Transcriptional Profiling of Lung Macrophages from Preterm Infants Identifies Disease Related Programs. Transcriptional Profiling of Lung Macrophages from Preterm Infants Identifies Disease Related Programs

To understand the molecular mechanisms of human lung macrophage development, function, and role in BPD pathogenesis, we conducted a clinical study using isolated tracheal aspirate macrophages from intubated preterm infants born before 30 wk gestation. One hundred twenty-eight patients intubated for respiratory distress syndrome and surfactant administration were consented for the study. Overall design: Tracheal aspirate cells were collected from intubated preterm infants as detailed below. After instillation of 0.5 ml of normal saline into the endotracheal tube, a suction catheter was introduced into the endotracheal tube and tracheal secretions were aspirated into a sterile mucus trap. The suction catheter was subsequently rinsed with an additional 1.5 ml of sterile saline to clear any remaining aspirate from the catheter into the mucus trap. Cells were collected by centrifugation and resuspended in DMEM with 10% fetal bovine serum, penicillin, and streptomycin. Equal aliquots of resuspended cells were divided between two separate tissue culture treated plates and incubated at 37 ˚C in a humidified environment of 95% air and 5% CO2 to allow macrophage attachment. After 30 min, nonadherent cells were removed by gentle washing and fresh cell culture media was applied. Lipopolysaccharide from E. coli (strain 055:B5, gel purified, Sigma-Aldrich L2637; 250 ng/ml) was added to one of the plates and both were incubated for 4 h at 37 ˚C. After incubation, the media was removed and cells were lysed in TRIzol monophasic solution containing phenol and guanidine isothiocyanate (Thermo Fisher). RNA was isolated using standard protocols for TRIzol extraction. RNA yield and quality were measured using High Sensitivity RNA ScreenTape Analysis (Agilent Tapestation).

为阐明人类肺巨噬细胞的发育、功能及其在支气管肺发育不良（bronchopulmonary dysplasia, BPD）发病机制中的作用，我们开展了一项临床研究，采用孕30周前出生的插管早产儿的分离气管抽吸物巨噬细胞作为研究材料。本研究共纳入128名因呼吸窘迫综合征接受气管插管并予以肺表面活性物质治疗的患者，均签署了研究知情同意书。
整体实验设计如下：从插管早产儿体内收集气管抽吸物细胞，具体操作如下：向气管导管内滴注0.5ml生理盐水后，将吸痰导管插入气管导管，将气管分泌物抽吸至无菌黏液收集器中。随后用额外1.5ml无菌生理盐水冲洗吸痰导管，将导管内残留的抽吸物全部冲入黏液收集器。通过离心收集细胞，并重悬于添加10%胎牛血清、青霉素与链霉素的DMEM培养基中。将重悬后的细胞等分为两份，分别接种于两个经组织细胞培养处理的培养板中，于37℃、95%空气与5%二氧化碳的湿润环境中孵育，以允许巨噬细胞贴壁。30分钟后，通过轻柔洗涤去除非贴壁细胞，并更换新鲜的细胞培养基。向其中一块培养板加入大肠杆菌（菌株055:B5，凝胶纯化，Sigma-Aldrich L2637）脂多糖（终浓度250ng/ml），随后将两块培养板均置于37℃孵育4小时。孵育结束后移除培养基，使用含苯酚与异硫氰酸胍的TRIzol单相裂解液（Thermo Fisher）裂解细胞。采用标准TRIzol提取法分离RNA，通过高灵敏度RNA ScreenTape分析（Agilent Tapestation）检测RNA的产量与质量。