HIF-1α结合活性及蛋白含量的表达变化在缺氧性肺动脉高压大鼠中的作用和意义

Determination of HIF-1α Binding Activity by EMSA in Pulmonary Arteries of Rats With Hypoxia-induced Pulmonary Hypertension

目的研究HIF-1α结合活性及蛋白含量的表达变化在缺氧性肺动脉高压(HPH)大鼠中的作用和意义。方法40只成年雄性SD大鼠随机分为对照组(C组)和缺氧3、7、14天和21天组(H3、H7、H14和H21组),每组8只,常压缺氧复制HPH大鼠模型。测各组大鼠平均肺动脉压(mPAP)、右室肥大指数(RVHI)、血管形态学指标;EMSA法检测HIF-1α结合活性的变化,免疫组化、Western blot检测HIF-1α蛋白质表达。结果(1)缺氧7天后,mPAP开始上升,与对照组比较差异有显著性(P<0.05)。缺氧14天达高水平并维持于此水平。缺氧14天后出现肺血管重塑,RVHI改变。(2)EMSA发现对照组HIF-1α结合活性水平较低,3天组迅速升高,14天组达到高峰。(3)Western blot发现,HIF-1α蛋白在对照组有一定的表达,缺氧3天组蛋白质明显升高,缺氧7天达高峰,此后保持高水平;免疫组化示HIF-1α蛋白在肺小血管表达不明显,缺氧3天明显升高(0.194±0.017,P<0.05),7天达到高峰,随后缺氧14天和21天组水平下降。结论HIF-1α的结合活性及蛋白含量在HPH大鼠均可见明显增高,HIF-1α在大鼠HPH的发病机制中发挥作用。

Objective To investigate the dynamic expression of hypoxia - inducible factor 1α binding activity and protein levels in pulmonary arteries of rats with hypoxia - induced pulmonary hypertension. Methods Male SD rats （n=40） were randomly divided into 5 groups and exposed to hypoxia for0, 3, 7, 14 or 21 day（C group, H3, H7, H14, H21）, respectively. Mean pulmonary arterial pressure （mPAP）, vessel morphometry and right ventricle hypertrophy index （RVHI） were measured. Electrophoretic mobility shift assay（EMSA） were used to determine the binding activity of HIF - 1α. Immunohistochemistry and Western blot were used to determine the expression of protein levels. Results mPAP increased significantly after 7 day of hypoxia （ P 〈 0.05 ）, reached its peak after 14 day of hypoxia, and then remained stable. Pulmonary artery remodeling developed significantly after 14 day of hypoxia. The binding activity of HIF- 1α was weak in control rats, increased dramatically after 3 day of hypoxia, and reached its peak after 14d of hypoxia. HIF- 1α protein was poorly positive in control, markedly upregulated after 3 day of hypoxia（0. 194 ± 0.017, P 〈 0.05）, and then declined slightly after 14 day and 21 day of hypoxia. Conclusion Both HIF - 1α binding activity and protein levels were up - regulated in pulmonary arteries of rats with hypoxia- induced pulmonary hypertension, and HIF- 1α is involved in the pathogenesis of hypoxic pulmonary＇hypertension in rats.