胆汁酸激活TGR5-EGFR-ERK1/2途径调控胃Lgr5+细胞分化参与反流性胃炎发病的机制研究

Chronic regurgitation of duodenal juice into the stomach is capable of damaging the gastric mucosa and causing symptoms including bloating, nausea, vomiting and loss of appetite. Pathological changes are characterized with foveolar hyperplasia, mucous cell hyperplasia, and smooth-muscle fibers proliferation in muscularis propria, together with only a minor increase in chronic inflammatory cells. Mechanism of these pathological changes and abnormal gastrointestinal motility is unclear. The current study basing on gastroid and mice model of bile reflux gastritis will use flow cytometry, immunofluorescence, Rt-PCR, Western-blot, EDU-click-it, stomach EMG and etc.to explore the effect of bile acids on stem cell patterning and fate, which ultimately leads to changes of the structure and function of antral mucosa. We will further confirm the consequence of TGR5-EGFR-ERK1/2 pathway activation by bile acids in antral Lgr5+ cells. Our hypothesis is that the activation of this pathway alters the fate of antral Lgr5+cells and drives stem cells differentiating into mucous cells rather than endocrine cells, which induces the decreased gastrin, ghrelin, somatostatin, 5-HT and other gastrointestinal hormones and gastrointestinal motility disorders. This study will offer fresh insights into the pathogenesis of reflux gastritis and provide useful target for the treatment of the disease.

胆汁反流入胃可引起的胃黏膜损伤，产生腹胀、恶心呕吐和食欲下降等症状。病理变化以胃小凹上皮增生为特征，伴腺颈黏液细胞增生、黏膜浅层血管扩张和固有层平滑肌纤维增生，但仅有少量淋巴细胞增生。目前胆汁反流造成胃小凹上皮及黏液细胞的增生和胃肠动力异常的机制尚不明确。本研究将使用三维培养gastroid及胆汁反流性胃炎动物模型，采用流式细胞、免疫荧光、Rt-PCR、Western-blot 、EDU-click-it方法、胃肌电图等技术，探索胆汁反流影响胃窦Lgr5+细胞命运变化导致胃窦黏膜结构和功能改变。进一步明确胆汁酸是否通过活化TGR5-EGFR-ERK1/2信号通路使胃窦Lgr5＋细胞向内分泌细胞分化减少而向黏液细胞分化增多，导致胃泌素、ghrelin、生长抑素、5-HT等胃肠激素分泌异常，引起食欲及胃肠动力改变。本研究将为胆汁反流性胃炎的治疗提供新的靶标。

胆汁反流性胃炎是由十二指肠内容物胃反流所致，胆汁反流入胃可引起的胃黏膜损伤，产生腹胀、恶心呕吐和食欲下降等症状。胆汁反流造成的胃小凹上皮增生和胃肠激素水平紊乱是否由胆汁成份刺激所致，目前相关研究很少。本项目在体外三维培养小鼠胃类器官 (gastroids)基础上，观察胆汁的成分之一鹅去氧胆酸（Chenodesoxycholic Acid，CDCA）对小鼠gastroids的生长、增殖、凋亡、分化及胃促生长素Ghrelin mRNA表达的影响。通过倒置显微镜观察gastroids的直径、形态变化；流式细胞术（PI染色、Annexin V/PI染色）检测gastroids中细胞增殖、凋亡情况；qPCR方法检测ghrelin mRNA表达变化；采用单细胞测序方法分析CDCA对gastroids中细胞分化的影响。结果发现CDCA浓度达200 μmol/L时，gastroids变小、死亡。CDCA浓度在0~100 μmol/L时，随着浓度增加，gastroids增殖活性无明显改变；而CDCA浓度在30、100 μmol/L时gastroids早期凋亡、晚期凋亡细胞比例增加。CDCA 10 μmol/L浓度刺激24h可导致gastroids小凹细胞的各个亚群比例发生变化，表现为促进处于细胞分裂周期的亚群凋亡。随着CDCA浓度增加，gastroids中Ghrelin mRNA表达水平逐渐下降。因此认为初级胆汁酸CDCA能够促进gastroids中小凹细胞群中处于细胞分裂周期亚群的凋亡，抑制小凹细胞的更新。同时，CDCA能使gastroids中内分泌细胞ghrelin mRNA的表达下调。这部分解释了长期胆汁反流中胆汁酸刺激胃黏膜上皮所致的小凹上皮细胞增生及胆汁反流性胃炎患者胃肠动力紊乱。