II型11β-羟基类固醇脱氢酶表观遗传变化在PPHN发病中的作用和可能机制

Persistent pulmonary hypertension of the newborn (PPHN), one of critical diseases in the newborn, is due to pulmonary vascular abnormalities caused by the intrauterine hypoxia and so on. Our previous studies have shown that intrauterine hypoxia and other environmental factors will lead to the epigenetic changes of some vascular tension regulatory factors. Excessive glucocorticoids can increase the effect of vascular vasoconstrictor.11β-hydroxysteroid dehydrogenase type 2 (HSD11B2), the key enzyme in corticosteroid metabolism, distributes in both lung tissues and blood vessels, and is involved in the regulation of glucocorticoids concentrations in systemic and local tissues. We noted that there is a CpG island in the HSD11B2 promoter. Herein, basis on the PPHN rat model established in our previous studies, we plan to clarify the effects and mechanisms of HSD11B2 gene epigenetic changes in PPHN by investigating the effects and mechanisms of intrauterine hypoxia on the pulmonary vascular development, glucocorticoid level, lung tissues and vessels HSD11B2 gene expression and epigenetics in the offspring using the cell culture, epigenetic and molecular biology technologies. It will help us to better understand the pathogenesis of PPHN, and provides new insights into the clinical diagnosis and intervention for PPHN.

新生儿持续肺动脉高压（PPHN）是由于宫内缺氧等导致肺血管异常引起新生儿常见危重症之一。我们前期研究显示宫内缺氧等环境可导致部分血管张力调控因子表观遗传学变化。过多的糖皮质激素会增加血管的缩血管效应，II型11β-羟基类固醇脱氢酶（HSD11B2）是体内皮质激素代谢关键酶，在肺组织和血管均有分布，参与全身和局部糖皮质激素浓度调节。生物信息学分析发现HSD11B2启动子其有1个CpG岛。本项目拟在前期建立的PPHN新生大鼠模型基础上，采用细胞培养、表观遗传学和分子生物学等技术，探讨缺氧等宫内环境对子代肺部血管发育、糖皮质激素水平、肺组织和肺血管HSD11B2基因表达和表观遗传学的影响；初步阐明HSD11B2基因表观遗传学变化在PPHN发生中的作用和机理。这将使我们更深入了解PPHN发病机制，为临床PPHN的诊治提供新思路。

新生儿持续肺动脉高压（PPHN）是新生儿重症监护室的一个重要的临床问题。本课题的目的是为了探讨II型11β-羟基类固醇脱氢酶（11β-hydroxysteroid dehydrogenase, type 2,11β-HSD2）参与PPHN发生的机制及相关信号分子在新生儿持续肺动脉高压（PPHN）发病中的作用。采用缺氧加吲哚美辛的方法建立PPHN大鼠模型，综合应用real time PCR、Westen Blot、免疫荧光、免疫组化等方法测定肺组织11β-HSD2的表达,分析比较PPHN与正常新生大鼠肺脏11β-HSD2之间的差别。RT-PCR测定肺组织中转录因子SP1 、cAMP反应元件结合蛋白(cAMP response element binding protein, CREB)mRNA的表达； ELISA方法检测PPHN大鼠血清中皮质醇、肺组织中前列环素（PGI2）、肾素、血管紧张素、醛固酮的浓度。硝酸还原酶法检测肺组织匀浆后血清中NO水平。结果发现 PPHN组新生大鼠右心室游离壁较对照组增厚，PPHN组新生大鼠肺小动脉中膜厚度/（中膜+内腔）厚度百分率较对照组增高。PPHN组新生大鼠肺组织中11β-HSD2表达水平降低。PPHN组肺组织中SP1、CREB mRNA、PGI2、NO及NOS较对照组均降低。PPHN组肺组织中肾素、血管紧张素、醛固酮及血清皮质醇水平较对照组均增高。11β-HSD2蛋白主要在肺泡上皮细胞、支气管上皮细胞和小肺血管内皮细胞的细胞质中表达。这些提示11β-HSD2参与了PPHN的发病机制，宫内缺氧可能通过降低11β-HSD2的表达增加肺组织中的糖皮质激素，引起肺血管功能障碍和血管收缩，从而诱发持续性肺动脉高压。皮质醇、PGI2、肾上腺素、血管紧张素、醛固酮在PPHN发生的肺血管收缩中起作用，且SP1、CREB 可能在缺氧诱导的PPHN发生的肺血管重建中发挥了作用。