CELL BIOLOGY OF SURFACTANT PROTEIN B (SP B)

先天性肺泡蛋白沉积症(CAP)是一种致命的家族性疾病。 足月新生儿。我们已经确定了一个原因：缺乏表面活性物质 蛋白B(SP-B)由II型病毒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 我们发现了SP-B基因的两个突变--更常见的是， 移码突变-121ins2，或3‘剪接连接缺失(增量 (儿科杂志：125：43-50,1994)。意义重大的是，我们的 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； 免疫金标记示SP-A、SP-C和膜性蛋白积聚 II型细胞与其基底膜之间的(脂)泡 提示II型细胞表面活性物质的顶端(极性)分泌丧失 脂类和蛋白质。(Am J Respir Cell Mol Biol 11：230-239,1994)。 这些功能提供了对SP-B在类型中的重要性的独特见解 II细胞生物学，并提出SP-B：1)是一种调节因子的假说 在合成、组装和分泌表面活性物质脂类和 蛋白质SP-A和SP-C；2)维持细胞必需的极性 以保持牙槽衬里的解剖和结构完整性。 为了检验这些假设，我们将使用我们不朽的、充分的 特化的、极化的、分化的II型细胞系T7， 分泌表面活性物质脂质和SP-A、SP-B和SP-C，以：1)产生 我们的SP-B反义逆转录病毒感染SP-B基因缺陷细胞系 Moloney鼠白血病病毒来源的载体；2)正常对照 和SP-B缺乏的II型细胞(SP-B+和SP-B-)合成， 表面活性物质脂类的组装、极性分泌、再利用和 SP-A、SP-B和SP-C蛋白与细胞极性的关系 胆碱和蛋氨酸与脉冲式磷脂结合 检测、免疫沉淀、免疫印迹和免疫定位 具有荧光、共聚焦和电子显微镜的表面活性剂。 这些研究将极大地扩大对SP-B在 调节表面活性物质的新陈代谢和维持细胞的完整性 肺泡上皮和表面活性物质衬里在许多情况下被破坏 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.