交感神经兴奋调控放射损伤后肠道干细胞微环境重建的作用及机制研究

Large dose of ionizing radiation usually causes severe mucosa damage in gastrointestinal tract, which would result in high mortality of patients. The reestablishment of intestinal stem cell (ISC) niche is the basis for the regeneration of ISC and whole GI epithelium. In mammals, activation of sympathetic nervous system is involved in the regulation of many adult stem cells and their niche. However, it still remains unknown that how sympathetic nerve regulates the niche of ISC. Sympathetic nervous system is frequently over activated after severe trauma and stress, which causes hypoxia and ischemia of intestinal mucosa, apoptosis of epithelial cells, and finally the breakdown of intestinal barrier. Our group has evidenced that epinephrine (EPI) and norepinephrine (NE) can promote the proliferation of intestinal epithelial cells through binding to β2-adrenergic receptors (β2-AR), and the expression of β2-AR in Paneth cells was significantly increased at 3d after radiation. So we propose that activation of sympathetic nerve could improve the function of Paneth cells by binding to their β2-AR and facilitate the reestablishment of ISC niche. In this project, we would employ transgenic mice, FACS, enteroid culture and RNA-Seq to validate this hypothesis and the potential mechanism(s). The results of this project would give us a further insight into the function of sympathetic nerve in the maintenance of ISC niche, and provide us a new potential target for the treatment of radiation caused GI symptoms.

大剂量电离辐射暴露引起的肠粘膜屏障破坏常导致患者死亡，重建干细胞微环境是放射损伤后肠道干细胞和肠上皮得以再生的重要基础。交感神经参与多种干细胞及其微环境的组成和调控，但交感神经对肠道干细胞微环境的调控作用尚不清楚。严重创伤可引起交感神经过度兴奋，进而造成肠粘膜缺血缺氧、肠上皮细胞凋亡和肠屏障功能破坏。最近本课题组发现肾上腺素和去甲肾上腺素可通过与肠上皮细胞表面的β2-肾上腺素受体（β2-AR）结合促进肠上皮细胞增殖，同时放射损伤后3天潘氏细胞内β2-AR表达水平显著增加，因此我们提出交感神经兴奋可通过作用于β2-AR调节潘氏细胞功能从而促进放射损伤后肠道干细胞微环境的修复与重建这一理论假说。本课题将运用转基因小鼠、流式分选、类器官体培养和RNA-Seq等技术方法验证该假说并寻找其具体作用机制。其研究结果将深化对肠道干细胞微环境调控的相关认识，并为胃肠道放射损救治提供潜在新靶点。

目前大剂量电离辐射暴露所致肠道放射损伤仍无有效救治手段。交感神经过度兴奋可造成肠粘膜缺血缺氧、肠上皮细胞凋亡和肠屏障功能破坏，而交感神经适时适度兴奋对于组织再生修复具有促进作用。本项目围绕交感神经兴奋在正常和放射损伤后肠干细胞微环境重建中的作用和机制进行了一系列研究工作，主要取得了如下发现:（1）肌层细胞和潘氏细胞表达酪氨酸羟化酶证实肠道内存在交感神经支配；（2）小肠组织可表达α1b-AR, α2a-AR、α2c-AR、β1-AR、β2-AR、β3-AR等6种ARs，肠隐窝细胞仅表达α2a-AR、β1-AR、β2-AR三种受体，潘氏细胞可表达β2肾上腺素受体；（3）放射损伤后潘氏细胞中β2肾上腺素受体表达水平升高，提示在肠道放射损伤修复的关键节点需激活该受体表达从而介导静息肠干细胞活化；（4）放射损伤后肠壁ADRB2表达迅速减少而潘氏细胞ADRB2表达迅速增加，5-FU处理后小肠肌层ADRB2并不减少，同时潘氏细胞也未出现ADRB2染色增强，提示电离辐射和化疗药物在引发肠道损伤后对交感神经及其受体的激活模式不同；（5）肾上腺素可促进正常和照射后肠上皮细胞增殖；（6）通过条件性敲除潘氏细胞ADRB2发现ADRB2条件性敲除组小鼠的肠道再生修复与对照组之间存在明显差异；（7）电离辐射暴露能够引起肠道干细胞微环境中HIF1a表达变化；（8）发现iNOS在正常和放射损伤后肠隐窝内的表达特点；（9）发现褪黑素能够调控放射损伤后静息肠干细胞活化；（10）发现不同肠干细胞亚群在维持肠上皮更新和再生修复能力方面具有显著差异；（11）发现铁死亡能够调控肠道放射损伤再生修复进程。在本项目资助下，课题组已发表各类论文9篇，招收培养硕士研究生3人，完成了本项目的预期研究目标。本项目揭示了交感神经等内分泌因素在肠道放射损伤再生修复中的作用机制，为肠道放射损伤临床救治提供了新的作用靶点。