更活跃。(3)细胞外基质作用；纤维连接蛋白介导肺泡II型细胞的体外粘附。单分子膜具有较高的电位差和电阻。纤维连接蛋白上II型细胞的荧光显微镜显示其周围有明显的肌动蛋白和角蛋白束聚集。纤维连接蛋白是细胞外基质的重要功能成分，可能在肺损伤后肺泡再上皮化过程中支持肺泡II型细胞。气管上皮的功能：气管上皮具有与肺泡II型细胞相似的功能，如离子转运和细胞增殖。少

1. Isolation and culture of human alveolar type II cells from resected human lungs: Specimens were obtained from ll patients undergoing lobectomy or pneumonectomy for primary tumors. Alveolar type II cells were isolated by a modification of the method for isolating rat type II cells with elastase and density gradients. The yield of type II cells was 2.7 x10^6 cells/g of tissue weigh. the percentage of type ii cells as determined by the papanicolaou stain after one day in culture was 71.0 3.4 % in 8 of ll isolations. These human cells have more numerous and smaller granules than do isolated rat type II cells. EM of the cells showed characteristic surface microvilli and intracytoplasmic lamellar bodies, of which some were multicentric. Examining the cytoskeleton structure by an avidin-biotin stain, they have fine intermediate filaments such as actin and keratin. We measured the secretion of pulmonary surfactant apoprotein from human type II cells over 3-6 hours after one day in culture. … More Apoprotein was secreted about 33.1 ng/10^6 cells in 3 hrs, and 35.3 ng/10^6 cells in 6 hrs. This secretion was stimulated almost 2-fold by ADP 10^<-3>M.2. Functions of alveolar type II cells: (1) effect of silica in vitro; Alveolar type II cells appear hypertrophic and hyperlasic in silica-treated lungs, we investigated type II cell proliferations, measuring tritiated thymidine incorporation. Co-culturing type II cells with alveolar macrophages stimulated by silica, using transwell membrane, thymidine incorporation was inhibited to 46.9 % of the control. (2) in vivo culture; Altough the cells are necessarily damaged and lose surface receptors to some extent by the isolation procedure and conditions in vitro, we develop a new system of in vivo culture, which isolated cells are again implanted into the peritoneum or subcutaneous region of another rat, using diffusion chambers. The cells in vivo culture more proliferated and had more active ion transport. (3) effect of extracellular matrix; Fibronectin mediates alveolar type II cells adherence in vitro. Monolayers on fibronection coated filter have higher pothtial difference and resistance. Fluorescence microscopy of type II cells on fibronectin showed a marked accumulation of actin and keratin bundles in their periphery. Fibronectin is an important functional component of the extracellular matrix that may support alveolar type II cells during the alveolar re-epithelization after lung injury.3. Function of tracheal epithelium: The tracheal epithelium have some similar functions to those of alveolar type II cells, such as ion transport and cell proliferation. Less