【摘要】目的探讨一氧化氮对缺氧性肺血管结构重建大鼠肺动脉中血小板源生长因子(PDGF)表达的影响。方法将大鼠随机分为4组：常氧组(6只)、低氧组(6只)、低氧+L-精氨酸组(低氧+L-Arg组)(7只)及低氧+N^ω-硝基- L-精氨酸甲酯组(低氧+L-NAME组)(6只)。以光学显微镜观测4组大鼠肺中、小肌型动脉的相对中膜厚度(RMT)。应用PDGF的单克隆抗体经免疫组织化学技术对4组大鼠肺动脉PDGF进行定位及含量分析。结果低氧组大鼠中型肺动脉及小型肺动脉RMT均高于常氧组和低氧+L-Arg组 (P＜0.01)；低氧+L-NAME组均高于低氧组 (P＜0.01)。4组大鼠各级肺动脉内皮细胞及平滑肌细胞中均有PDGF的表达，低氧组较常氧组表达增强(P＜0.01)，L-Arg使其表达明显抑制(P＜0.01)，而L-NAME使其表达明显增强(P＜0.01)。中型肺动脉内皮细胞及平滑肌细胞中PDGF的表达与相应中型肺动脉RMT之间呈正相关(r分别为0.863 6、0.826 4,P均＜0.01)；小型肺动脉内皮细胞及平滑肌细胞中PDGF的表达与相应的小型肺动脉RMT之间呈正相关(r分别为0.889 8、0.868 8，P均＜0.01)。结论一氧化氮对缺氧性肺血管结构重建大鼠肺动脉中PDGF表达有抑制作用。

Impact of nitric oxide on platelet-derived growth factor expression in pulmonary arteries of rats with hypoxic pulmonary vascular remodeling

ZHOU Bin，DU Junbao（Department of Pediatrics，First Hospital，Beijing Medical University，Beijing 100034, China）

【Abstract】ObjectiveThe pathological basis of chronically hypoxic pulmonary hypertension is pulmonary vascular structural remodeling. Nitric oxide (NO), a new typical messenger of gas molecule, plays an important role in regulating hypoxic pulmonary vascular remodeling. However, the regulatory mechanism is unknown. Our present experiment aimed at examining the impact of NO on platelet-derived growth factor-B（PDGF-B）expression in intrapulmonary arteries of rats with hypoxic pulmonary vascular remodeling, and thereby to investigate the role of PDGF in the mechanisms by which NO regulates hypoxic pulmonary vascular remodeling. MethodsTwenty-five rats were randomly divided into four groups: normoxic group (n=6), hypoxic group (n=6)，hypoxic+L-arginine group (hypoxic + L-Arg group, n=7) and hypoxic +N^ω -L-nitro-arginine methyl ester group (hypoxic + L-NAME group, n=6). Relative medial thickness (RMT) of small and median muscularized pulmonary arteries was observed in lung sections of rats by using a light microscope. An immunohistochemical technique was used to assess the abundance and localization of PDGF in pulmonary arteries of rats in four groups. ResultsRMT of small and median intrapulmonary arteries in rats of hypoxic group was markedly higher than that of normoxic group and hypoxic+L-Arg group (P＜0.01). Whereas RMT of small and median intrapulmonary arteries in rats of hypoxic + L-NAME group was obviously higher than that of hypoxic group (P＜0.01). pDGF-B was expressed by pulmonary artery endothelial cells and smooth muscle cells in rats of 4 groups. In rats of normoxic group, hypoxic group, hypoxic + L-Arg group and hypoxic + L-NAME group, PDGF expression score was 35.0, 58.3, 42.1 and 75.8, respectively, in endothelial cells of median pulmonary arteries, and 31.7, 50.8, 38.6 and 73.3，respectively in smooth muscle cells of median pulmonary arteries. The score was 38.3, 57.5, 47.1 and 78.3 respectively, in endothelial cells of small pulmonary arteries, and 37.5, 60.8, 45.7 and 80.8, respectively, in smooth muscle cells of small pulmonary arteries. There was a positive correlation between RMT and PDGF expression by endothelial cells and smooth muscle cells of median pulmonary arteries (r=0.863 6, 0.826 4, P＜0.01, respectively). There was also a positive correlation between RMT and PDGF expression by endothelial cells and smooth muscle cells of small pulmonary arteries (r=0.889 8, 0.868 8, P＜0.01, respectively). ConclusionNitric oxide inhibited PDGF expression by pulmonary arteries of rats with hypoxic pulmonary vascular remodeling.

【Key words】Nitric oxide;Platelet-derived growth factor;Anoxia;Pulmonary artery

缺氧性肺动脉高压(hypoxic pulmonary hyper-tension, HPH)是与许多呼吸系统疾病紧密相关的一种常见并发症^［1］。肺血管结构重建是肺动脉高压形成的重要病理基础^［2］。一氧化氮（nitric oxide，NO）对缺氧性肺血管结构重建具有重要的调节作用，但其调节机制尚未十分清楚。血小板源生长因子(platelet-derived growth factor，PDGF)是自1974年以来才被认识的一种刺激细胞增殖的肽类生长因子^［3］。近年来，人们逐渐认识到PDGF在低氧性肺动脉高压的肺血管结构重建中发挥重要作用^［4］。L-精氨酸（L-arginine，L-Arg）是NO合成的前体，N^ω-硝基-L-精氨酸甲酯（N^ω-nitro-L- arginine methyl ester，L-NAME）是一氧化氮合酶抑制剂。我们运用免疫组织化学方法，通过研究低氧时以及经L-Arg、L-NAME处理的低氧大鼠中型肺动脉及小型肺动脉内皮细胞及平滑肌细胞中PDGF表达的变化及其与血管结构变化之间的相关性，探讨NO对缺氧性肺血管结构重建大鼠肺动脉中PDGF表达的影响，进一步了解缺氧性肺动脉高压的发生机制。

材料和方法

一、 实验对象

雄性Wistar大鼠25只，体重200～300 g，购自北京医科大学第一医院实验动物中心。将大鼠随机分为4组，即：常氧组(6只) ，低氧组(6只), 低氧+L-Arg组 (7只)，低氧+L-NAME组 (6只)。

二、 实验方法

1．大鼠缺氧方法：将大鼠置于CG1型常压低氧舱内，保持控制舱内氧浓度在(10.0±0.5)%。每天连续低氧6 h，共低氧2周。对低氧+L-Arg组在每天低氧前，予腹腔注射L-Arg 500 mg/kg，对低氧+L- NAME组在每天低氧前，予腹腔注射L- NAME 5 mg/kg。常氧组大鼠呼吸室内空气，4组大鼠的常规饲养及饮食条件相同。

2．标本取材处理：4组大鼠在上述条件下饲养2周后，取两侧肺叶，一侧肺叶做成5μm厚冰冻切片，应用PDGF的单克隆抗体经免疫组织化学方法(ABC法)对大鼠肺动脉中PDGF进行定位及定量分析；另一侧肺叶做成5 μm厚石蜡切片，进行Weight弹力纤维染色，Van Geieson复染，并进行光学显微镜观测。

三、 观测指标

1．肺动脉病理结构指标：中型肺动脉及小型肺动脉的相对中膜厚度(relative medial thickness, RMT)的测定：应用Q550LW图像处理与分析系统对整个石蜡切片中的内弹力层清晰、形状较规则的中型肺动脉及小型肺动脉进行分析，运用Leica Qwin软件测量每个血管的如下指标：通过血管轴心外弹力层之间的最长外径(D₁)和最短外径(D₂)、内弹力层长度(L_IEL)及外弹力层长度(L_EEL)。应用上述指标，根据Barth等^［5］的方法，首先对血管进行理想化转换，使之成为完全圆形、中膜厚度一致的血管，然后再计算不同切面角度(0°≤α＜90°)及处于不同舒缩状态的血管RMT。RMT为转化后肺理想血管中膜厚度(T)与外弹力层所包绕的理想血管半径(R)之比，而T为R与内弹力层所包绕的理想血管半径(r)之差。R值可用公式R=L_EEL/{π×［1.5×(1+1/Cos α)-(1/Cos α)^1/2］}求得。r值可用公式r=L_IEL/{π×［1.5×(1+1/Cos α)-(1/Cos α)^1/2］}求得，其中，Cos α=D₂/D₁，α为血管实际切面与水平切面之夹角。每只大鼠测量5～10个中型肺动脉及小型肺动脉,并求其均值。

2．中型肺动脉和小型肺动脉PDGF的含量与分布：采用免疫组织化学染色方法对中型肺动脉和小型肺动脉PDGF的含量进行测定^［6］。在光学显微镜下，棕黄色颗粒为其阳性信号。采用半定量法分析PDGF的含量。首先，根据血管外径选择肺动脉：中型肺动脉 (管径50～150 μm)和小型肺动脉(管径15～50 μm)。根据每条肺动脉内皮细胞和平滑肌细胞呈现PDGF阳性表达的百分比将其免疫活性表达分为三个等级：肺动脉内皮细胞或平滑肌细胞中无PDGF表达者为(-)；肺动脉有1%～50%内皮细胞或平滑肌细胞中有PDGF表达者为(+)；肺动脉有51%～100%内皮细胞或平滑肌细胞中有PDGF表达者为(++)。肺动脉中PDGF含量进一步用积分值表示，即：以PDGF某一表达强度的肺动脉百分比乘以表达强度加权值(“-”者，表达强度加权值为0；“+”者，表达强度加权值为0.5；“++”者，表达强度加权值为1)，每份标本各级血管至少检测10条。

四、统计学处理

分别对大鼠肺动脉PDGF表达积分值、中型肺动脉及小型肺动脉RMT在各组之间差异运用单因素方差分析（F检验）进行统计学处理。在两组之间的差异用 q检验。大鼠中型肺动脉和小型肺动脉PDGF表达积