基于“邪毒致变”从Wnt/β-catenin信号通路探讨Hp相关胃病良恶性病理演变的证候特征

Our studies have shown that Helicobacter pylori-related gastric disease (HPGD) Hp infection, inflammation, and gastric mucosa atrophy with intestinal metaplasia, heterotypic hyperplasia pathological change tendency obvious different in different syndrome. The abnormal expression of NF-κB inflammatory pathways and IL1-B-511CT and TNF-α-308AG mutation genotype can be reflected the characteristics of spleen deficiency and splenogastric hygropyrexia different syndrome at a certain extent. "viscous damp evil" is the pathogenic key of "both inside and outside pathogenic factor" on Hp infection and inflammation factor abnormal expression. Through high-throughput second generation sequencing transcriptome qualitative analysis also suggests that the HPGD involves differences genes and function change on the Wnt/β-catenin signal pathways. Continuing " evil poison lead to change " viewpoint and characteristic of "viscous damp evil", this project from the regulate differentiation and risk of tumor associated genes, with Hp infection and β-catenin, CDX2 and COX2 candidate genes of Wnt signaling pathways in gastric mucosa as the breakthrough point, through genetic sequencing polymorphism personalized analysis, protein qualitative positioning and mRNA quantitative validation testing, expanded the pattern of syndromes further and included in cases of gastric cancer explore HPGD the pathological basis of the formation of different syndrome stage on splenogastric hygropyrexia, liver stomach discord, stagnation of phlegm turbidity, stagnation of blood stasis and spleen deficient damp accumulated. The research is helpful to find HPGD from benign to malignant pathological evolution in different syndrome phenotypic characteristics of related gene and risk gene polymorphic groups, for clinical targeted susceptible early warning of category homoplasy on syndrome, and individualized diagnosis and treatment provide basis.

前期研究表明，Hp相关胃病(HPGD)虚实证候胃黏膜Hp感染、炎症和萎缩伴肠化生、异型增生倾向性不同，NF-κB通路异常表达及IL1-B-511和TNF-α-308突变基因型可体现脾胃湿热与脾气虚证不同发病特点，“湿邪黏滞”为Hp感染与炎症因子表达“内外合邪”致病的关键；高通量测序转录组定性分析提示，HPGD差异基因与功能改变涉及Wnt/β-catenin信号通路。本项目延续“邪毒致变”观点，从细胞分化和肿瘤风险基因不同角度，以Hp感染与β-catenin、CDX2和COX2为切入点，通过基因多态性测序个性化分析及蛋白定性定位、mRNA定量验证检测，扩大证型并纳入胃癌病例探讨HPGD脾胃湿热、肝胃不和、痰浊凝滞、瘀毒内结和脾虚湿蕴不同证候形成的病理基础；研究有利于寻找HPGD良恶性病理演变中不同证候相关基因生物学表型特征和风险基因多态群，为证候群类趋同性发生的早期预警和个体化诊疗提供依据。

为探讨Hp相关胃病（HpGD）良恶性病理改变与Wnt/β-catenin信号通路中β-catenin基因rs1880481、CDX2基因rs1805107和COX2-899基因rs20417多态性改变和mRNA转录、蛋白表达差异以及与脾胃湿热、脾气虚、肝胃不和、瘀血内阻、痰浊凝滞不同证候的关联。本研究采用HE、快速尿素酶和美兰染色检测HpGD受试者幽门螺旋杆菌（Hp）感染与病理改变，一代直接测序法检测基因多态性，荧光定量PCR和ElivisionTM plus进行mRNA转录水平定量和蛋白定性定位表达验证检测。结果发现，① HpGD和不同证候形成与环境和情志因素、遗传易感性、Hp感染、胃黏膜病理改变等多因素相关；HpGD发病以脾胃湿热证最多，肝胃不和证次之，Hp感染程度及感染率则以瘀血内阻证最高，肠化生、异型增生程度也最重；HpGD病理改变与年龄、性别密切相关，高龄、男性可能是防治胃癌的不利因素。Hp感染是引发或加重HpGD不同证候病理改变的重要原因，尤以瘀血阻滞证最为明显，其次为痰浊凝滞证、肝胃不和证、脾胃湿热证及脾气虚证；在胃黏膜炎症→萎缩→肠化生→异型增生病理演变中，其病变程度随着Hp感染率及其程度的增加而加重；胃黏膜发生癌变后，Hp感染率及其程度却呈下降趋势；提示作为HpGD发生发展的先导因素，Hp感染贯穿于胃黏膜良恶性病理演变的始终；Hp感染阳性高龄男性且为痰浊凝滞或瘀血内阻证者，应注意早期胃癌的筛查。② HpGD痰浊凝滞证携带β-catenin基因rs1880481位点C等位基因、瘀血内阻和痰浊凝滞证携带CDX-2基因rs1805107位点T等位基因和瘀血内阻证携带COX-2-899基因rs1880481位点G等位基因且Hp感染阳性者及其mRNA转录水平高表达可能是胃黏膜恶性病变的高风险因素；CDX-2蛋白由胞浆入核强阳性异位表达预示着胃黏膜恶化的可能；当胃癌前病变转变为胃癌后，Hp感染率及其程度有所降低，COX-2 mRNA转录和蛋白表达水平亦降低，提示胃黏膜癌变后炎症反应可能不再是主导矛盾。在由实至虚的证候演变中，COX-2 mRNA转录和蛋白表达水平也降低，提示COX-2基因可能参与了“邪毒”Hp致病，机体抵御外邪的发生过程。