HIF-2α在反流性食管炎发病新机制中的研究

Reflux esophagitis (RE) is well known as one of major risk factors contributing to the development of esophageal adenocarcinoma. Prevention and treatment of RE can prevent the development of Barrett's esophagus and esophageal adenocarcinoma. However, about forty percent of the patients with gastro-esophageal reflux disease are not relieved from the PPI treatment, which has presented a great challenge to the widely accepted theory that reflux esophagitis develops from a refluxed-acid-induced, caustic chemical injury. Recent studies suggested that RE could result from an injury response mediated by inflammatory cytokines. It is well established that hypoxia exerts a substantial impact on the inflammatory process via HIF regulation Previous study reported that HIF2a expression increases gradually from normal squamous and metaplastitc epithelium, to adenocarcinoma. In our preliminary study, we found HIF2a is transcriptional activated in response to acid and bile treatment in the RE patient-derived squamous epithelial cells. We propose that HIF2a-mediated-inflammatory response plays a pivotal role in this novel mechanism of RE pathogenesis. That is, reflux activates HIF2a, and HIF2a then mediated the synthesis and secretion of inflammatory cytokines and promotes hyperplasia and progression of RE. Our technical strategy includes the use of in-vitro cell culture model, reporter gene assays, immune-precipitation to tackle the mechanism of HIF2a activation, the RNA and protein synthesis and interferences to evaluated the effects of HIF2a on chemokine activation and cell proliferation, and through clinical investigation to determine the correlation between HIF2a and the degree of RE, chemokine expression, and cell proliferation in patient tissues. Our study will provide a novel insight into how acid reflux may promote the development of RE while at the same time explore new targets for treatment and prevention of this disease.

胃食管反流引起的反流性食管炎（RE）是食管腺癌的危险因素。防治食管炎症明显降低食管腺癌的发生。约40%的食管反流病抑酸治疗仍迁延不愈，挑战传统的酸损伤机制。最新研究提出细胞因子介导的炎性损伤是RE的发病机制，并发现HIF-2α在食管正常鳞状上皮、肠化上皮和腺癌中表达依次增高。我们前期研究证实RE食管鳞状上皮细胞中HIF-2α被活化，因而推测HIF-2α介导的炎症反应是RE发病新机制的重要环节：反流导致HIF-2α激活下游通路促进炎症因子分泌和上皮增生，导致RE演进。我们以体外RE模型，报告基因、免疫沉淀等技术研究酸和胆盐刺激下HIF-2α活化机制；再通过阻断和增加HIF-2α表达，研究其对炎性因子活化和细胞增生的影响；最后，采用临床标本研究HIF-2α与食管炎严重度，炎性因子表达，细胞增殖及PPI疗效的相关性。本研究通过探讨RE新机制有望为治愈RE、预防其炎性增生和癌变提供新思路和靶点。

胃食管反流引起的反流性食管炎（RE）是食管腺癌的危险因素。防治食管炎症可明显降低食管腺癌的发生。然而传统的抑酸治疗仍有40%患者迁延不愈，对传统的酸损伤机制提出挑战。最新研究提出细胞因子介导的炎性损伤是RE的发病机制，并发现 HIF-2α 在食管正常鳞状上皮、肠化上皮和腺癌中表达依次增高。我们前期研究证实 RE 食管鳞状上皮细胞中 HIF-2 α 被活化，因而推测 HIF-2α 介导的炎症反应是 RE 发病新机制的重要环节：反流导致 HIF-2 α 激活下游通路促进炎症因子分泌和上皮增生，导致 RE 演进。我们以体外 RE 模型，报告基因、免疫沉淀等技术研究酸和胆盐刺激下 HIF-2α 活化机制；再通过阻断和增加 HIF-2α 表达，研究其对炎性因子活化和细胞增生的影响；最后，采用临床标本研究 HIF-2α 与食管炎严重度，炎性因子表达，细胞增殖及 PPI 疗效的相关性。本研究通过探讨 RE 新机制有望为治愈 RE、预防其炎性增生和癌变提供新思路和靶点。.项目推进过程中，我们在NES-G2T细胞中发现：①该细胞存在完整的HIF通道；②在正常氧浓度下， HIF-2α在细胞核中有基础表达，酸和胆盐的刺激可使细胞核中HIF-2α蛋白持续增高；③证明酸和胆盐可增加HIF-2α的表达，且早期可能是通过减少蛋白降解，晚期可能是通过增加蛋白合成。④上调HIF-2α后，Dcytb1，Cox-2等目的基因的蛋白表达上调；⑤敲低细胞中的HIF-1α， 酸和胆盐仍然可以增加HRE报告基因的活性；而敲低HIF-2α可则抑制细胞生长；⑥发现酸和胆盐的刺激可使COX-2、IL-1β、IL-8等促炎因子的蛋白表达增加，过表达HI F-2α可使这一改变的幅度加大，而敲低HIF-2α可使这一改变的幅度减小，提示酸和胆盐可增加NES-G2T细胞中促炎因子的表达，并且这一功能受HIF-2α的调 节。然而在后续研究中，我们未能在NES-G4T细胞及反流性食管炎患者食管鳞状上皮的原代细胞中重复出上述结果，但为保证科研基金的合理有效利用，我们将在课题进行过程中所收集的胃食管反流病患者临床病理资料，用于两篇文章的书写并公开发表于Neurogastroenterol Motil (2016) 及 J Gastroenterol Hepatol (2016)，为反流性食管炎的防治提供了策略指导。