肺上皮性干细胞对放射性肺损伤的应答及其分化调控机制

Studies have demonstrated that the lung is particularly vulnerable to radiation. Lung fibrosis is a major late side effect of radiation damage characterized by loss of alveolar epithelium, as well as deposition of excess collagen and extracellular matrix (ECM). It is a serious consequence after radiotherapy. Unfortunately, all current therapies for this life-threatening complication are palliative at best, since its early phase usually displays nonspecific symptoms; subsequently it progresses to irreversible fibrosis. .Normally, lung epithelial stem cells (LESCs) are responsible for repopulation of damaged epithelial cells after tissue injury occurs. Therefore, regeneration of injured tissue critically depends on normal differentiation of LESCs. Currently, fibrosis is believed to be the consequence of aberrant transition from the regenerative to the fibrotic phase. Why does this LESC population fail to normally repopulate injured parenchymal cells during fibrogenesis? To date, reports regarding the source of collagen- and ECM-producing cells cannot exclude the effect of LESC on development of fibrosis. Our results showed that the expression of LESC marker and Wnt signaling pathway were enhanced after mice exposed IR, and mesenchymal cell markers and collagen were also increased. Recent study showed that downregulation of let-7 miRNA by TGF-β induced lung AEC II to express mesenchymal markers, resulting in fibrosis, indicating that let-7 miR is essential for LESC normal differentiation. It is known that let-7 is inhibited by LIN28, a downstream target of, activated by Wnt pathway. Thus, we hypothesize that inhibition of let-7 miRNA by IR causes LESC transdifferentiation into mesenchymal cells through evaluating the expression of TGF-β1, which stimulates Wnt/β-catenin signaling pathway, as a result, Lin28, a suppressor of let-7 miRNA, is activated. The mesenchymal cells are source of fibrotic components. In addition, graphene, a single atom thick sheet of carbon atoms arranged in two-dimensional honeycomb structure with unique physical, chemical and mechanical properties, and its derivatives have been reported to promote stem cell fate changes, and the reprogramming of mouse somatic fibroblasts into induced pluripotent stem cells (iPSCs). It also improved cellular reprogramming efficiency by inducing mesenchymal-epithelial-transition. The major goal of this study is to get insight into the new mechanism for radiation-induced lung fibrosis (RILF) and confirm our hypothesis by identifying TGF-β-Wnt-Lin28-let-7 miR asix and tracing LESC dynamic phenotypes and fates in radiation-induced lung injury models. Additionally, graphene will be used as a carrier for let-7 miRNA deliver allowing transfected LESCs epigenetically expressing let-7 or to transfect siRNA to knockdown let-7 to further confirm the essential for let-7 miRNA in LESC normal differentiation.

肺极易受离子辐射(IR)损伤，其后果可发生肺纤维化。照射后小鼠肺上皮干细胞标志物表达和分化信号通路Wnt被上调, 间质细胞标志物增强。正常和肺癌细胞经照射后let-7 miR改变最为显著。TGF-β1可显著下调let-7 miR，导致肺干细胞表达间质标记物，提示let-7 为肺上皮干细胞正常分化所必需。Wnt通过激活Lin28 蛋白抑制let-7 miR。故此，我们假设是IR导致TGF-β1大量产生，启动Wnt 信号，Lin28激活，let-7 miRNA 下调，肺干细胞异分化为肌成纤维细胞，导致纤维化。石墨烯可诱导干细胞分化，也能通过激活MET促进成纤维细胞重编程为iPSC。本课题通过放射肺损伤模型，追踪肺损伤时肺干细胞动态表型验证我们的假设。用石墨烯作为基因载体，通过沉默干细胞 let-7，进一步确认let-7为肺干细胞正常分化所必需。评估过表达let7逆转干细胞异分化可能性。

已知肺是对电离辐射最敏感的器官之一，放疗患者经多次照射后，致肿瘤细胞死亡的辐射剂量也严重损伤了正常肺上皮细胞，可致肺纤维化发生。目前对放射诱导的肺纤维化确切机制，尤其是对肺损伤修复如何演变为纤维化的过程缺乏了解，这使临床治疗不能对症下药。.本研究首先确认放射肺损伤再生修复致纤维化之间的转化点，追踪了具有修复肺上皮损伤功能的II型肺上皮干细胞(AECII)表型的动态变化，明确该细胞在放射肺损伤不同阶段的作用，同时也对细胞分化相关调控因子及信号传导途径进行分析，为继后肺纤维化发生的预防研究提供理论和实验依据。本研究主要亮点在于首次从放射损伤各个阶段的肺组织分离AECII进行研究，其价值在于获得的结果直接反映了该细胞的真实面貌，避免了AEC II体外培养时细胞表型的自发改变所造成的假象。.本课题主要部分已完成，已在SCI期刊发表2篇英文论文：FASEB Journal 和Journal of Radiation Research；一篇中文论文：中华放射肿瘤学杂志。一项专利申请已被接受。主要贡献：1. 从放射肺损伤后的肺组织直接分离AECII用于该研究，实验设计为首创；2. 首次用上皮干细胞标志物与AECII特异标志物明确AECII干细胞特性；3. 首次引用Lin28/let-7比率(ratio)作为分析AECII分化趋向的指标，即分化为肺上皮细胞还是间质细胞；4.首次提出新的干细胞分化调控信号传导网络（GSK-3β/Wnt/β-catenin/Lin28，Lin28/let-7 miRNA）。待完成部分：低剂量/低剂量率电离辐射预照射预防放射诱导肺损伤可能性及其机制研究。.重要结果和发现：1. AECII 表达上皮干细胞标志物；2. 照射后AECII即表达上皮细胞标志物，也表达间质细胞标志物；3.分离的AECII细胞纯度达91.2%；4.辐射后3个月为肺损伤修复关键期，5.辐射后GSK-3β, lin28与let-7 表达在AECII中被上调；6.AEC II Lin28/let-7 比率随辐射后时间而逐渐升高；7. GSK-3β/β-catenin/Lin28 通路诱导II型细胞异分化为间质细胞。.意义：肺损伤可逆修复关键时间点的确定和 AECII分化表型的干预对防止纤维化发生具有重要意义。