Basic Study on Lung Injuries and Neutrophils-Isolation and Purification of Alveolus-Derived Neutrophil Inhibitory Factor and Its Effects on The Development of Lung Injuries-

在肺损伤的发生发展过程中，主要涉及浸润性的中性粒细胞及其损伤诱导物，如蛋白酶或活性氧自由基。大多数治疗方法对这些疾病状态都没有效果，这些患者的预后仍然很差。我们主要关注在肺泡中发现的中性粒细胞抑制因子。在一系列这些研究中，我们报道了大鼠肺表面活性物质和大鼠肺泡II型细胞目录抑制人中性粒细胞超氧化物的产生。我们还在大鼠支气管肺泡灌洗液的可溶性部分中发现了另一种中性粒细胞抑制因子。这些抑制活性在100ﾟC处理10min后被破坏，表明抑制来自蛋白质组分。这些抑制因素也不同于以前描述的那些因素，如超氧化物歧化酶，有望成为治疗L…患者的新药物。在1996年的研究中，我们已经证明这种抑制作用不依赖于表面活性物质的特异性蛋白，如SP-A或SP-D。在接下来的一年里，我们将重点放在抑制细胞因子之一，转化生长因子-β(TGF-β)作为中性粒细胞抑制因子的候选。我们尝试了(1)显示人转化生长因子-β是否能抑制人中性粒细胞功能的实验，(2)测定肺灌洗液中转化生长因子-β的浓度，以及(3)中和抗转化生长因子-β抗体的抑制活性。然而，由于人和大鼠物种差异的原因，这种细胞因子的非特异性吸附活性很高，结果并不令人满意。虽然我们确定了这些抑制活性的大约分子量在200-300 kDa左右，但我们无法纯化这些抑制蛋白，因为在纯化过程中失去了抑制活性。目前本研究的目的尚未达到，但需要进一步纯化这些抑制性分子，并建立识别这些分子的特异性单抗。较少

In the development of lung injury, the infiltrating neutrophils and their injury-causative agents, such as protease or active oxygen radicals are mainly involved. Most of the therapies have not been effective for those disease states and the prognosis of these patients are still poor.We have focused on the neutrophil inhibitory factors found in the pulmonary alveolus. In a series of these studies, we reported that rat lung surfactant and rat alveolar type II cells directory inhibited human neutrophil superoxide production. We also found another neutrophil inhibitory factors in the soluble fraction of the bronchoalveolar lavage fluids in rats. These inhibitory activities were destroyed by treating the samples at 100ﾟC for 10min, suggesting that inhibition was derived from the protein components. The inhibitory factors were also shown to be different from those previously described, such as superoxide disumutase (SOD) and were expected to be new therapeutic agents for the patients with l … More ung injuries.In the researches in 1996, we have shown that the inhibitory effect was independent of that by surfactant specific proteins, such as SP-A or SP-D.In the next year, we focused on one of the inhibitory cytokines, transforming growth factor-beta (TGF-beta) as the candidate of neutrophil inhibitory factors. We tried the experiments (1) showing whether human TGF-beta can inhibit human neutrophil functions, (2) measuring the TGF-beta concentration in the lung lavage fluids, and (3) neutralizing the inhibitory activities by anti-TGF-beta antibodies. The results, however, were not satisfactory by the reasons of the species difference between human and rats, the high nonspecific adsorbent activity of this cytokine. Although we determined the approximate molecular weight of these inhibitory activities around 200-300 kDa, we were unable to purify these inhibitory proteins since inhibitory activities were lost during the purification procedures. The goal of the present study has not been achieved at this point, but we further need to purify these inhibitory molecules and to establish the specific monoclonal antibody recognizing these molecules. Less