【作者】 陈晴清；

【导师】 林培政；

【作者基本信息】 广州中医药大学 ， 中医临床基础， 2007， 博士

【摘要】 研究目的脾胃湿热作为湿热证的一个常见证型，同时也是中医脾胃理论中的一个重要实证，尤其在南方地区尤为多见，涉及的病种广泛，与消化系统疾病关系最为密切，尤以慢性胃炎占首位。脾为后天之本，脾胃功能的健旺，是保证机体抵御外邪的重要因素。目前研究显示脾胃湿热证患者机体内出现免疫异常，部分细胞因子如IL-2、IL-10、TNF-a等介导了机体内的炎症反应，局部胃粘膜免疫及体液免疫增强。脾胃湿热证中的局部组织炎症改变及免疫功能紊乱可能与幽门螺杆菌（Hp）感染及其导致的免疫反应有关，Hp感染可能启动了胃粘膜局部免疫反应，激活IFN-γ等细胞因子，诱使周围上皮细胞表达HLA-Ⅱ类分子，导致辅助性T淋巴（Th）细胞亚群平衡失调，出现Th1细胞反应占优。在一定程度上，免疫系统可以认为是阴阳平衡的调节系统，免疫调节也相当于是对阴阳平衡的调节。Th1／Th2细胞平衡包括其功能性的细胞因子之间的相互作用、相互制约在一定程度上体现了机体内阴阳相互对立制约、相互作用。Th1／Th2细胞平衡与脾胃湿热证的阴阳合邪、正邪相争、阴阳的动态平衡存在着一定的联系。HLA-Ⅱ类基因多态性在一定程度上影响着Th细胞亚群的分化方向和Th细胞亚群活性及增殖。目前研究显示脾胃与人体免疫系统密切相关，且认为中医证型和体质与HLA基因有关，脾虚证可能有一定的免疫遗传学基础，素体脾胃盛衰不同对疾病的易感性及转化存在明显的差异。因此，通过研究慢性浅表性胃炎和消化性溃疡的脾胃湿热证患者与Hp感染的相关性及血清细胞因子IL-2、IL-4、IL-10、IFN-γ水平及IFN-γ／IL-4比值的情况，以及脾胃湿热证与HLA-Ⅱ类基因多态性的相关性，了解脾胃湿热证机体内Th1／Th2细胞免疫平衡状态，寻找脾胃湿热证形成的易感基因，进一步揭示脾胃湿热证的生物学基础及发生机理，探讨脾胃湿热证的形成实质，从而指导临床脾胃湿热证的诊断和治疗。研究方法病例组及对照组均为同时期广东地区籍贯的汉族人，病例组为经胃镜检查诊断为慢性浅表性胃炎及消化性溃疡病人，共57例，来源于广州中医药大学第一附属医院内科病房住院和门诊病人，经辨证分为脾胃湿热证（33例）及脾气虚证（24例）两组；对照组15例，为同时期健康人。采用¹⁴C-尿素呼气试验检测病例组的Hp感染；采用双抗体夹心酶联免疫吸附测定（ELISA）法测定三组血清细胞因子IL-2、IL-4、IL-10和IFN-γ值，并比较三组细胞因子水平和IFN-γ／IL-4比值；采用聚合酶链反应-序列特异性引物（PCR-SSP）基因分型方法检测三组人全血的HLA-Ⅱ类基因中的HLA-DRB1和-DQA1的等位基因。结果脾胃湿热组Hp阳性率显著高于脾气虚组（P＜0.05），OR=3.733，显示脾胃湿热证与Hp感染相关。脾胃湿热组和脾气虚组IL-2水平均较正常对照组增高（P＜0.05），但脾胃湿热组和脾气虚组IL-2水平比较无显著差异（P＞0.05）；脾胃湿热组IFN-γ水平和IFN-γ／IL-4比值均较脾气虚组和正常对照组显著升高（P＜0.05）；而脾胃湿热组IL-4水平均较脾气虚组和正常对照组显著降低（P＜0.05），但IL-10水平仅较脾气虚组显著降低（P＜0.05），而与正常对照组相比无显著差异（P＞0.05）；脾气虚组IL-10水平较正常对照组显著升高（P＜0.05）；另外，Hp（+）组IFN-γ水平和IFN-γ／IL-4比值均较Hp（-）显著升高（P＜0.05），但是IL-2、IL-4及IL-10水平两组之间无显著差异（P＞0.05）。DQA1*0103基因型在脾胃湿热组显著高于正常对照组（P＜0.05），OR=4.48，提示DQA1*0103与脾胃湿热证相关，但与脾气虚组比较无明显差异；DQA1*0601基因型在脾胃湿热组显著低于正常对照组及脾气虚组（P＜0.05），但OR=0.156，提示DQA1*0601与脾胃湿热证的相关度较强。但HLA-DRB1各基因型在三组之间无显著性差异（P＞0.05）。结论结果显示慢性浅表性胃炎和消化性溃疡中脾胃湿热证患者和Hp感染相关，机体出现了Th1／Th2细胞免疫失衡，以Th1细胞反应占优势，表现为IL-2、IFN-γ水平的升高，IL-4水平的降低，IL-10水平的异常，细胞因子网络紊乱。脾胃湿热证中Th1／Th2平衡偏倚，免疫调节失衡，正是机体内阴阳失调、邪正相争、互相消长的体现。脾胃湿热证中IL-2、IFN-γ等促炎细胞因子水平升高，抑炎因子IL-4降低，引起持续的免疫应答，造成炎症病理损害，这与湿热证中热邪致病之性相似；但同时通过IL-10的抗炎作用，从而抑制免疫免疫反应，减缓炎症反应，导致脾胃湿热证病变缓慢。Th1型细胞因子和Th2型细胞因子共同作用，相互制约，导致脾胃湿热证起病缓慢，病势缠绵，这可能是湿热证缠绵难愈的病理机制之一。而Hp感染导致Th1／Th2免疫平衡偏移，也可能造成脾胃湿热证机体内Th1细胞反应占优的原因之一。脾胃湿热证与HLA-DQA1*0103基因型呈正相关，与HLA-DQA1*0601呈负相关，脾胃湿热证存在着一定的免疫遗传学基础，DQA1*0103基因型可能是脾胃湿热证的易感基因，DQA1*0601可能是脾胃湿热证的保护性基因。正是HLA-Ⅱ类易感基因通过编码的HLA-Ⅱ类分子识别和结合导致脾胃湿热证的感染因子或抗原刺激物，递呈给Th细胞，调控Th细胞的增殖和分化，影响免疫应答，从而形成脾胃湿热证。这提示脾胃湿热证可能存在着免疫遗传易感因素和病原体等感染的病因，故容易导致“脾土衰弱，失健运之堤防，湿气停聚不化”，再加遭受湿热外邪的入侵，内外合邪，从而诱发免疫应答，导致免疫平衡失调，细胞因子网络调节紊乱，正是这种免疫异常导致湿热证阴阳失调的病理改变及缠绵难愈的病程变化。更多还原

【Abstract】 ObjectiveGastrosplenic damp-heat syndrome （GDS） is a common syndrome type of damp-heatsyndrome （DHS）, as well as an important sthenia syndrome of Chinese medicine theory ofspleen and stomach. With the change in the way of life, particularly in the southern regionin China, GDS was particularly prevalent. GDS has extensive clinical basis, involvesvarious diseases, and most closely related to the diseases of the digestive system, especiallychronic gastritis for the first. Strong and vigorous spleen and stomach function is to ensurethat the organism to resist evils in virtue of spleen and stomach acquired for the foundationof life. Spleen and stomach closely related to the immune system. The current study showsthat the etiology of GDS is linked to Helicobacter pylori （H. pylori） infection. This paperstudies the local tissue inflammation and immune dysfunction in GDS may be related to H.pylori infection and immunoreaction subsequently. H. pylori infection may be activatedgastric local immune response and interferon （IFN）-gama and other cytokines, therebyinducing the surrounding epithelial cell expression of HLA classⅡmolecules. That leads tolymphatic T helper （Th） cell subsets imbalance, there Thl cell response dominant. H. pyloristrains may also through gastric epithelial human leukocyte antigen （HLA） classⅡmolecules-mediated host happen with closer interaction which led to a more significantgastric epithelial injury, as well as promote the immune response. To some extent, theimmune system can be considered to balance the Yin and Yang-conditioning systems,immune regulation also equivalent to the yin and yang balance adjustment. Th cells work akey role in the regulation of cellular and humoral immunity and specific immune andnon-specific immunity. Th1/Th2 balance, including its functions of cytokines interactionsbetween and mutual restraint, to some extent, reflects yin and yang to each other, mutualrestriction and generation and working together. Th1/Th2 balance of immune systemregulation reflected the pathology of GDS that varied by an association with yin evil andyang evil and the struggle healthy qi and pathogenic factor in organisms. And Th1/Th2balance also accorded with homeostasis between yin and yang opposing constraints,mutually restricting, eating and flowing in the process of GDS.The major histocompatibility complex （MHC） containing the human leukocyte antigen（HLA） is recognized as one of the most important genetic regions in relation to common human disease. The allelic and genetic structure of the MHC is complex. These extremelevels of polymorphism and dense genetic organization, including highly reiteratedsequences, correspond to so many differences of the individual constitution. And HLAallele frequencies also influenced by the geographical environment and so on. MeanwhileHLA classⅡgene polymorphism in a certain extent affected polarization, activity andproliferation of Th cell subsets. The current studies showed that spleen and stomach oftraditional Chinese medicine （TCM） are closely related to the immune system, and that thesyndrome types of TCM and body constitution were related to HLA genes. SpleenDeficiency Syndrome （SDS） may have some basis of immunogenetics. The constitutionalups and downs of spleen and stomach affect the susceptibility to disease and the conversionin the course of diseases.Therefore, we researched the relativity between the stomach-heat syndrome and H.pylori-related infection that diagnosed chronic superficial gastritis or peptic ulcer. And wemensurated the serum levels of interleukin （IL）-2, IL-4, IL-10 and IFN-gama, andcalculated IFN-gama/IL-4, so we may find out Th1/Th2 balance of immune cells in GDSfrom the results. At the same time we also studied the relativity between GDS and HLAclassⅡgene polymorphism, to search the gene type susceptible to GDS. Accordingly, wemay further reveal the biological basis and the mechanism of GDS, and know the formationof substance of GDS which to guide the diagnosis and treatment of GDS.MethodsWe selected 57 patients as case group, which diagnosed chronic superficial gastritisand peptic ulcer by gastroscopy from the medical wards in First Affiliated Hospital ofGuangzhou University of Chinese Medicine, and 15 normal cases as control group. Casegroup and control group were Guangdong native people of the Han nationality. Accordingto syndrome differentiation, case group was devided into two groups, which were GDSgroup （33cases） and SDS group （24 cases）. The patients in GDS group and SDS group werechecked H. pylori infection by ¹⁴C-urea breath test. The serum levels of IL-2, IL-4, IL-10and IFN-gama in the three groups were mensurated by double-antibody sandwichenzyme-linked immunosorbent assay （ELISA）, and were compared statistically. The allelesof HLA-DRB1 and HLA-DQA1 in three blood groups were detected by polymerase chainreaction-sequence specific primers （PCR-SSP） genotyping method.ResultThe infective rates of H. pylori in SDS group was remarkably higher than the rate inSDS group （P＜0.05）and the odds ratio （OR） is 3.733. That showed H. pylori infection wasrelated to GDS. The levels of IL-2 in GDS group and SDS group were higher than the levelin control group （P＜0.05）, but the level of IL-2 was no significant difference in GDS groupand SDS group（P＞0.05）. The level of IFN-gama and IFN-gama/IL-4 were higher in GDS group than in SDS group and control group （P＜0.05）. Contrarily, the level of IL-4 waslower in GDS group than in SDS group and control group （P＜0.05）. The level of IL-10 wasonly lower in GDS than in SDS （P＜0.05）, but showed no significant difference with thelevel in control group （P＞0.05）. In addition, the levels of IFN-gama and IFN-gama/IL-4were higher in H. pylori（+） group than in H. pylori（-） group （P＜0.05）, but IL-2, IL-4 andIL-10 levels were not significantly different （P＞0.05）.The gene frequency of DQA1^*0103 in SDS group was remarkably higher than innormal contrast group （P＜0.05）, and OR was 4.48, but no statistical significant differencewas found between SDS group and SDS group（P＞0.05）. On the contrary, the genefrequency of DQA1^*0601 in SDS group was remarkably lower than the other tow groups（P＜0.05）, OR was 0.156. The data suggest that DQA1^*0103 and DQA1^*0601 is closelyassociated with SDS. But no statistical significant differences in carrier frequencies ofHLA-DRB1 alleles were evident （P＞0.05）.ConclusionThe results showed that GDS in chronic superficial gastritis and peptic ulcer wasrelated to H. pylori infection. It was Th1/Th2 cellar immune imbalance that emerged in theorganism of GDS. There was Th1 cell response bias and cytokines network disorders as theresult of the increase of IL-2 level and IFN-gama level, the reduction of IL-4 level, andabnormity of IL-10 level. The bias of Th1/Th2 balance, embodied imbalance of immuneregulation, reflected the vivo imbalance between yin and yang, the struggle healthy qi andpathogenic factor. It may be one of lingering and refractory pathological mechanism ofdamp-heat syndrome that the Th1-type cytokines and Th2-type cytokines interactions andmutual constraints lead to the slow onset and potential lingering GDS. Moreover, theimbalance of Th1/Th2 immune deviation leaded by H. pylori infection may also cause Th1cell response domination in the organism of GDS. The data showed that HLA-DQA1^*0103was positively correlated genotype with GDS, and HLA-DQA1^*0601 was negativelycorrelated genotype. It suggested that GDS has a certain immunogenetic basis. HLA-DQA1^*0103 may be the susceptible gene to SDS, and DQA1^*0601 may be the protectivegene. HLA classⅡmolecules encoded by HLA-susceptibility gene can identify andcertificate GDS infectious factor or antigenic irritant leading to GDS, submit to Th cells,and regulate Th cell proliferation and polarization, thus affects the immune response. It isthe cocontribution of HLA classⅡ-susceptibility gene and Th1/Th2 cell immune balancethat result in GDS.Above all, because GDS may have immunogenetic susceptible factors and theinfectious pathogens, it will easily lead to weak functions of spleen and stomach and vivoaccumulation of dampness. So if damp-heat evils invade, it will combine with the vivodampness and consequently lead to imbalance of yin and yang and induce immunologicalresponse and result in immune imbalance and cytokine network disorders. It is these immune disorders that induce the imbalance between yin and yang and the lingering andrefractory pathological changes in GDS.更多还原