乏氧诱导的microRNA-210对放射性直肠纤维化的影响与机制的研究

Hypoxia has been found well not only in solid tumors but also in radiation-induced normal tissue injury. However, the relation between hypoxia and radiation-induced rectal fibrosis is still unclear. Our previous studies have demonstrated radiation-induce tissue hypoxia contributed the development of rectal fibrosis, and microRNA-210 (miRNA-210) increased in murine rectum after radiation, which were demomstrated to be related with hypoxia-inducible factors family. These results suggested miRNA-210 and hypoxia maybe play a role in radiation-induced fibrosis. In this study, we will study the role of hypoxia in induction and development of fibrosis, and the relation of hypoxic signal and miRNA-210 in vitro and in vivo by hypoxic culture, miRNA-210 transfection, ELISA, mice model of radiation-induced rectal fibrosis and so on. Our study may be good to know the radiation-induced rectal fibrosis well and to improve the quality of life of patients who received pelvic radiotherapy.

乏氧是肿瘤的特征，同时也影响放射导致的正常组织损伤。迄今对乏氧和放射性直肠纤维化的关系还不明确。我们前期研究证实照射后的组织乏氧促进放射性直肠纤维化发展。另外预实验提示，和乏氧相关的miRNA-210在放射性直肠晚期损伤组织中表达增加，但二者影响放射性纤维化的机制还有待了解。为此我们提出假设，放射后的组织乏氧和放射性直肠纤维化密切相关，且很可能受miRNA-210及其相关基因的调节。为验证这一假设，我们将通过体外细胞模型，放射性直肠纤维化小鼠模型，采用质粒转染，乏氧培养，ELISA等手段，从分子，细胞，组织及动物等多方面探讨照射后的组织乏氧对放射性直肠损伤的影响，明确miRNA-210及乏氧微环境与放射性直肠纤维化的相互关系。本研究将从放射引起的组织乏氧这个新视点为揭示放射性直肠纤维化发生机制奠定基础，进而为放射性直肠炎的防治提供新思路。

我们前期研究证实照射后的组织乏氧是促进放射治疗后组织损伤的重要因素之一，但其详细机制仍未阐明。组织乏氧是放疗后组织的显著特征，研究证实乏氧本身是促进细胞损伤的刺激因素。MicroRNA-210是乏氧特征性miRNA，前期对C57BL/6N小鼠直肠部位进行单剂量25Gy照射建立了放射性直肠损伤模型。分别于照射后不同时间段取直肠组织，通过免疫染色检测组织损伤及纤维化程度，乏氧诱导因子-1alpha（HIF-1alpha）等，同时检测了microRNA-210表达。实验提示，和乏氧相关的microRNA-210在放射性直肠晚期损伤组织中表达增加。本研究中我们详细研究乏氧条件下microRNA-210对细胞的影响机制。我们利用人皮肤成纤维细胞GM0639，利用自噬抑制剂，ROS清除剂等手段，进一步探讨了乏氧条件下，microRNA-210与细胞自噬，凋亡，ROS清除，抗氧化等方面相互关系。结果显示，乏氧条件下和常氧条件下相比，细胞凋亡，自噬以及ROS水平显著提高。乏氧条件下，siRNA干扰microRNA-210表达后，细胞凋亡以及ROS水平进一步显著升高，同时伴随着BDNF（Brain-derived neurotrophic factor）表达增加。BDNF抑制可以促进NF-kB的表达及转录，进而促进自噬相关的LC3-II /LC3-I比例增加。综合以上提示乏氧条件下，细胞自噬保护细胞降低乏氧导致的细胞凋亡，其影响可能与通过microRNA-210/BDNF/NF-κB通路降低乏氧条件下的ROS水平密切相关。microRNA-210对放射治疗后，组织乏氧促进的直肠损伤具有潜在的保护价值，其机制有必要进一步深入研究。