Method for the isolation of surfactant from homogenates and lavages of lung of adult, newborn, and fetal rats.
Method for the isolation of surfactant from homogenates and lavages of lung of adult, newborn, and fetal rats.
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从成年、新生和胎鼠的肺匀浆和灌洗液中分离表面活性剂的方法。
DOI：
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发表时间：
1977
期刊：
影响因子：
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通讯作者：
B. Lombardi
中科院分区：
文献类型：
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作者：
S. Katyal;L. Estes;B. Lombardi
本文介绍了一种用差速不连续密度梯度离心法从大鼠肺匀浆和灌洗液中分离肺表面活性物质的方法。分离的表面活性剂，其特征在于，通过电子显微镜，酶标记物测定不同的亚细胞器，其化学组成。从肺匀浆中分离的表面活性剂似乎不含其他污染性细胞器。从整个匀浆中分离的表面活性剂的电子显微镜检查显示存在板层体和管状髓鞘图，因此，代表总肺表面活性剂。另一方面，从肺灌洗液中制备的表面活性剂大多具有管状髓鞘图，因此代表细胞外表面活性剂。这两种制剂都富含磷脂，特别是卵磷脂和磷脂酰甘油，并显示出高磷脂与蛋白质的比例。用该方法研究了大鼠肺发育过程中肺表面活性物质的含量和组成。从妊娠第19至21天的胎仔以及新生和成年大鼠的肺匀浆中分离出表面活性剂。在妊娠19至21天期间，蛋白质和磷脂的量分别增加10倍和20倍（毫克/克湿肺）。出生后再增加2倍。在第20天，磷脂酰胆碱占总磷脂的65%，在第21天占71%，在足月时占81%。在此期间，磷脂酰胆碱与鞘磷脂的比例和磷脂酰甘油的量逐渐增加。肺表面活性物质中二饱和卵磷脂的量（以总磷脂酰胆碱的百分比表示）从出生第20天的49%增加到出生时的53%。新生儿阶段后，不饱和卵磷脂的量没有变化。本发明用于分离肺表面活性剂的方法是可重复的，耗时较少，并且可用于从小的肺等分试样中定量分离表面活性剂。
A method is presented for the isolation of pulmonary surfactant from rat lung homogenates and lavages using differential discontinuous density gradient centrifugation. The isolated surfactant was characterized by electron microscopy, assay of enzyme markers for different subcellular organelles, and its chemical composition. Surfactant that was isolated from lung homogenates appeared to be free of other contaminating cellular organelles. Electron microscopic examination of surfactant isolated from whole homogenates showed the presence of lamellar bodies and tubular myelin figures and, hence, represented total lung surfactant. On the other hand, surfactant prepared from lung lavages had mostly tubular myelin figures and, thus, represented extracellular surfactant. Both preparations were rich in phospholipids, especially lecithins and phosphatidylglycerol, and showed high phospholipid to protein ratios. The method was used to study the quantity and composition of surfactant during lung development in rats. Surfactant was isolated from lung homogenates of fetuses at days 19 to 21 of gestation and from newborn and adult rats. During the period of 19 to 21 days of gestation, there is a 10- and 20-fold increase in the amount of protein- and phospholipids (milligrams per gram of wet lung), respectively. A further 2-fold increase occurs after birth. Phosphatidylcholines account for 65% of the total phospholipids at day 20, 71% on day 21, and 81% at term. A progressive increase in the phosphatidylcholine to sphingomyelin ratios and in the amount of phosphatidylglycerol occurs during this period. The amount of disaturated lecithins (expressed as percentage of total phosphatidylcholines) in the lung surfactant increases from 49% at day 20 to 53% at birth. There is no change in the amount of disaturated lecithins after the newborn stage. The present method for the isolation of lung surfactant is reproducible, is less time consuming, and can be used to isolate quantitatively surfactant from small lung aliquots.