生后肺发育过程中NMDA受体NR2不同亚基程序化表达模式的变化及在高氧性肺损伤中的作用

Oxygen is the most commonly used therapy in neonatal nurseries as an integral part of all respiratory support. However, the prolonged hyperoxia can induce bronchopulmonary dysplasia (BPD) in newborn. Our laboratory has found that N-methyl-D-aspartate receptor (NMDAR) antagonist MK-801 can decrease hyperoxia-induced acute andchronic lung injury. This introduces a novel and potentially more successful therapeutic strategy for BPD. Mesenchymal interstitial fibroblast has important functions in lung development and injury repair after acute lung injury. Previous study had shown that hyperoxia stimulated the upregulation of NR2A and NR2D subunits of NMDAR in newborn rat lung fibroblast, and NMDAR activation play an important role in hyperoxia induced lung fibroblast phenotypic transformation. NR2 subunits A to D are expressed differently from different stage of lung development and control the electrophysiological properties of the NMDA receptor. NR2A participate in lethal excitotoxicity and that NR2D have excitoprotective functions. So, it is not clear that which subunit.of NR2A and NR2D take part in the lung development and hyperoxia induced lung injury. In this study, we further observe the different role of NR2A and NR2D in lung development and hyperoxia induced lung injury. In addition, the applicant proposes to determine the regulation mechanism of NR2A and NR2D genes expression, and the role of NR2A or NR2D activation in the regulation of thymocyte differentiation antigen 1(Thy-1) expression. We further examine the epigenetic mechanism and significance of Thy-1 gene expression regulation in the level of Histone acetylation, in order to reveal the different role and molecular mechanisms of NR2A and NR2D in the hyperoxia induced newborn rat lung dysplasia. This project aims to confirming which NR2s play a key role in lung development and hyperoxia induced lung injury through lung fibroblast. Which will provide an exact therapeutic target by using NR2 antagonist to treatment BPD in clinic.

前期研究显示谷氨酸及其NMDAR过度激活介导高氧致新生大鼠的急慢性肺损伤;高氧通过上调新生大鼠肺成纤维细胞NMDA受体NR2A和2D亚基表达,诱导肺肌成纤维细胞表型转化。研究还发现大鼠不同肺发育阶段NMDA受体NR2亚基表达不同。由于NMDAR的功能由NR2不同亚基决定,尚不能明确何种NR2亚基参与了生后肺发育及高氧导致的肺发育不良。申请者将在整体及肺成纤维细胞水平,进一步研究在生后肺发育过程中NR2A及2D亚基的时序性表达在高氧致肺损伤过程中中的作用。同时观察调节NR2A及2D基因表达的上游转录因子,及其激活后胸腺细胞分化抗原-1（Thy-1）基因表达及组蛋白去乙酰化水平的改变，从基因转录调控及表观遗传角度探讨NR2A或2D亚基在肺发育及高氧性肺损伤中的机制。在整体及细胞分子水平明确何种NR2亚基介导了高氧所致的生后肺发育不良,为临床上抗NMDAR防治高氧性肺损伤寻找更为确切的治疗靶点。

前期研究发现NMDAR激活参与了新生大鼠高氧所致急慢性肺损伤，大鼠不同肺发育阶段NMDA受体NR2亚基表达不同。由于NMDAR的功能由不同NR2亚基决定,尚不能明确何种NR2亚基参与了新生大鼠生后肺发育及高氧导致的肺发育不良。本项目分别在整体及肺成纤维细胞水平观察不同NR2亚基在新生大鼠生后肺发育及高氧性肺损伤的不同阶段的作用；同时在表观遗传修饰方面，通过整体及肺成纤维细胞，研究不同NR2亚基激活对Thy-1基因启动区组蛋白乙酰化水平的影响。结果发现1.NR2A与NR2D亚基在新生大鼠肺发育过程中存在时间程序化表达；2.高氧促进SD大鼠胎肺、生后肺组织以及肺成纤维细胞NMDA受体NR2A与NR2D亚基表达增加，谷氨酸通过促进NMDA受体介导的胎肺成纤维细胞表型转化，改善“高氧”对体外培养SD大鼠胎肺发育的抑制作用；3.NR2A和NR2D过表达均可以促进胎肺成纤维细胞表型转化；4.NR2A 亚基不参与新生大鼠高氧性肺损伤慢性阶段肺成纤维细胞的表型转化，NR2D 激活是高氧诱导新生大鼠肺成纤维细胞细胞表型转化的主要 NR2 亚基，NR2A 受体激活可能参与了细胞的急性损伤，而 NR2D 激活参与新生大鼠肺成纤维细胞的细胞增值；5.肺成纤维细胞NR2D激活后，促进Thy-1基因启动区组蛋白去乙酰化，抑制Thy-1基因表达，促进细胞增殖，并减少细胞凋亡，NR2A激活则无此作用；6.高氧可以导致新生大鼠肺组织CREB和STAT3表达增加，但细胞验证显示并非NR2各亚基的特异性启动因子，存在其他细胞因子干扰可能性大。通过以上研究结果，充分验证了NMDA受体NR2D亚基在高氧所致新生大鼠急慢性损伤中的组织修复，细胞增殖中发挥了重要的作用，为临床上新生儿支气管肺发育不良提供了精确的防治靶点。