低氧和高二氧化碳作用下肺动脉高压形成过程中骨桥蛋白表达与分布的实验研究

Expression and distribution of osteopontin in the development of pulmonary hypertension induced by hypoxia-hypercapnia

目的:观察慢性低O2高CO2大鼠肺动脉骨桥蛋白(OPN)及其基因的表达与分布情况,探讨OPN在肺动脉高压发病机制中的作用。方法:48只雄性SD大鼠随机分为4组:即正常对照组(NC组),低O2高CO21周、2周和4周组(1HH、2HH和4HH)。采用RT-PCR法测定不同低O2高CO2组大鼠离体肺动脉和肺组织骨桥蛋白(OPN) mRNA,免疫组织化学染色法测定肺细小动脉骨桥蛋白分布表达情况,ELISA法定量测定肺组织匀浆OPN水平,Western blotting法测定离体肺动脉OPN表达水平。结果:①低O2高CO2各组大鼠mPAP和RV/LV+S均明显高于NC组(P<0.01),4组大鼠间mCAP比较无显著差异(P>0.05);②低O2高CO2各组大鼠肺组织和离体肺主动脉OPN mRNA表达水平显著高于对照组(均P<0.01);③免疫组化法显示NC组大鼠肺组织OPN表达主要见于支气管、肺泡上皮细胞内,1HH组大鼠肺细小动脉内皮细胞、平滑肌细胞以及周围巨噬细胞均见有OPN的表达,其中以中膜平滑肌细胞最为明显,而2HH和4HH组大鼠OPN表达更加明显,低O2高CO2各组肺细小动脉OPN相对含量与对照组比较有显著差异(均P<0.01),且随缺氧时间的延长,骨桥蛋白表达也呈增高趋势;④低O2高CO2各组大鼠肺组织匀浆OPN含量分别为正常对照组的1.69倍、2.28倍和2.87倍(均P<0.01);⑤Western blotting分析1HH、2HH和4HH各组大鼠离体肺动脉OPN表达明显增强,与NC组比较有显著差异(均P<0.01)。结论:慢性低O2高CO2能够刺激大鼠肺组织和肺动脉OPN及其基因表达增强,OPN可能在慢性低O2高CO2性肺动脉高压发病的病理和病理生理过程中起着重要的作用。

AIM： To study the expression and distribution of osteopentin （OPN） in lungs and pulmonary arteries in pulmonary hypertensive rats induced by hypoxia - hypercapnia, and to explore the role of OPN in pathogenesis of pulmonary hypertension. METHODS ： Forty - eight male Sprague - Dawley rats （ Weight 180 g - 220 g） were randomly divided into four groups： normal control group （ NC）, hypoxic hypercapnia 1 - week, 2 - week and 4 - week group （ 1HH, 2HH and 4HH）. The expressions of OPN mRNA and protein in lungs and pulmonary arteries were detected by RT - PCR and immunohistochemistry. ELISA was used to detect the concentration of OPN in lung homogenates. The content of OPN in pulmonary arteries was detected by Western blotting. RESULTS： ① The mean pulmonary artery pressure （mPAP） and weight ratio of right ventricle to left ventricle and septum [ RV/（ LV ＋ S） ] in all hypoxic hypercapniac groups were higher than those in normal control group （P 〈0. 01 ）, respectively. Differences of mean carotid artery pressure （mCAP） among these four groups were not significant （ P 〉 0. 05 ）.② The expression of OPN mRNA was significantly increased in pulmonary arteries and lung tissues in hypoxic hypercapnic groups compared with normal control group （ P 〈 0. 01 ）. ③ The result of immunohistochemistry showed that OPN was only detected in bronchus and alveolar epithelium, but not detected in pulmonary arterioles of normal control group. In contrast,OPN expression was evident in pulmonary arterioles o! IHH rats,especiaUy in media. Moreover, the expression of OPN was markedly increased in group 2HH and 4HH.④ OPN levels in lung homogenates in 1HH, 2HH and 4HH were increased by 69%, 128% and 187% （ P 〈 0. 01 ）, respectively, compared with control rats. ⑤ Western blotting analysis showed that the contents of OPN were significantly higher in all hypoxic hypercapnic groups than those in NC group （ P 〈 0. 01 ）. CONCLUSION ： The expressions of OPN in pulmonary arteioles and lung are increased in rats with pulmonary hypertension. OPN might play an important role in the pathogenesis of pulmonary hypertension induced by chronic hypoxia and hypereapnia.