Panos R J, Patel R, Bak P M
Pulmonary Division, Northwestern University Medical School, Chicago, Illinois 60611, USA.
Am J Respir Cell Mol Biol. 1996 Nov;15(5):574-81. doi: 10.1165/ajrcmb.15.5.8918364.
Alveolar epithelial injury occurs universally in common respiratory illnesses associated with diffuse lung damage. After alveolar injury, type II cells proliferate and reestablish epithelial integrity, thereby restoring normal lung structure and function. However, the regulation of type II cell proliferation and alveolar epithelial repair is poorly understood. Hepatocyte growth factor/scatter factor (HGF/SF) is a heparin-binding growth factor that has been shown to be mitogenic for cultured alveolar type II cells. In this study, we determined the effect of intratracheal instillation of rhHGF/SF on type II cell proliferation in vivo. To quantify the alveolar type II cell proliferative response, we developed a double-label immunohistochemical technique to detect replicating alveolar type II cells in formalin-fixed lung sections that utilized the identification of proliferating cells by bromodeoxyuridine (BrdUrd) incorporation into DNA and alveolar type II cells by 3F9 immunoreactivity. BrdUrd detection was optimized by enzymatic antigen recovery and silver intensification of the horseradish peroxidase reaction product. Intratracheal instillation of rhHGF/SF induced a time- and dose-dependent increase in type II cell proliferation. The type II cell labeling index increased to 12.3 +/- 6.0% 48 h after 1.0 mg/kg rhHGF/SF administration, compared with 2.6 +/- 0.9% after PBS instillation. To compare the normal type II cell reparative response with the level of proliferation after exogenous rhHGF/SF administration, we measured the specific alveolar type II cell labeling index in rat lung sections obtained from animals exposed to hyperoxia for 50 h and then allowed to recover in room air. After 1 day of recovery, the alveolar type II cell labeling index was 0.45 +/- 0.2%. The specific labeling index increased to 5.4 +/- 1.3% at 2 days and then declined to 0.31 +/- 0.16% 5 days after hyperoxia exposure. In animals not exposed to hyperoxia, the alveolar type II cell labeling index was 0.6 +/- 0.14%. These studies demonstrated that intratracheal instillation of rhHGF/SF promoted alveolar type II cell proliferation in vivo. The maximal level of type II cell proliferation after rhHGF/SF administration was more than twice that reached during recovery from hyperoxia exposure. Thus, intratracheal instillation of HGF/SF may provide a potential strategy to promote type II cell proliferation and augment alveolar epithelial repair after lung injury.
肺泡上皮损伤普遍发生于与弥漫性肺损伤相关的常见呼吸系统疾病中。肺泡损伤后，Ⅱ型细胞增殖并重新建立上皮完整性，从而恢复正常的肺结构和功能。然而，Ⅱ型细胞增殖和肺泡上皮修复的调控机制尚不清楚。肝细胞生长因子/分散因子（HGF/SF）是一种肝素结合生长因子，已被证明对培养的肺泡Ⅱ型细胞具有促有丝分裂作用。在本研究中，我们测定了气管内滴注重组人HGF/SF（rhHGF/SF）对体内Ⅱ型细胞增殖的影响。为了量化肺泡Ⅱ型细胞的增殖反应，我们开发了一种双标记免疫组织化学技术，用于检测福尔马林固定肺切片中正在复制的肺泡Ⅱ型细胞，该技术利用溴脱氧尿苷（BrdUrd）掺入DNA来鉴定增殖细胞，并通过3F9免疫反应性鉴定肺泡Ⅱ型细胞。通过酶促抗原修复和辣根过氧化物酶反应产物的银增强来优化BrdUrd检测。气管内滴注rhHGF/SF可诱导Ⅱ型细胞增殖呈时间和剂量依赖性增加。给予1.0mg/kg rhHGF/SF后48小时，Ⅱ型细胞标记指数增至12.3±6.0%，而滴注PBS后为2.6±0.9%。为了比较正常Ⅱ型细胞的修复反应与外源性rhHGF/SF给药后的增殖水平，我们测量了从暴露于高氧50小时然后在室内空气中恢复的动物获得的大鼠肺切片中特定的肺泡Ⅱ型细胞标记指数。恢复1天后，肺泡Ⅱ型细胞标记指数为0.45±0.2%。高氧暴露后2天，特定标记指数增至5.4±1.3%，然后在5天后降至0.31±0.16%。在未暴露于高氧的动物中，肺泡Ⅱ型细胞标记指数为0.6±0.14%。这些研究表明，气管内滴注rhHGF/SF可促进体内肺泡Ⅱ型细胞增殖。rhHGF/SF给药后Ⅱ型细胞增殖的最大水平是高氧暴露恢复期间达到水平的两倍多。因此，气管内滴注HGF/SF可能提供一种促进Ⅱ型细胞增殖和增强肺损伤后肺泡上皮修复的潜在策略。
