新生兔机械通气肺损伤MMP-9与白细胞相关性的研究

目的:研究新生兔机械通气肺损伤中基质金属蛋白酶与灌洗液中白细胞相关关系及机械通气肺损伤的发生机制。方法:对生后日龄1～5天的新生兔72只,按2×2×3析因设计,随机分配在高浓度氧、低浓度氧两组,每组又分为高吸气峰压、低吸气峰压共4组进行机械通气,通气中选取1h、3h、6h3个时间点活杀取肺,左肺灌洗液细胞计数,沉渣涂片细胞分类,测湿干比,并结合病理片分析。ELISA法检测肺组织匀浆中MMP-9的浓度。结果:MMP-9与白细胞总数、单核巨噬细胞正相关,与湿干比呈正相关,与淋巴细胞、分叶核细胞不相关。结论:新生兔机械通气肺损伤,巨噬细胞可能是MMP-9的主要来源,巨噬细胞通过分泌MMP-9参与肺损伤的发生。

新生兔机械通气肺灌洗液中白细胞的动态变化

目的:观察新生兔机械通气肺损伤中白细胞的变化规律。方法:实验于2006-03在南方医科大学儿科实验室进行。将72只新生新西兰兔,按2×2×3析因设计随机分配在高氧(FiO2=1.0)、低氧(FiO2=0.45)组,每组又分为高吸气峰压(2.45kPa)、低吸气峰压(0.98kPa)共4组进行机械通气,选取1,3,6h3个时间点处死动物取肺,每个时间点每组6只兔。取左肺灌洗液进行细胞计数,沉渣涂片细胞分类,测肺湿质量/干质量比,结合病理片分析,同时采用双变量Pearson相关分析法分析各指标间相关性。另取3只兔不干预,为正常对照组。结果:75只兔均进入结果分析。①灌洗液中白细胞计数:在高氧组略高于低氧组,高吸气峰压组略低于低吸气峰压组,差异均不显著(P>0.05);但随时间延长而增高,1,3,6h比差异显著[(11.04±4.00)×109,(16.05±3.99)×109,(15.63±4.83)×109L-1,P<0.001]。②肺湿质量/干质量比:高氧组和低氧组比、高吸气峰压组和低吸气峰压组比差异均不显著(P>0.05),均高于正常对照组(P=0.001);随时间延长其值增高,1,3,6h分别为5.49±0.22,5.64±0.23,5.71±0.22,P=0.003。③肺灌洗液白细胞分类:高氧组分叶核细胞百分率高于低氧组[(21.4±9.9)%,(15.4±7.6)%,P=0.000];高吸气峰压组淋巴细胞百分率高于低吸气峰压组[(53.0±16.3)%,(47.2±14.9)%,P=0.005],而巨噬细胞百分率则低于低吸气峰压组[(28.2±11.0)%,(35.2±12.5)%,P=0.001];随通气时间的延长分叶核细胞百分率下降,而巨噬细胞变化不大。④肺湿质量/干质量与灌洗液中白细胞总数呈正相关(r=0.356,P=0.002),与分叶核计数呈负相关(r=-0.247,P=0.036)。结论:白细胞计数变化同机械通气肺损伤程度一致,与肺水肿有相关性,分类计数的改变随通气策略不同而异。

Change and role of heme oxygenase-1 in injured lungs following limb ischemia/reperfusion in rats

Objective: To study the change and role of heme oxygenase-1 (HO-1) in injured lungs following limb ischemia/reperfusion in rats. Methods: A total of 96 healthy male Sprague-Dawley rats, weighing 250-300 g, were used in this study. Hind limb ischemia was made on 40 rats through clamping the infrarenal aorta for 2 hours with a microvascular clip, then limb reperfusion for 0, 4, 8, 16 and 24 hours (n=8 in each time point) was performed, respectively. Other 8 rats undergoing full surgical operation including isolation of the infrarenal aorta without occlusion were taken as the sham operation group. Lung tissues were obtained from the 48 animals and Northern blotting and Western blotting were employed to measure the changes of HO-1 mRNA and protein expression, respectively. Immunohistochemistry technique was used to determine the cell types responsible for HO-1 expression after limb ischemia/reperfusion. Then hind limb ischemia was made on other 12 rats through clamping the infrarenal aorta for 2 hours with a microvascular clip, among whom, 6 rats were given zinc protoporphyrin (ZnPP), an inhibitor of HO. Then limb reperfusion for 16 hours was performed on all the 12 rats. And other 12 rats underwent full surgical operation including isolation of the infrarenal aorta without occlusion, among whom, 6 rats were then given ZnPP. Then lung tissues were obtained from the 24 animals and lung injury markers, lung histology, polymorphonuclear leukocyte (PMN) count and malondialdehyde (MDA) content were detected, respectively. HO activity was determined through measuring the carboxyhemoglobin (COHb) level in artery blood with a CO-oximeter after limb ischemia/reperfusion. And the animal mortality was observed on the other 24 rats. Results: Northern blotting analysis showed that HO-1 mRNA increased significantly at 4 hours after reperfusion, peaked at 16 hours, and began to decrease at 24 hours. In contrast, no positive signal was observed in the sham and simple ischemia animals. Increased HO-1 mRNA levels were accompanied by similar increases in HO-1 protein. Lung PMNs and MDA content increased significantly at 4, 8, 16 and 24 hours after reperfusion, compared with the sham controls (P< 0.001), while they decreased in rats with reperfusion for 16 hours when compared with rats with reperfusion for 4 hours (P< 0.001). Immunohistochemical studies showed that HO-1 was expressed in a variety of cell types, including the airway epithelia, alveolar macrophages and vascular smooth muscular cells. The blood COHb level and animal mortality increased significantly after limb ischemia/reperfusion compared with the sham controls (P< 0.001). ZnPP administrated to the ischemia/reperfusion animals led to a decrease in the COHb level and an increase in lung PMN number, MDA content and animal mortality (P< 0.001 compared with ischemia/reperfusion group), and the lung injury was aggravated. Conclusions: Limb ischemia/reperfusion up-regulates pulmonary HO-1 expression, which serves as a compensatory protective response to the ischemia/reperfusion-induced lung injury in rats.