REACTIVITY OF ALVEOLAR EPITHELIAL-CELLS IN PRIMARY CULTURE WITH TYPE-I CELL MONOCLONAL-ANTIBODIES
REACTIVITY OF ALVEOLAR EPITHELIAL-CELLS IN PRIMARY CULTURE WITH TYPE-I CELL MONOCLONAL-ANTIBODIES
复制标题
DOI：
10.1165/ajrcmb/6.3.296
复制
发表时间：
1992-03-01
影响因子：
6.4
通讯作者：
CRANDALL, ED
中科院分区：
文献类型：
--
作者：
DANTO, SI;ZABSKI, SM;CRANDALL, ED
了解肺泡上皮细胞生长和分化的过程需要能够追踪和分析细胞经历的表型转变。该分析需要针对 II 型，尤其是 I 型肺细胞的特异性表型探针。为此，使用一种旨在增强从粗制肺膜制剂中产生肺特异性克隆的方法，产生了针对 I 型肺泡上皮细胞的单克隆抗体。通过酶联免疫吸附测定和免疫组织化学技术相结合来筛选单克隆抗体，I型细胞特异性的确定主要依靠免疫电镜定位。 I 型肺细胞表型的两个新标记（II F1 和 VIII B2）用于分析在标准组织培养塑料和据报道影响这些培养细胞形态的各种基质上生长的 II 型细胞的原代培养物。在组织培养塑料上，抗体无法与早期（第 1 至 3 天）II 型细胞培养物发生反应。随着培养时间的延长，细胞的反应性逐渐增强，到第 8 天时达到大约 6 倍背景的平台，第 3 天和第 5 天之间增加率最大。这一发现与原代培养物中的 II 型细胞至少部分分化为 I 型细胞的假设是一致的。在层粘连蛋白上生长的 II 型细胞（据报道可延缓 II 型细胞外观的丧失）和在纤连蛋白（据报道可促进细胞扩散和 II 型细胞特征的丧失）上生长的 II 型细胞在体外培养过程中形成 I 型细胞标记，其动力学与在未包被的组织培养塑料上相似。据报道，I 型胶原和组织培养处理的 Nuclepore 过滤器上的细胞支持具有 I 型细胞样形态的单层，在第 3 至 5 天左右也会增加 II F1 和 VIII B2 表位的表达。结合现有的形态学信息，这些数据表明，原代培养物中 II 型细胞对不同肺泡上皮细胞表型标记物的表达可能是独立调节的。本报告中描述的单克隆抗体探针应该有助于继续研究肺泡上皮细胞分化的机制和调节。
An understanding of the process of alveolar epithelial cell growth and differentiation requires the ability to trace and analyze the phenotypic transitions that the cells undergo. This analysis demands specific phenotypic probes to type II and, especially, type I pneumocytes. To this end, monoclonal antibodies have been generated to type I alveolar epithelial cells using an approach designed to enhance production of lung-specific clones from a crude lung membrane preparation. The monoclonal antibodies were screened by a combination of enzyme-linked immunosorbent assay and immunohistochemical techniques, with the determination of type I cell specificity resting primarily on immunoelectron microscopic localization. Two of these new markers of the type I pneumocyte phenotype (II F1 and VIII B2) were used to analyze primary cultures of type II cells growing on standard tissue culture plastic and on a variety of substrata reported to affect the morphology of these cells in culture. On tissue culture plastic, the antibodies fail to react with early (days 1 to 3) type II cell cultures. The cells become progressively more reactive with time in culture to a plateau of approximately 6 times background by day 8, with a maximum rate of increase between days 3 and 5. This finding is consistent with the hypothesis that type II cells in primary culture undergo at least partial differentiation into type I cells. Type II cells grown on laminin, which reportedly delays the loss of type II cell appearance, and on fibronectin, which has been reported to facilitate cell spreading and loss of type II cell features, develop the type I cell markers during cultivation in vitro with kinetics similar to those on uncoated tissue culture plastic. Cells on type I collagen and on tissue culture-treated Nuclepore filters, which have been reported to support monolayers with type I cell-like morphology, also increase their expression of the II F1 and VIII B2 epitopes around days 3 to 5. Taken together with available morphologic information, these data suggest that expression of different alveolar epithelial cell phenotypic markers by type II cells in primary culture may be independently regulated. The monoclonal antibody probes described in this report should prove useful in the continued investigation of the mechanisms and regulation of alveolar epithelial cell differentiation.