CELL BIOLOGY OF SURFACTANT PROTEIN B (SP B)

先天性肺泡蛋白沉积症（CAP）是一种家族性疾病， 足月新生儿 我们已经确定了一个原因：缺乏表面活性剂 蛋白质B（SP-B），由II型糖尿病分泌的四种表面活性蛋白之一 肺细胞（NEJM 328：406-410，1993）。 在研究的前十个婴儿中， 我们在SP-B基因中发现了两种突变， 移码突变-121ins2，或3'剪接点缺失（δ Ex 4）（J Pediatr：125：43-50，1994）。 意义重大，我们 SP-B缺陷肺的免疫染色显示， II型细胞SP-C和肺泡腔SP-A和SP-C; 免疫胶体金定位显示SP-A、SP-C和膜性聚集 II型细胞与其基底膜之间的（脂质）囊泡 表明II型细胞顶端（极性）表面活性剂分泌的丧失 脂质和蛋白质。 (Am J Respir Cell Mol Biol 11：230-239，1994）。 这些特征为SP-B在类型中的重要性提供了独特的见解 II细胞生物学，并提出假设，SP-B：1）是一个调节 表面活性剂脂质的合成、组装和分泌中的因子， 蛋白质SP-A和SP-C; 2）维持细胞的极性， 以确保牙槽膜的解剖和结构完整性 为了验证这些假设，我们将使用我们不朽的，完全 其特征在于，极化的分化的II型细胞系T7， 分泌表面活性剂脂质和SP-A、SP-B和SP-C，以：1）产生 用我们的SP-B反义逆转录病毒感染SP-B缺陷细胞系 来源于Moloney鼠白血病病毒的载体; 2）在正常小鼠中比较 和SP-B缺陷型II细胞（SP-B+，& SP-B-）的合成， 组装、极性分泌、表面活性剂脂质的再利用， 蛋白SP-A、SP-B和SP-C与细胞极性的关系，使用放射性标记的 胆碱和蛋氨酸掺入与脉冲追踪，磷脂 免疫沉淀、蛋白质印迹和免疫定位 表面活性剂与荧光，共聚焦和电子显微镜。 这些研究将大大扩展对SP-B在以下方面作用的了解 调节表面活性剂代谢，并维持 肺泡上皮细胞和表面活性物质衬里被破坏， 新生儿的重要疾病和多种形式的肺损伤。

Congenital alveolar proteinosis (CAP) is a familial disorder fatal in full term newborns. We have identified a cause: deficiency of surfactant protein B (SP-B) one of four surfactant proteins secreted by the Type II pneumocyte (NEJM 328:406-410, 1993). In the first ten infants studied we identified two mutations in the SP-B gene - the more common, a frameshift mutation - 121ins2, or a 3' splice junction deletion (delta Ex 4) (J Pediatr: 125:43-50, 1994). Of great significance, our immunostaining of SP-B deficient lungs revealed an abnormal increase in Type II cell SP-C and alveolar luminal SP-A and SP-C; ultrastructure with immunogold localization showed accumulation of SP-A, SP-C and membranous (lipid) vesicles between Type II cells and their basement membrane indicating loss of Type II cell apical (polar) secretion of surfactant lipids and proteins. (Am J Respir Cell Mol Biol 11:230-239, 1994). These features provide unique insight into the importance of SP-B in Type II cell biology and suggest the hypotheses that SP-B: 1) is a regulatory factor in the synthesis, assembly and secretion of surfactant lipids and proteins SP-A and SP-C; 2) maintains the polarity of the cell essential for anatomical and structural integrity of the alveolar lining. To test these hypotheses we will use our immortalized, fully characterized, polarized, differentiated type II cell line T7, that secretes surfactant lipids and SP-A, SP-B, and SP-C, to: 1) produce an SP-B deficient cell line by infection with our SP-B anti-sense retroviral vector derived from Moloney murine leukemia virus; 2) compare in normal and SP-B deficient Type II cells (SP-B+, & SP-B-) the synthesis, assembly, polar secretion, reutilization of surfactant lipids and proteins SP-A, SP-B, and SP-C and the cell polarity, using radiolabelled choline and methionine incorporation with pulse-chase, phospholipid assay, immunoprecipitation, Western blotting and immunolocalization of surfactant with fluorescence, confocal and electron microscopy. These studies will greatly expand understanding of the role of SP-B in regulating surfactant metabolism and in maintaining the integrity of the alveolar epithelial and surfactant lining that are disrupted in many important diseases of the newborn and many forms of lung injury.