Pharmacologic reduction in tumor necrosis factor activity of pulmonary alveolar macrophages.
Pharmacologic reduction in tumor necrosis factor activity of pulmonary alveolar macrophages.
复制标题
药理学降低肺泡巨噬细胞的肿瘤坏死因子活性。
DOI：
10.1165/ajrcmb/8.2.169
复制
发表时间：
1993
影响因子：
6.4
通讯作者：
Fowler3rd,AA
中科院分区：
文献类型：
--
作者：
Leeper-Woodford,SK;Fisher,BJ;Sugerman,HJ;Fowler3rd,AA
材料和方法PAM的分离将体重为15至25 kg的约克郡猪用肌内氯胺酮（25 mg/kg）和硫酸阿托品（0.4 mg）预先麻醉，并以仰卧位放置。通过静脉内给予戊巴比妥钠（10 mg/kg）麻醉动物，用带套囊的气管内导管（National catheter）进行气管内插管，并用泮库溴铵（0.2 mg/min）麻痹。使用大型动物呼吸机（哈佛;潮气量，15 ml/kg; 0.5 FI 02）以产生40 mm Hg动脉Pco的频率进行机械通气。在所有动物中使用呼气末正压（5em H2O）。使用纤维支气管镜（町田VT-5100 C，4 mm;奥兰治堡，NY）通过气管内导管进行支气管肺泡灌洗。将支气管镜的远端楔入右肺中叶和下叶的三级或四级支气管，并使用25 ml无菌0.9% NaCl等分试样进行75 ml灌洗。收集回收的灌洗液部分并离心（400 × g，4 ℃，10分钟）.为了定义肺泡细胞群，使用改良的Wright-Giemsa染色剂（Diff Quik; American Scientific Products，McGaw Park，IL）在细胞离心（Cytospin 2; Shandon，Pittsburgh，PA）制备物上进行肺泡巨噬细胞、单核细胞、中性粒细胞和淋巴细胞的分类计数。将细胞级分在磷酸盐缓冲盐水中洗涤，计数，并以106个细胞/ml悬浮在含有1%青霉素-链霉素和5%胎牛血清（DMEM）的Dulbecco培养基（GIBCO，Grand Island，NY）的培养基中。使PAM粘附在塑料培养皿（康宁，24孔板）上1小时（37 ℃，5%CO2），然后倒出培养基和未粘附的细胞，并将1 ml新鲜DMEM铺在粘附的PAM上。该方法产生> 95%的PAM，如使用非特异性酯酶染色（Sigma Chemical Co.，获得PAM条件培养基的方案所有实验均在来自四头猪中每头的单独PAM群体上进行。将粘附的PAM在随机指定的实验条件下孵育（37 ℃，5%CO2）指定的时间间隔（1、2、4、6、24或48小时）。猪PAM产生TNF的能力通过在单独的DMEM或DMEM中孵育粘附的细胞来评估。
Materials and MethodsIsolation of PAM Yorkshire pigs weighing 15 to 25 kg were pre-anesthetized with intramuscular ketamine (25 mg/kg) and atropine sulfate (0.4 mg) and placed in a supine position. Animals were anesthetized by intravenous administration of sodium pentobarbital (10mg/kg), endotracheally intubated with a cuffed endotracheal tube (National catheter), and paralyzed with pancuronium bromide (0.2 mg/min). Mechanical ventilation was performed using a large animal ventilator (Harvard; tidal volume, 15 ml/kg; 0.5 FI02) at a frequency that produced an arterial Pco, of 40 mm Hg. Positive end expiratory pressure (5 em H20) was used in all animals. Bronchoalveolar lavage was performed using a fiberoptic bronchoscope (Machida VT-5100C, 4 mm; Orangeburg, NY) via the endotracheal tube. The distal end of the bronchoscope was wedged into a third-or fourth-order bronchus of the middle and lower lobes of the right lung, and a 75-mllavage using 25-ml aliquots of sterile 0.9% NaCl was performed. The recovered lavage fractions were pooled and centrifuged (400 x g, 4 C, 10 min). To define the alveolar cell population, differential counts of the alveolar macrophages, monocytes, neutrophils, and lymphocytes were performed on cytocentrifuge (Cytospin 2; Shandon, Pittsburgh, PA) preparations using modified Wright-Giemsa stain (Diff Quik; American Scientific Products, McGaw Park, IL). Cell fractions were washed in phosphate-buffered saline, counted, and suspended in culture medium at 1Q6 cells/ml of Dulbecco's medium (GIBCO, Grand Island, NY) containing 1% penicillin-streptomycin and 5% fetal bovine serum (DMEM). The PAM were allowed to adhere to plastic culture dishes (Corning, 24-well plates) for 1 h (37 C, 5% COz), the medium and nonadherent cells were then decanted, and 1 ml of fresh DMEM was layered over the adhered PAM. This method yielded> 95% PAM, as demonstrated using nonspecific esterase stain (Sigma Chemical Co., St. Louis, MO) on selected preparations.Protocol for Obtaining PAM Conditioned Medium All experiments were performed on individual PAM populations from each of four pigs. Adhered PAM were incubated (37 C, 5% CO2) for designated time intervals (1, 2, 4, 6, 24, or 48 h) with one of the randomly assigned experimental conditions. The ability of the porcine PAM to produce TNF was assessed by incubating adhered cells in DMEM alone or