【作者】 田华；

【导师】 戴爱国；

【作者基本信息】 南华大学 ， 内科学， 2010， 硕士

【摘要】 背景:缺氧诱导因子是细胞适应缺氧的关键转录因子,在缺氧性肺动脉高压(HPH)的发生发展中起十分重要的作用,是使缺氧性肺血管重塑形成中调控目标基因表达的“分子开关”。低氧诱导因子1(HIF-1)是异二聚体的转录因子,由氧敏感的α亚基(HIF-1α)和在核内稳定表达的β亚基(HIF-1β)组成。HIF-1α是功能性亚基,调控100多种涉及低氧应激下细胞适应与存活的靶基因,从而在低氧应答反应中起核心作用。最近几年发现的泛素样蛋白家族成员小泛素蛋白样修饰蛋白-1(SUMO-1)能与HIF-1α共价结合,并对其进行翻译后修饰作用,该过程称为SUMO化。SUMO化是一个可逆的动态过程,可被特异性蛋白酶SENP-1将其从底物上去除,此过程称为去SUMO化。可逆的SUMO化修饰能在低氧下调节HIF-1α的稳定性及转录活性。目的:研究低氧性肺动脉高压(HPH)大鼠模型和患者肺动脉高压形成过程中SUMO-1、SENP-1、HIF-1α及其靶基因血管内皮生长因子(VEGF)在肺小动脉表达变化规律及相互关系,以及肺组织内SUMO-1对HIF-1α的SUMO化修饰情况,阐明SUMO-1、SENP-1调控HIF-1α的SUMO化修饰在HPH发生、发展过程中的作用,为HPH发病机制提供新的理论依据。方法:实验共分两部分。第一部分为动物实验部分:将40只成年雄性Wistar大鼠随机分为常氧对照组和低氧3 d、7 d、14 d、21 d组,每组8只,常压间断低氧复制HPH大鼠模型。用右心导管法测定大鼠平均肺动脉压(mPAP),称量法计算右心室肥大指数(RVHI),并进行HPSR指标观察。原位杂交法检测肺小动脉HIF-1α、VEGF mRNA表达,免疫组织化学法检测其蛋白水平;原位杂交、逆转录-聚合酶链反应(RT-PCR)法检测肺内SUMO-1、SENP-1 mRNA表达水平及部位,免疫组织化学、Western blot法检测其蛋白表达水平及部位;免疫共沉淀法(CO-IP法)检测SUMO-1对HIF-1α的SUMO化修饰水平。第二部分为临床实验部分:将36名患者分为慢性阻塞性肺疾病(COPD)并肺动脉高压组(PH)组(PH组)、COPD非PH组(COPD组)、非COPD非PH组(对照组),行肺功能、血气分析、多普勒超声心动图等检查。收集他们的肺组织,观察其肺血管重塑指标,原位杂交法检测肺小动脉壁HIF-1α、VEGF、SUMO-1、SENP-1 mRNA的表达水平,免疫组织化学法检测肺小动脉壁HIF-1α、VEGF、SUMO-1、SENP-1蛋白表达水平。CO-IP法检测SUMO-1对HIF-1α的SUMO化修饰水平。结果:(1)低氧7 d后,大鼠肺小动脉出现血管重塑,且mPAP明显上升。低氧14 d后,肺小动脉重塑更明显,mPAP达高峰。RVHI在低氧14 d后明显增加。HIF-1αmRNA在对照组表达阳性,低氧14 d后表达明显增高。HIF-1α蛋白在对照组表达不明显,低氧3 d后明显升高,低氧7 d后达高峰,此后表达稍下降。VEGF mRNA和蛋白在对照组表达均不明显,低氧7 d后表达升高,低氧14 d后达高峰。对照组SUMO-1 mRNA呈弱阳性表达,低氧3d后显著增高,低氧14天后达高峰,此后表达稍下降但仍高于对照组。SUMO-1蛋白在对照组呈弱阳性表达,低氧3d后显著增高,低氧14 d后达高峰,缺氧21d继续持平。SENP-1 mRNA在对照组呈阳性表达,低氧后其相对量无明显变化。SENP-1蛋白在对照组呈阳性表达,低氧后其表达量呈进行性下降。CO-IP结果显示:对照组肺组织内SUMO-1没有对HIF-1α发生SUMO化修饰,低氧3d后SUMO-1对HIF-1α发生了明显的SUMO化修饰,低氧14d达高峰,低氧21d维持于高水平。直线相关分析表明,SUMO-1 mRNA及蛋白与SUMO-1对HIF-1α的SUMO化修饰程度呈正相关;SENP-1蛋白与SUMO-1对HIF-1α的SUMO化的呈负相关;SUMO化的HIF-1α与HIF-1αmRNA、HIF-1α蛋白均呈正相关;HIF-1α蛋白与VEGF mRNA、VEGF蛋白均呈正相关。HIF-1α、VEGF蛋白与mPAP、肺小动脉重塑指标均呈正相关。SUMO化的HIF-1α与VEGF mRNA、VEGF蛋白、mPAP、RVHI、WA%、WT%均成正相关。(2)COPD组患者肺小动脉出现血管重塑,PH组患者肺小动脉重塑更明显。对照组患者肺小动脉壁HIF-1αmRNA呈弱阳性、HIF-1α蛋白呈阴性;COPD组患者肺小动脉壁HIF-1αmRNA及蛋白表达明显上调;PH组患者肺小动脉壁HIF-1αmRNA表达进一步增加,而HIF-1α蛋白表达较COPD组无显著变化。VEGF mRNA、VEGF蛋白在对照组患者肺小动脉壁均呈弱阳性表达,COPD组表达明显上调,PH组表达则进一步增加。SUMO-1 mRNA及蛋白在对照组患者肺小动脉壁呈弱阳性表达,COPD组呈阳性表达,PH组呈强阳性表达。SENP-1 mRNA在对照组患者肺小动脉壁呈弱阳性表达,COPD组及PH组其相对量无明显变化,与对照组比较差异无统计学意义。SENP-1蛋白在对照组呈阳性表达,COPD组表达下降,PH组下降更加明显。CO-IP法结果显示:对照组患者肺组织没有检测到SUMO-1对HIF-1α的SUMO化修饰,COPD组检测到SUMO-1对HIF-1α发生了明显的SUMO化修饰,而PH组该修饰变得更加明显。直线相关分析表明,SUMO-1 mRNA及蛋白与SUMO-1对HIF-1α的SUMO化修饰程度呈正相关;SENP-1蛋白与SUMO-1对HIF-1α的SUMO化的呈负相关;SUMO化的HIF-1α与HIF-1αmRNA、HIF-1α蛋白均呈正相关;HIF-1α蛋白与VEGF mRNA、VEGF蛋白均呈正相关。HIF-1α、VEGF蛋白与肺小动脉重塑指标、肺动脉收缩压均呈正相关。SUMO化的HIF-1α与VEGF mRNA、VEGF蛋白、肺血管重塑指标WA%、WT%,肺动脉收缩压均成正相关。结论:(1)HIF-1α、VEGF均参与HPH发病过程,HIF-1α可能以转录激活的形式上调VEGF表达,导致HPH的发生和发展。(2)慢性低氧诱导肺小动脉壁SUMO-1表达增加,SENP-1蛋白降解, SUMO化修饰的HIF-1α明显增加,HIF-1α稳定性和转录活性增加,导致VEGF等HIF-1α靶基因转录激活,从而参与HPH的发生、发展。更多还原

【Abstract】 Background The hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor composed of an oxygen-sensitiveαsubunit (HIF-1α) and a constitutively expressedβsubunit. HIF-1α, as a functional subunit, regulates the expression of more than 100 genes involved in cellular adaptation and survival and then plays a crucial role in the cellular response to the stress of hypoxia. HIF-1αis also a transcriptional regulator which plays a key role during the development of hypoxic pulmonary vascular remodeling (HPSR). Recently, people have found small ubiquitin-related modifier-1 (SUMO-1), which can covalently attach to HIF-1α. The process that SUMO covalently attach to proteins is known as SUMOylation. The SUMOylation is a dynamic process reversible, which can be removed from the substrate by specific protease SENP-1 as de-SUMOylation. The reversibly SUMOylational modification of HIF-1αcan change the stability and transcriptional activity of HIF-1αunder hypoxic conditions..Objective To evaluate the role of SUMO-1 and SENP-1 in regulating HIF-1αduring the development of hypoxic pulmonary hypertension(HPH)and provide the theoretical basis of mechanism for HPH. We investigated SUMOylation of HIF-1αby SUMO-1, and the expression patterns of SUMO-1, SENP-1, HIF-1α, and vascular endothelial growth factor (VEGF), a well-characterized target gene of HIF-1α, as well as their relationship to each other in the pulmonary arterioles of rats and patients at different phases of HPH development.Methods The study consisted of two parts. (1) Forty adult male Wistar rats were randomly divided into 5 groups, 8 rats in each group, and exposed to normoxia (control group) or exposed to hypoxia for 3, 7, 14 or 21 d, respectively. The mean pulmonary artery pressure (mPAP) was measured by right-heart catheterization, while right ventricular hypertrophy index (RVHI) was calculated by the ratio of right ventricle to the left ventricle plus septum, and hypoxic pulmonary vascular remodeling (HPSR) was observed with morphmetric analysis. The mRNA and protein expression of HIF-1αand VEGF in pulmonary arterioles were detected by in situ hybridization and immunohistochemistry, respectively. Reverse transcription- polymerase chain reaction (RT-PCR) and in situ hybridization were used to determine the mRNA expression of SUMO-1 and SENP-1. Immunohistochemistry and Western blot were adopted to determine the protein expression of SUMO-1 and SENP-1. Co-Immunoprecipitation were adopted to determine SUMOylation of HIF-1αby SUMO-1. (2) Thirty-six patients were divided into three groups: chronic obstructive pulmonary disease (COPD) with pulmonary pertension (PH) group (PH group), COPD without PH (COPD group) and control group. Their lung tissues were collected from surgically resected specimens. The HPSR was observed with morphmetric analysis too. The expression of HIF-1α, VEGF, SUMO-1 and SENP-1 in pulmonary arterioles was examined by in situ hybridization and immunohistochemistry. The SUMOylation of HIF-1αby SUMO-1 was examined by Co-Immunoprecipitation.Results (1) mPAP in hypoxic rats increased significantly after 7 d of hypoxia, reached its peak after 14 d of hypoxia, and then remained stable. The hypoxic rats developed HPSR after 7 d of hypoxia, and more significantly after 14 d of hypoxia. RVHI in hypoxic rats was markedly increased after 14 d of hypoxia. HIF-1αmRNA is positively stained in control and increased dramatically after 14 d of hypoxia. HIF-1α?protein was poorly positive in control, up-regulated markedly after 3 d and 7 d of hypoxia, and then declined slightly after 14 d and 21 d of hypoxia. VEGF mRNA and protein were poor positively stained in control, increased markedly after 7 d of hypoxia, and then reached their peak after 14 d of exposure to hypoxia. SUMO-1 mRNA and protein expression in pulmonary arteriole walls was markedly increased in pulmonary arteriole walls after 3 d of exposure to hypoxia, reached its peak after 14 d of hypoxia, then weakened after 21 d of hypoxia, but was still higher than that in the control. SENP-1mRNA and protein were positively stained in control. SENP-1 mRNA expression had little changes after exposure to hypoxia compared with the control, however, SENP-1 protein expression was increased Slightly after 3d of hypoxia, and declined gradually after 7 d of hypoxia. The results of Co-immunoprecipitation showed that SUMOylation of HIF-1a almost did not happen in lung tissues of control rats. But it was markedly positive after 3 and 7 d of hypoxia in lung tissues of rats, and reached its peak after 14 d of hypoxia, then remained stabilized. Linear correlation analysis showed that SUMO-1 mRNA and protein was positively correlated with SUMOylation of HIF-1αby SUMO-1. SENP-1 protein was negatively correlation with SUMOylation of HIF-1αby SUMO-1. While SUMOylation of HIF-1αby SUMO-1 was positive correlation with HIF-1αmRNA and HIF-1αprotein,HIF-1αprotein was positively correlated with VEGF mRNA and VEGF protein, HIF-1αprotein and VEGF protein were positively correlated with mPAP and pulmonary arteriole remodeling index. SUMOylation of HIF-1αby SUMO-1 was positive correlation with VEGF mRNA and VEGF protein、mPAP、RVHI、WA%、WT%.(2) The HPSR developed in patients from COPD group, and more significantly in patients from PH group. HIF-1αmRNA and protein were poorly stained in pulmonary arteriole walls in control group, increased markedly in COPD group and then HIF-1αmRNA expression increased further while HIF-1αprotein expression changed little in pulmonary arteriole walls in PH group compared with COPD group. VEGF mRNA and VEGF protein were poorly stained in pulmonary arteriole walls in control group, up-regulated markedly in COPD group, and increased further in PH group. SUMO-1 mRNA and SUMO-1 protein were poorly stained in pulmonary arteriole walls in control group, up-regulated markedly in COPD group, and increased further in PH group. SENP-1 mRNA expression remained unchanged in pulmonary arteriole walls between different groups, while SENP-1 protein was strongly stained in pulmonary arteriole walls in control group, weakened remarkably in COPD group and decreased further in PH group. The results of Co-immunoprecipitation showed that SUMOylation of HIF-1a almost did not happen in lung tissues of control group. But it was markedly positive in COPD group, and increased further in PH group. Linear correlation analysis showed that SUMO-1 mRNA and SUMO-1 protein was positively correlated with SUMOylation of HIF-1αby SUMO-1. SENP-1 protein was negative correlation with SUMOylation of HIF-1αby SUMO-1. While SUMOylation of HIF-1αby SUMO-1 was positive correlation with HIF-1αmRNA and HIF-1αprotein,HIF-1αprotein was positively correlated with VEGF mRNA and VEGF protein, HIF-1αprotein and VEGF protein were positively correlated with pulmonary arteriole remodeling index and pulmonary artery systolic pressure. SUMOylation of HIF-1αby SUMO-1 was positively correlated with VEGF mRNA and VEGF protein、pulmonary arteriole remodeling index、pulmonary artery systolic pressure.Conclusion (1) Both HIF-1αand VEGF are involved in the pathogenesis of HPH. HIF-1αmay up-regulate the expression of VEGF via transactivation, resulting in the development of HPH. (2) The dynamic expression of SUMO-1 may play a role in stabilizing protein and action of HIF-1a by SUMOylation, then being involved in the development of PH; SENP-1 protein can be degradated in hypoxia, The dynamic expression of SENP-1 protein may play a role in implicating in the development of PH.更多还原