菌群失调介导的粘液层损伤与消化性溃疡的关系

PUD (peptic ulcer disease) is one of the common upper gastrointestinal diseases. The etiopathogenesis of PUD is still under investigation. Currently, the most recognized etiology research mainly confined to the acidity in stomach and HP infection, however, ulceration does indeed not develop spontaneously in healthy gastroduodenal mucosa despite being bathed in strong corrosive acid-peptic solution or HP high infective rate. Also, it cannot deny the clinical truth that PUD patients always bothered by repeated recurrence after HP eradication and acid suppression therapy. Our project presents an innovative hypothesis that PUD belongs to a mucous disease which is induced by the dysbacteriosis in the gastric mucous layer. Ulceration is the ultimate consequence of a disequilibrium between the degeneration and synthesis of gastric mucus proteins. The over effect of proteolytic and deglycosylation enzyme in bacterial cell lead to mucous layer injure, which weaken the mucous membrane barrier. The aggression factor such as gastric acid, pepsin and Hp can result in secondary damage of endothelial cells . In our study, we use human gastric mucosa samples to analysis the bacterial flora change, mRNA expression of bacterial enzymes and integrity of gastric mucous layer among the HP -positive, idiopathic peptic ulcer and normal control. Bacterial Gene sequencing, real-time PCR, SDS-PAGE electrophoresis, ,biological mass spectrometry and animal validation models can be needed. The purpose of this study was to find the relation between the gastric mucous layer damage induced by gastric dysbacteriosis and the pathogenesis of PUD, also try to establish a better cure for PUD patients.

无论是胃酸、胃蛋白酶还是Hp感染均无法作为原发性因素阐明PUD的发病机制，因为这些因素必须穿过胃粘液层才能引起黏膜上皮损伤而致病。所以除胃酸和Hp感染之外，影响胃黏膜完整性的因素才是PUD发病的起始原因。本项目提出以下假说：PUD患者胃粘液存在菌群失调，细菌对胃粘液蛋白的过度降解导致局部粘液屏障受损，胃酸、胃蛋白酶以及Hp等攻击因素得以穿过胃粘液层达到黏膜上皮引起病理损伤，可作为PUD发病的继发性因素，最终导致溃疡发生。本研究主要采取高通量测序和Real-time PCR分析证明PUD患者胃粘液细菌种群构成异常，SDS-PAGE和质谱技术进行分析胃粘液层的完整性。并构建细菌干预动物验证模型。通过对比Hp感染性、原因不明性PUD和正常对照组的细菌差异、细菌蛋白水解酶和脱糖基酶表达水平，以及其与粘液完整性的关系。阐明胃内菌群失调介导的粘液损伤在PUD发病机理中作用。

背景：胃酸和病因Hp是目前普遍认为引起PUD的两大因素，但无论Hp还是胃酸和胃蛋白酶都无法穿过完整的胃粘液层到黏膜上皮产生损伤。胃粘液层菌群失调可能参与局部黏膜屏障受损。.研究内容：1.胃粘液菌群分析：收集了140个患者（包括慢性胃炎、胃溃疡、十二指肠溃疡、胃癌患者）的胃粘液层样本，分析并提取细菌，测定粘液层完整性；2. 差异细菌与PUD的关系：将粘液中发现的差异细菌进行培养；3.建立差异菌群小鼠感染模型，使用抑酸治疗和接种Hp干预。对比各组Hp定植率与病理组织学差异。.重要结果：1.患者胃粘液层样本分析发现，在属水平上，对比胃炎组，胃十二指肠溃疡组Roseburia和Oscillospira明显增加，Bacteroides和Parabacterioides相对减少；与胃癌组相比，Parabacterioides明显减少。在门的水平上分析，与正常组相比较，胃十二指肠溃疡组及胃癌组的拟杆菌明显增加，厚壁菌丰度明显减少。胃粘液主要黏蛋白MUC5AC和MUC6的完整性被破坏。2. 前期得到的差异菌群进行分离培养,对不可培养细菌，提取粘液中总DNA及mRNA，同样行real time-PCR分析种或属水平的细菌在不同人群和胃各部分的分布，验证结果同测序结果相一致。差异性细菌与PUD相关3.使用差异细菌建造动物模型，测定Hp的定植率：造模组接种Hp4周、8周后胃黏膜Hp的定植率均达100%，PPI加Hp对照组的定植率分别为50%和37.5%，直接给予Hp对照组的定植率分别为37.5%和25%。生理盐水对照组为0。造模组与各组比较有显著性差异（p<0.05）。病理组织学结果：接种Hp后4周、8周，造模组小鼠胃黏膜均出现慢性炎症改变, 且炎症程度随时间增加而加重，胃黏膜炎症评分明显高于PPI加Hp对照组和单用Hp对照组（p<0.05）。.科学意义：胃粘液层的菌群失调与消化道溃疡有一定相关，PUD患者胃黏膜菌群存在差异，进而使胃酸、胃蛋白酶以及Hp等攻击因素作用于粘膜上皮。为预防及治疗消化道溃疡疾病提供新的思路及方向。