MICROSCOPIC IMAGING OF ALVEOLAR FUNCTION

肺泡功能的显微成像

• 批准号: 6637528
• 负责人: Jahar Bhattacharya
• 金额: $ 25.72万
• 依托单位: ST. LUKE'S-ROOSEVELT INST FOR HLTH SCIS
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-10 至 2004-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6637528
• 关键词: bioimaging /biomedical imaging; calcium flux; calcium indicator; confocal scanning microscopy; fluorescent dye /probe; fluorimetry; immunofluorescence technique; intravital microscopy; laboratory rat; lung alveolus; pulmonary surfactants; respiratory airway volume; respiratory epithelium; secretion

Aims: Our overall objective is to understand the in situ cell biology of lung alveoli. Here, we will determine the role of the cytosolic free calcium concentration ([Ca2+]i) in alveolar secretion stimulated by mechanical stress and soluble stimulants. The specific aims are to quantify for the first time, [Ca2+]i and secretion rates in type II cells of intact alveoli. Secretion will be stimulated by different amounts cf lung inflation, and different surfactant secretagogues. The new hypotheses to be tested are that (1) type II cell secretion is regulated by intercellular communication of oscillatory [Ca2+]i signals, and (2) type II cells exhibit polarity in their agonist- induced secretion responses. Procedures: (I) [Ca2+]i quantification. Fluorometric imaging of [Ca2+]i- sensitive dyes will be conducted intravitally in single alveoli of the isolated, blood-perfused rat lung, using both conventional and confocal microscopy. Amplitude and phase locking of [Ca2+]i oscillations will be analyzed in the frequency domain by fast Fourier transforms (FFT). (2) Immunoimaging. Type I and type II cells will be identified by imaging cell-specific immunofluorescence. (3) Secretion. Type II cell secretion will be determined in single cells by the loss of cell fluorescence attributable to the acidotropic dye LysoTracker Green, and by the alveolar fluorescence of the phospholipid.binding dye FM 1-43. (4) 3-D reconstruction. Dimensional changes in type I and type II cells, [Ca2+]i and secretion rates will be determined in 3-D reconstructions of alveoli inflated to different extents. Significance: This proposal addresses a new understanding of type II cell secretion. Type II cell physiology is basic to lung function, but remains inadequately understood. It is important to know the role of [Ca2+]i in type II cell secretion, because dysregulation of alveolar [Ca2+]i nay be common to many mechanisms that affect type II cell function and thereby, promote lung injury. Sustained [Ca2+]i increases, or [Ca2+]i oscillations in pathological conditions, may constitute a potent signal for gene transcription and consequently, lung remodeling. If preliminary data bear out, this research will prove for the first time that in alveolar cells, prolonged [Ca2+]i oscillations markedly extend beyond duration of stimulus. These oscillations may regulate type II cell secretion by intercellular intercommunication. The presence of polarity in type II cell responses may lead to different secretion responses depending on whether secretagogues are delivered by the blood or the airborne route. No previous understanding of such mechanisms exist in lung alveoli. These proposed studies are therefore, outstandingly novel and important.

目的：我们的总体目标是了解肺肺泡的原位细胞生物学。在这里，我们将确定胞质游离钙浓度（[Ca2+] i）在机械应力和可溶性刺​​激剂刺激的肺泡分泌中的作用。具体的目的是首次量化[Ca2+] I和完整肺泡II型细胞中的分泌速率。分泌物将受到不同量的CF肺通胀和不同的表面活性剂促分泌物的刺激。要测试的新假设是（1）II型细胞分泌受振荡性[Ca2+] I信号的细胞间通信，（2）II型细胞在其激动剂诱导的分泌反应中表现出极性。程序：（i）[Ca2+] i量化。 [Ca2+] i-敏感染料的荧光成像将使用常规显微镜和共聚焦显微镜在分离的，血液per的大鼠肺的单个肺泡中进行室内进行。 [Ca2+] I振荡的振幅和相位锁定将通过快速傅立叶变换（FFT）在频域中分析。 （2）免疫影像。 I型和II型细胞将通过成像细胞特异性免疫荧光鉴定。 （3）分泌。 II型细胞分泌将在单个细胞中通过可归因于酸性染料lysotracker绿的细胞荧光以及磷脂的肺泡荧光。结合染料FM 1-43。 （4）3-D重建。 I型和II型细胞的尺寸变化，[Ca2+] I和分泌速率将在膨胀到不同范围的肺泡的3-D重建中确定。意义：该提案解决了对II型细胞分泌的新理解。 II型细胞生理学是肺功能的基础，但仍然不充分理解。重要的是要知道[Ca2+] I在II型细胞分泌中的作用，因为肺泡[Ca2+]的失调I对许多影响II型细胞功能的机制而言是常见的，从而促进肺损伤。持续的[Ca2+] I增加，或者在病理条件下的[Ca2+] I振荡可能构成基因转录的有效信号，因此肺部重塑。如果初步数据含有，这项研究将首次证明在肺泡细胞中，延长[Ca2+] I振荡显着延伸超出了刺激的持续时间。这些振荡可能通过细胞间间断调节II型细胞分泌。 II型细胞反应中极性的存在可能会导致不同的分泌反应，具体取决于促分子是由血液还是空中途径传递的。肺肺泡中没有先前对这种机制的理解。因此，这些提出的研究是实用新颖和重要的。