电离辐射对神经突起生长发育的抑制作用及其发生机制中NFAT3/NFATc4所起作用的研究

Radiotherapy plays an irreplaceable role in therapy of primary and metastatic malignant brain tumors. The majority of patients with cranial irradiation suffered radiation-induced cognitive dysfunction. Radiation-induced impairment of Dentate Gyrus neurite outgrowth plays a significant role in cognitive dysfunction, the underlying molecular mechanisms are yet to be exploited. Published studies have shown that the nuclear factor of activated T-cells (NFAT) family of transcription factors are expressed in hippocampal neurons, where they play important roles in neurite outgrowth. Our group has developed the rat model of hippocampus damage induced by irradiation, and has indentified DG neurite outgrowth impairment in irradiated rat brain. In the current project, we will investigate the potential role of NFAT3/NFATc4 in radiation-induced DG neurite outgrowth impairment. By using various methods including hippocampus HE staining, immuno-fluorescence, confocal microscopy, electron microscopy, RT-PCR and western bolt as well as whole cell patch clamp, electrophysiology and calcium imaging, we will first exam neuronal functions, neurite outgrowth, NFAT signaling level in green fluorescent protein (GFP)-labeled hippocampal neurons before and after different dosage of irradiation. The neurite outgrowth ability will also be examined in hippocampus neurons treated with NFAT3/NFATc4 activators or inhibitors, or in hippocampus neurons with altered NFAT3/NFATc4 expression levels . The results of this research hopefully provide a better understanding of pathogenesis of radiation-induced cognitive dysfunction.

本课题组在已建立放射性海马依赖神经功能损害大鼠模型、已检测到神经元DNA断裂与修复异常和神经突起生长发育异常等工作的基础上，应用神经科学中关于认知功能形成、维持与障碍发生等相关研究工作的新知识与先进技术，提出了电离辐射导致神经元突起生长与发育异常、转录因子NFAT3/NFATc4在这一过程中起重要作用的假说。拟在一定剂量与观测时间条件下，运用常规病理、免疫荧光、共聚焦显微镜、电子显微镜、RT-PCR、Western Blot、全细胞膜片钳、神经电生理、钙离子成像等技术手段，对于离体与在体状态下逆转录病毒标记绿色荧光蛋白的神经元，进行成熟神经元功能、突起生长发育形态、NFAT及其信号通路中主要蛋白质分子进行系统性地检测。确定照射后神经元、神经突起、NFAT及下游关键信号分子的变化特征，并使用特异性刺激与抑制剂来验证其作用的相关性。为放射性认知功能障碍发病机制的研究开拓思路、提供新的理论依据。

放射治疗是头、颈部恶性肿瘤的重要治疗手段。在放射治疗之后，部分病人会出现以学习、记忆及注意力等下降为特点的放射性认知功能障碍。解析放射性认知功能障碍的发生机制，寻找有效的防治方法非常必要。.项目中观察到全脑照射后大鼠海马NFATc4/3、 CaN、GSK-3β蛋白表达水平在不同的剂量下均降低，p-NFATc4/3水平则升高。单次电离辐射暴露后，大鼠海马内新生成的神经元及离体培养的原代神经元的突起生长发育均受到抑制。照射组大鼠在照射后2周和4周海马内新生神经元树突总长度显著减少，神经元分支数在照射后2周显著减少。照射后4周和8周海马区新生神经元数量显著降低。而外源性BDNF可以通过调节NFATc4/3信号通路缓解电离辐射对神经元生长发育的抑制作用。与2Gy组相比,2Gy+BDNF组神经元分支点数在照射后2周明显增加，神经元树突总长度在照射后4周后出现显著提高。BDNF处理后，NFATc4/3在细胞核内的表达水平较单纯照射组明显升高；而CsA处理后，NFATc4/3在细胞核内的表达水平较单纯照射组明显降低。证实了NFATc4/3与电离辐射抑制神经元突起生长的相关性。电离辐射可导致大鼠海马内TrkA含量的升高以及TrkB含量的减少。对大鼠海马区p75基因进行敲减可减轻电离辐射对海马DG区树突棘密度的抑制，改善射线所致树突棘形态的异常，改善放射性认知功能障碍。P5-TAT多肽通过抑制电离辐射诱导的CDK5过度激活减轻放射性认知功能障碍。.本项目确定了NFATc4/3在电离辐射抑制神经突起生长发育中所起的作用，同时探讨了通过干预NFATc4/3信号通路治疗放射性认知功能障碍的可行性，为放射性认知功能障碍的治疗提供了新思路。