生后营养不足致肺血管功能障碍的传代效应的DNA甲基化机制及干预研究

Perinatal nutritional insult may exert transgenerational cardiovascular outcome in later life, which is related to changed DNA methylation. In our previous study, we found extrauterine growth retardation (EUGR) caused development of an increased pulmonary artery pressure in male Sprague – Dawley rats. In the present study, we have found that female offspring following EUGR male parents showed a significant reduction in pulmonary artery pressure later in life compared with those sex-matched control group. The DNA methylation mechanism involved in the transgenerational effect remains unknown. To demonstrate the interesting result, we analyzed DMR (different methylated region) between F0 and F1. We found vasoconstrictive methylation in F0 induced by EUGR such as hypermethylated Ace2, hypomethylated VIP, which wasn‘t in F1. We found vasodilative methylation such as hypermethylated Notch4 and hypomethylated Pdgfb in F1. But the methylated loci of these genes need to be elucidated. Furthermore we hypothesized that early appropriate interventions such as methyl-donor supplementation might reverse different methylation of pulmonary hypertension-related genes. The intervention of folate supplementation might have a protective effect on the development of pulmonary hypertension later in life and even in progeny. The plan would explain the mechanisms of the transgenerational effects induced by perinatal insult, providing a new insight into preventing and controlling the fetal origin of adult diseases.

生命早期营养打击所致成年期心血管疾病可遗传至子代，DNA甲基化发挥了重要作用。我们前期已发现生后早期营养不足致雄性父代大鼠成年期发生肺动脉高压，但其繁衍的雌性子代成年期肺动脉压较对照组却显著降低。证实了生后早期营养不足致父代肺血管功能障碍可影响子代，但其中的DNA甲基化机制尚不清楚。本项目运用甲基化芯片结合生物信息学，对比分析后发现父代中Ace2高甲基化、VIP低甲基化等正调控肺动脉压的甲基化修饰未遗传至子代，而子代中则发现如Notch4高甲基化、Pdgfb低甲基化的负调控肺动脉压的修饰。具体的甲基化位点和程度尚不清楚，拟进一步验证明确。另外本项目通过在父代雄性大鼠生后早期补充叶酸，增加体内甲基化水平，明确可否通过逆转关键基因及位点的甲基化修饰以达到调控血管舒缩，预防肺动脉高压的发生、阻断传代效应。本研究对阐明生命早期营养打击所致传代效应的机制有重要意义，为疾病的早期干预提供新思路。

项目背景：生命早期营养不足所致成年期心血管不良效应可延续至子代，DNA甲基化发挥了重要作用。该课题研究内容：通过构建生后早期营养不足传代模型，明确代际肺动脉压效应，同时通过全基因组甲基化芯片+生物信息学技术筛选出参与调节该效应的功能基因。重要结果：我们发现生后早期营养不足（EUGR）致雄性父代大鼠成年期发生肺动脉压增高（PAP）效应，但其繁衍的雌性子代成年期肺动脉压较对照组却显著降低。运用甲基化芯片对比分析差异区域后发现部分与肺血管内皮细胞中血管张力调节基因的DNA甲基化改变有关。具体表现为F1代雄性个体的X染色体上有16个差异ROI基因，在F2代雌性个体中有86个差异ROI基因。同样，F1代有105个DEPS，F2代有38个DEPS。结合5个共同的ROI和14个共同的DEP，18个基因被认为是EUGR模型中与PAP可遗传变异相关的关键候选基因。富集分析后发现，突触和神经递质相关通路可能参与EUGR所致PAH形成。在MalaCard数据库中的102个PAH相关基因中分析最终发现Smad1和SERPINE1两个关键基因。科学意义：在EUGR诱导PAP表型中，X染色体启动子甲基化改变存在代际遗传，证实了生后早期营养不足致父代肺血管功能障碍可影响子代，并且涉及突触和神经递质相关通路的参与。Smad1和SERPINE1启动子区的甲基化程度降低可能参与生命后期PAP表型产生。以上结果已在SCI杂志中发表。为进一步探索在生命早期阻断或减轻早期不良打击对肺组织、肺血管生成损伤的有效干预措施，我们在课题后期开展了通过原代培养脐带来源的间充质干细胞WJ-MSC后提取细胞外囊泡EV，在生后早期气道内注入可减轻后期高氧对肺实质肺泡稀疏化、肺血管生成的损伤，并开始进行下游EV内活性递质的机制研究。

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