妊娠糖尿病所引起的DNA过甲基化导致胚胎神经管畸形的机理研究

Maternal diabetes-induced embyonic malformations occur up to 3 to 10 times more than babies of nondiabetic mothers. Neural tube defects (NTDs) are among the most common and severe malformatons under maternal diabetes. Numerous studies have revealed that oxidative stress and kinase signaling are intimately involved in diabetic embryopathy. However, many of the molecular intermediates downstream of hyperglycemia remain to be elucidated. Epigenetic regulation has been proved to be implicated in diabetes-induced embryopathy; while the underlying mechanism is completely unknown. Our preliminary studies have shown that maternal diabetes increases the expression of three major DNA methyltransferases (DNMT) leading to DNA hypermethylation in neuroepithelial cells. DNA methylation mainly occurs in a CpG dinucleotide context. Increased methylation level of a CpG island containing CpG clusters in a gene promoter coincides with transcriptional repression of that gene. We found that maternal diabetes increases CpG island methylation in genes that are essential for neural tube closure (NTC genes) and, consequently, suppressed the expression of these genes. In addtion, we have shown that blocking the oxidative stress-c-Jun-N-terminal kinase 1/2 (JNK1/2) pathway significantly suppresses NTDs formation under diabetes condition, and a potential DNA methylation inhibitor, EGCG, ameliorates high glucose in vitro-induced NTD formation. Based on our findings, we hypothesize that maternal diabetes-induced DNA hypermethylation suppresses the expression of genes that are essential for neural tube closure and that the oxidative stress-JNK1/2 pathway is responsible for DNA hypermethylation. In the curent study, we employ a streptozotocin-nduced type I diabetes mouse model and high-fat diet-induced pre-type II diabetes mouse model to investigate the mechanism of DNA hypermethylation in maternal daibetes-induced NTDs. Our study will provide scientific evidence for understandng diabetic embryopathy and new cellular mediators for therapeutic treatments of NTDs.

妊娠糖尿病所引起的胎儿畸形率是正常胎儿的3到10倍，其中神经管畸形（NTDs）最为常见。研究显示，妊娠糖尿病可引起氧化应激、DNA过甲基化，通过诱导细胞凋亡和抑制神经管发育关键基因的表达而导致神经管畸形。本课题组预实验结果显示，阻断氧化应激-JNK 信号通路有效地抑制神经管畸形的发生；妊娠糖尿病显著上调三个重要的DNA甲基化酶（DNMT）的表达水平，抑制神经管闭合基因（NTC）的表达；用DNA甲基化抑制剂表没食子儿茶素没食子酸酯(EGCG）在体外可显著抑制神经管畸形的发生率。基于以上实验结果，我们推测"妊娠糖尿病所引起的氧化应激促进了DNMT的活性，通过抑制NTC的表达，最终导致神经管畸形发生"。本研究采用链脲佐菌素（STZ）诱导的I型糖尿病模型和高脂食物诱导的前II型糖尿病模型，通过SOD1转基因鼠以JNK1/2敲除鼠来研究妊娠糖尿病致神经管畸形的分子机理，为该病的防治提供重要科学依据。

妊娠糖尿病所引起的胎儿畸形率是正常胎儿的3到10倍，其中神经管畸形（NTDs）最为常见。本课题实验结果显示，敲除氧化应激激动酶JNK1或者JNK2基因抑制母体糖尿病引起的神经管畸形。这两个基因的敲除，也抑制了DNA高甲基化和保证了神经管闭合基因（NTC）的正常表达。在去氧化酶SOD1转基因鼠中，母体糖尿病引起胚胎DNA高甲基化酶和NTDs也得到显著抑制。母体糖尿病引起DNA甲基化酶DNMT3a和DNMT3b在胚胎中的高表达，选择性地敲除DNMT3a或3b基因在神经发育组织中抑制了母体糖尿病引起的神经管畸形，同时恢复了NTC基因的正常表达。用DNA甲基化抑制剂儿茶素没食子酸酯(EGCG)可显著抑制母体糖尿病引起的高甲基化，高DNMT3a和3b基因表达，以及阻止神经管畸形的发生率。母体糖尿病也抑制胚胎中去甲基化酶Tet1的表达，用Tet1转基因鼠恢复Tet1表达,也抑制DNA高甲基化酶和NTDs。基于以上实验结果，我们相信"妊娠糖尿病所引起的氧化应激促进了DNMT的活性和抑制Tet1的活性，通过抑制NTC的表达，最终导致神经管畸形发生"。本研究得到的以上結论，在两个糖尿病小鼠模型（链脲佐菌素（STZ）诱导的I型糖尿病模型和高脂食物诱导的前II型糖尿病模型）中都得到验证，本研究提供了最新的妊娠糖尿病致神经管畸形的表观遗传基理，为该病的防治提供重要科学依据。