bdnf基因转录的表观遗传调控在放射性神经发生障碍中的作用机制研究

The impariment of neurogenesis is invovled in radiation-induced cognitive dysfunction, but the underlying molecular mechanisms remain elusive. Brain derived neurophic factor (BDNF) has been implicated in regulating several aspects of hippocampal neurogenesis, including proliferation of neural progenitors and development of newborn neurons. However，there is no evidence of the key role of BDNF on radiation-induced neurogenesis impairment.Using a brain radiation injury model, our preliminary results demonstrated whole brain radiation significantly inhibited neuron regeneration and long-term survival. Meanwhile, RT-PCR and western-blot showed the expression of BDNF and its receptor Trk-B in hippocampus decreased significantly after radiation. And these results are accompanied by histone H3 acetylation levels significantly decreased. Based on the facts that epigenetic plays a key role in on gene transcrption and our previous study,we here propose that radiation might inhibit bdnf gene transcription activation by affecting DNA methylation, histone acetylation and other epigenetic mechanisms, thus affecting neurogenesis .To test this hypothesis,we would perform the works as follows:(1)To demonstrate the role of BDNF in radiation-induced neurogenesis impairment by injiecting exogenous BDNF to radiated rats;(2)Alterations of DNA methylation and histone acetylation at bdnf promotor will also be examined by chromatin immunoprecipitation assay (ChIP), methylation-specific PCR (MSP), and by bisulfite DNA sequencing (BSP);(3)To reveal the key factors affecting bdnf transcription by a intervention trial with epigenetic inhibitors - sodium butyrate and Zebularine.(4)Primary hippocampal neurons will be cultured and in vitro experiment will be performed to verify the above results.This issue might provide a better understanding of the pathogenesis of radiation-induced neurogenesis impairment.

海马区神经发生损伤在放射性认知障碍中起关键作用，但机制仍不清楚。脑源性神经营养因子（BDNF）可能与之相关，但尚无明确证据。我们前期实验发现全脑照射显著抑制了海马区神经发生及BDNF、TrkB的表达，并伴随组蛋白H3乙酰化显著下降。基于表观遗传学对基因表达调控的关键作用，我们假设：照射可能通过影响DNA甲基化、组蛋白乙酰化等表观遗传机制抑制bdnf转录活化，从而影响神经发生。为验证假设，我们将：（1）对照射组大鼠注射外源性BDNF，明确BDNF对放射性神经发生损伤的作用；（2）分析bdnf启动子DNA甲基化、组蛋白乙酰化及转录因子HDAC1/HDAC2/MeCP2等表观遗传机制的变化。（3）利用表观遗传修饰的抑制剂进行干预实验，明确影响bdnf转录的关键因素。（4）培养海马原代神经元，通过体外实验验证。本课题将阐明放射性神经发生损伤的机制，从而为放射性脑损伤的早期干预和治疗提供新的理论基础

认知功能障碍是颅脑放疗后最严重的并发症，最近几年的研究发现海马区神经发生损伤在放射性认知障碍中起关键作用。但是放疗导致海马神经发生损伤的机制仍不清楚。本项目在既往研究的基础上，给予实验SD大鼠30Gy全脑照射，通过免疫荧光激光共聚焦、western blot、RT-PCR的方法分析照射后海马区BDNF及其下游信号通路变化对神经发生的影响，并进行干预实验，验证BDNF对放射性神经发生障碍的关键作用。应用染色质免疫共沉淀（ChIP）、甲基化特异PCR（MSP）、亚硫酸盐修饰基因测序方法（BSP）方法分析电离辐射后bdnf启动子区表观遗传调控机制的变化，随后进行立体定位侧脑室注射表观遗传学修饰因子抑制剂丁酸钠（SB）和Zebularine进行药物干预实验验证。研究结果发现：1、bdnf mRNA在照射后7天显著下降，较对照组相比下降44% (P<0.05)，western blot显示其蛋白表达水平在第7天下降33%。在第30天，BDNF的表达进一步降低，较对照组下降51%。在中枢神经系统中，BDNF主要在神经元内合成，由轴突运输，与其特异性受体酪氨酸激酶受体B (TrkB) 结合，激活下游相关信号通路作用于靶组织发挥作用。因此，我们也检测了全脑照射后Trkb的表达，weatern blot显示TrkB在照射后7天和30天分别下降了24% and 35%，与BDNF的表达呈现相一致的变化。2、组蛋白去乙酰化酶HDAC1在照射后7天表达量升高，照射后30天明显高于对照组。注射HDAC1抑制剂TSA后，海马区神经发生得到了明显保护。HDAC1依赖的H3乙酰化的降低在电离辐射所致海马神经发生损害过程中具有重要作用。3、DNMT1在10Gy和30Gy照射后分别下降26% 和 41%，DNMT3a则分别下降20% 和 53%。DNMT抑制剂5-Aza-dc展现了同样的结果腹腔注射5-Aza-dc明显抑制了实验大鼠海马区神经发生，这从侧面验证了DNMT1/DNMT3a的作用。本课题的成果首次报道了BDNF在放射性神经发生损伤过程中的关键作用，其后世界范围内的几项报道证实了我们研究的可靠性。在此基础上我们进一步揭示了bdnf基因表达的表观遗传调控机制，从而为放射性脑损伤的早期干预和治疗提供新的理论基础。