转录因子CREB在妊娠母体亚临床甲减致子代海马神经发育损伤中的作用机制研究

Thyroid hormone plays a key role in the differentiation and development of central nervous system. Thyroid hormone deficiency can cause the delayed neuropsychological development, even cretinism. Recently more attention was paid by specialists to the impact of maternal thyroid insufficiency in early pregnancy on fetal neuropsychological development of phase 1. The main etiology of thyroid insufficiency is hypothyroidism, especially subclinical hypothyroidism. Some investigators reported the prevalence of subclinical hypothyroidism during pregnancy was 2% to 3%. These indicate that the effect of maternal thyroid insufficiency on fetal brain development is common. Clinical trials have showed that mild thyroid insufficiency such as subclinical hypothyroidism and hypothyroxinemia during pregnancy can affect fetal neuropsychological development.CREB is a central transcription factor that mediates cyclic AMP (cAMP) and calcium-dependent gene expression and Ser-133 phosphorylation is considered to be a critical event that mediates the initiation of transcription，since mutation of Ser-133 to alanine abolishes transcription. Hippocampus is the key brain region related with learning and memory. CREB pathway plays an important role in signal transduction associated with learning and memory. CREB regulates transcription of many pro-survival genes, including BDNF and Bcl-2. The ability of calcium influx to induce transcription of brain-derived neurotrophic factor (BDNF) is regulated by the route of calcium entry into the cell, by the pattern of phosphorylation induced on the transcription factor cAMP-response element (CRE) binding protein (CREB). The present study aimed to explore whether the neurodevelopment damage was associated with the expression of CREB signaling pathway in the rats offspring.

国际上,几项大样本、多中心的关于孕期妇女亚临床甲状腺功能减退(SCH)致后代智力发育障碍的临床前瞻性研究报告，使该方向日益受到极大关注。但SCH如何致子代脑发育障碍的机制不详。本立项前期研究发现，核转录因子CREB的下游重要靶基因BDNF在SCH孕鼠子代海马表达下调，呈动态演变。结合子代学习记忆障碍，因此推测探究与BDNF表达密切相关的CREB信号网络有助于深入揭示孕期SCH对子代影响的作用机制。本立项在利用手术植入微渗泵建立稳定SCH孕鼠模型的基础上，应用电生理学、细胞凋亡检测、BrdU示踪标记及蛋白免疫印迹、Real-time qPCR等技术，从组织形态学、细胞水平、基因的调控、信号的转导等几个方面全面探讨母体SCH对后代脑发育影响在分子水平的致病机制，结合在孕期L-T4替代干预的效果，明确孕期母体SCH启动药物干预对改善后代脑发育障碍的时效性，具有重要的科学意义和实际的临床应用价值。

cAMP反应序列结合蛋白(CREB)是一种重要的核转录因子,调节启动子中具有环磷酸腺苷反应单元(CRE)的基因转录,在神经元再生、突触形成及学习记忆等方面具有重要的调节作用,是细胞内多种信号通路的一种关键成分。为了探讨亚临床甲减孕鼠对胎鼠以及仔鼠脑发育过程的影响及其分子机制，120只SPF级雌性Wistar大鼠随机分成：正常对照组（n=30)、手术完全切除甲状腺的甲减组（n=30)；将微渗泵（Osmotic Minipump）植入甲减鼠背部皮下，L-T4装入泵中，以1.0µg/（100g·d）浓度持续泵入建立的亚临床甲减组（n=30）以及亚临床甲减治疗组（n=30）即于亚临床甲减孕鼠孕13d（E13）对SCH大鼠进行补充L-T4治疗，剂量为1.25µg/（100g·d）。选取造模成功的雌鼠与正常的雄鼠合笼交配，于仔鼠出生1天（P1)、出生后7天（P7)及生后28天（P28)分别留取脑组织。孕鼠E10、E13、E17、P1留眶后静脉丛血，采用美国DPC公司固相化学发光酶免疫分析试剂盒检测甲状腺激素水平。P40仔鼠行HE、Nissl染色光镜观察CA1区病理变化及神经细胞内尼氏小体改变。Real-timePCR检测P1、P7、P28仔鼠海马中Bcl-2、rap-1mRNA的表达量，Western blot方法检测E13胎鼠海马PKA、BDNF、CREB蛋白表达量，并与同日龄对照组比较。P40行Morris水迷宫测试实验对象的空间学习、短时记忆、长时记忆以及学习新事物的能力。结果显示：母鼠亚临床甲减组在E10、E13、E17以及P1TSH明显高于对照组，TT4无明显差异（P>0.05）提示建模成功。2.与对照组相比，妊娠期亚临床甲减仔鼠出生后P40海马尼氏小体染色的平均光密度、积分光密度均明显减少。亚临床甲减组也可以引起后代的空间学习记忆能力的下降。3.测定P1、P7、P28 Bcl-2、rap-1mRNA水平与正常对照组相比差异显著,有统计学意义(P<0.05）；而Westernblot测定E13胎鼠脑组织PKA、BDNF、CREB蛋白表达量明显低于正常对照组(P<0.05)。结论：上述研究结果充分验证了我们提出的假说，即PKA/CREB/BDNF信号通路可能是调节亚临床甲减子代海马神经发生,改善学习记忆和认知功能缺陷的重要分子机制。