CD44/透明质酸偶联激活PI3K/Akt信号通路对提高肠干细胞修复放射性肠损伤的作用机制研究

In our preliminary work, the system for both sorting and culturing murine CD44-positive intestinal stem cells had been established. However, no one reports the results indicating whether the intestinal stem cells have therapeutic potentials for radiation-induced intestinal injury. To this end, we plan to do this research aiming at determining the therapeutic effects of CD44-positive intestinal stem cells on injured epithelium induced by ionizing irradiation, and exploring the intrinsic mechanisms by which intestinal stem cells repair epithelial injuries. CD44, a surface antigen, is the receptor of hyaluronic acid. Coupling between CD44 and hyaluronic acid will initiate the adhensive process of cancer cells and increase their anti-apoptotic potentials, which is predominantly driven by PI3K/Akt signalling pathway. On this basis, we attempt to establish the hyaluronic acid based models of 'Cell-migration' and ‘Anti-apoptosis’ in vitro for mimicking the adhension of intestinal stem cells to injured sites in vivo, and exploring mechanism by which PI3K/Akt enhances capability of intestinal stem cells in defensing injured milieu-induced cell-apoptosis. In vivo, we try to establish a NOD/SCID mouse model of radiation-induced intestinal injury to determine the therapeutic effects of CD44-positive intestinal stem cells on epithelial injuries. By comparing to the intestinal stem cells of Cd44 gene knock-out, we try to evaluate the advantages of CD44-positive intestinal stem cells in the following aspects, including their engrafting efficacies in injured sites, their apoptosis and the expressions of apoptosis-related molecules downstreaming of PI3K/Akt signalling pathway.

我们前期已建立小鼠CD44阳性肠干细胞的分选培养体系，但目前尚未见使用该细胞修复放射性肠损伤的研究报道。为此，本项目将对这一问题展开研究并探索CD44阳性肠干细胞在损伤环境下抵御凋亡的机制。CD44为透明质酸的受体。二者偶联所介导的细胞黏附及抗凋亡作用在肿瘤转移模型中被证实；其中，细胞凋亡抗性提高系PI3K/Akt通路激活所致。这是开展本项目的理论基础。我们通过建立以透明质酸为基础的‘细胞迁徙’和‘细胞抗凋亡’体外模型，分别研究CD44/透明质酸偶联对促进肠干细胞定植的作用；及其激活PI3K/Akt通路对提高肠干细胞凋亡抗性的分子机制。另外，建立放射性肠损伤NOD/SCID小鼠模型，以Cd44基因敲除肠干细胞为对照；通过比较CD44阳性肠干细胞和CD44阴性肠干细胞在损伤部位的定植效率、凋亡数量和表达PI3K/Akt通路下游相关凋亡分子水平的差异，评价CD44阳性肠干细胞修复肠损伤的优势。

放射性肠损伤属于临床常见疾病，而利用肠干细胞对其进行修复并未见报道。研究表明：CD44和其配体透明质酸的相互作用能够提高干细胞的抗凋亡能力；同时，PI3K/Akt是著名的抗凋亡通路。基于这一点，我们开展了本项目研究。项目主要围绕"CD44/透明质酸对提高肠干细胞抗辐射诱导凋亡能力”的功能学以及“PI3K/Akt如何介导这一抗凋亡作用”的机制两方面展开研究。发现：①低-中分子量的透明质酸对辐射诱导的小肠隐窝细胞凋亡产生拮抗作用，而高分子量产生促进作用；②低分子量透明质酸的辐射抗凋亡作用较中等分子量持久；③极低分子量透明质酸较低分子量透明质酸的抗辐射凋亡作用具有浓度依赖性；④透明质酸的抗凋亡作用与Akt分子Thr308和Ser473位点的磷酸化水平正相关；⑤低分子量透明质酸能够抑制DNA的损伤，主要表现为降低γH2AX蛋白的表达。另外，我们成功构建了CD44-mCherry荧光报告基因小鼠和CD44基因敲除小鼠。通过mRNA测序分析，发现：CD44基因敲除能够显著上调细胞自噬相关基因NoxA等的表达。这是本项目创新性发现之一。NoxA基因的表达调控受p53蛋白影响；同时，CD44蛋白的胞内段存在iASPP的结合域，iASPP又是抑制p53功能的关键蛋白。因此，我们进一步提出假：CD44蛋白与iASPP相互作用能够促进p53蛋白向iASPP蛋白结合，进而抑制p53蛋白向细胞核内迁移。该部分研究工作目前正在进行。本项目相关结果的预期为放射性肠损伤的干细胞治疗提供依据。