Lakritz Jeffrey, Marsh Antoinette E, Cockrell Mary, Smith Michael F, Tyler Jeff W
Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211, USA.
Am J Vet Res. 2004 Feb;65(2):163-72. doi: 10.2460/ajvr.2004.65.163.
To characterize gelatinases in bronchoalveolar lavage fluid (BALF) and gelatinases produced by alveolar macrophages of healthy calves.
Samples of BALF and alveolar macrophages obtained from 20 healthy 2-month-old calves.
BALF was examined by use of gelatin zymography and immunoblotting to detect gelatinases and tissue inhibitor of metalloproteinase (TIMP)-1 and -2. Cultured alveolar macrophages were stimulated with lipopolysaccharide (LPS), and conditioned medium was subjected to zymography. Alveolar macrophage RNA was used for reverse transcriptase-polymerase chain reaction assay of matrix metalloproteinases (MMPs), cyclooxygenase-2, and inducible nitric oxide synthase.
Gelatinolytic activity in BALF was evident at 92 kd (14/20 calves; latent MMP-9) and 72 kd (18/20; latent MMP-2). Gelatinolytic activity was evident at 82 kd (10/20 calves; active MMP-9) and 62 kd (17/20; active MMP-2). Gelatinases were inhibited by metal chelators but not serine protease inhibitors. Immunoblotting of BALF protein and conditioned medium confirmed the MMP-2 and -9 proteins. Endogenous inhibitors (ie, TIMPs) were detected in BALF from all calves (TIMP-1) or BALF from only 4 calves (TIMP-2). Cultured alveolar macrophages expressed detectable amounts of MMP-9 mRNA but not MMP-2 mRNA.
Healthy calves have detectable amounts of the gelatinases MMP-2 and -9 in BALF Endogenous inhibitors of MMPs were detected in BALF (ie, TIMP-1, all calves; TIMP-2, 4 calves). Lipopolysaccharide-stimulated alveolar macrophages express MMP-9 but not MMP-2 mRNA. The role of proteases in the pathogenesis of lung injury associated with pneumonia has yet to be determined.
鉴定支气管肺泡灌洗液（BALF）中的明胶酶以及健康犊牛肺泡巨噬细胞产生的明胶酶。
从20头健康的2月龄犊牛获取BALF和肺泡巨噬细胞样本。
使用明胶酶谱法和免疫印迹法检测BALF中的明胶酶以及金属蛋白酶组织抑制剂（TIMP）-1和-2。用脂多糖（LPS）刺激培养的肺泡巨噬细胞，对条件培养基进行酶谱分析。肺泡巨噬细胞RNA用于基质金属蛋白酶（MMP）、环氧化酶-2和诱导型一氧化氮合酶的逆转录聚合酶链反应检测。
BALF中的明胶酶活性在92 kd（14/20头犊牛；潜伏型MMP-9）和72 kd（18/20；潜伏型MMP-2）处明显。明胶酶活性在82 kd（10/20头犊牛；活性MMP-9）和62 kd（17/20；活性MMP-2）处明显。明胶酶被金属螯合剂抑制，但不被丝氨酸蛋白酶抑制剂抑制。BALF蛋白和条件培养基的免疫印迹证实了MMP-2和-9蛋白。在所有犊牛的BALF中均检测到内源性抑制剂（即TIMP）（TIMP-1），或仅在4头犊牛的BALF中检测到TIMP-2。培养的肺泡巨噬细胞表达可检测量的MMP-9 mRNA，但不表达MMP-2 mRNA。
健康犊牛的BALF中可检测到明胶酶MMP-2和-9。在BALF中检测到MMP的内源性抑制剂（即TIMP-1，所有犊牛；TIMP-2，4头犊牛）。脂多糖刺激的肺泡巨噬细胞表达MMP-9，但不表达MMP-2 mRNA。蛋白酶在与肺炎相关的肺损伤发病机制中的作用尚待确定。
