【作者】 赖逸贵；

【导师】 刘友章；

【作者基本信息】 广州中医药大学 ， 中医学， 2009， 博士

【摘要】 目的:慢性萎缩性胃炎(Chronic atrophic gastritis,CAG)是一种以胃粘膜上皮和腺体萎缩、粘膜变薄、粘膜肌层增厚,常伴有肠上皮化生、不典型增生为主要病理特征的慢性胃病,是消化系统疾病中难治病之一。根据胃癌发生的“连锁学说”,即胃癌的组织病理学演变经历了“慢性浅表性胃炎—慢性萎缩性胃炎—肠上皮化生—不典型增生—胃癌”的发展过程,慢性萎缩性胃炎伴肠化生或异型增生作为癌前病变已经被广泛接受。目前现代医学虽能通过根除HP及抗酸及营养支持,可以治愈胃黏膜炎症,但对已经进展至萎缩性病变患者,却无理想药物来逆转已经发现萎缩、化生或异型增生的病变,而中医根据整体观进行辨证论治,对本病有很好疗效。因此,对中医药治疗慢性萎缩性胃炎的机制进行深入研究,有着重要的科学意义及社会效益。导师刘友章教授根据本病的临床症状以胃脘部饱胀、胀满或胀痛不适,伴有食少纳呆、嗳气,大便溏或排便不爽,消瘦乏力等为主要表现,多将其归属于“胃痞”来进行辨证论治。其基本病机是由于素体禀赋不足、脾胃素虚;或饮食不节,损伤脾胃;或抑郁伤肝,横犯脾胃等多种因素导致脾胃虚弱,脾失健运,胃失和降,气血不足,中焦气机不利,升降失常,致使脾胃生理功能失调,迁延日久则致本病。故在治疗上,刘教授多以健脾益气、养血和胃及生肌为法。胃炎Ⅰ号是刘教授在几十年的临床实践中总结出来的治疗慢性萎缩性胃炎的有效方剂,具有健脾益气,和胃生肌之功效,并已经临床研究及实验研究初步证实胃炎Ⅰ号对脾胃虚弱型慢性萎缩性胃炎胃粘膜修复有一定的疗效,并有一定的抗上皮化生的作用。本实验研究以基质金属蛋白酶-1(matrix metalloproteinases,MMP1)和基质金属蛋白酶抑制因子-1(TIMP1)及整合素β1(integrin-β1)为切入点,对胃炎Ⅰ号治疗大鼠慢性萎缩性胃炎的作用机制进行更深入探讨,为胃炎Ⅰ号的临床疗效提供科学的实验依据,可以为开发临床治疗慢性萎缩性胃炎有效中成药胃炎Ⅰ号提供理论依据。本实验首次同时研究慢性萎缩性胃炎模型大鼠胃黏膜组织MMPI及TIMP1mRNA表达及酶活性相互关系,以及两者在大鼠慢性萎缩性胃炎形成中的协同作用机制。并对细胞外基质成分及细胞间黏附分子介导的细胞信号转导机制在大鼠慢性萎缩性胃炎形成中的作用机制进行初步探讨。方法:本研究包括文献研究和实验研究两部分。中医学无慢性萎缩性胃炎这一病名,故文献研究首先从本病与中医对应病名及其沿革进行整理,并对古代历代医家及现代历代中医家对本病病因病机、辩证分型以及治疗原则进行了系统论述。随着科学技术发展,现代医学对本病的病因、发病机制及诊疗技术等方面研究不断深入,本文对其进展也进行了综述。实验研究部分,选用健康SD大鼠84只,雌雄各半。共分六个组:模型组24只,空白组12只,高剂量组12只,中剂量组12只,低剂量组12只,西药对照组12只。采用50μg/ml甲基-硝基-亚硝基胍(MNNG)避光自由饮用、3mg/ml雷尼替丁灌胃每天一次2ml、加上2天给食1天停食的饥饱失常综合造模法构建大鼠慢性萎缩性胃炎模型,造模20周。经病理检查模型构建成功后,高剂量治疗组给予5g/(kg·d)胃炎Ⅰ号,中剂量治疗组给予2.5g/(kg·d)胃炎Ⅰ号,低剂量组给予1.25g/(kg·d)胃炎Ⅰ号,西药对照组给予0.86 g/(kg·d)维酶素,空白组正常饮食。采用明胶酶谱法检测MMP1酶活性,反向明胶酶谱法检测TIMP1酶活性,荧光定量PCR检测MMP1和TIMP1 mRNA表达,免疫组化定量检测整合素β1表达。结果:①光镜观察结果:模型大鼠胃黏膜腺体大小不一,结构紊乱,出现广泛的腺体壁变薄、腺体萎缩,腺体腔扩张、囊性变,未见肠化生及异型增生等病变。空白对照大鼠胃黏膜腺体排列整齐紧密,未见胃黏膜坏死、腺体萎缩、肠化生及异常等病变。中剂量胃炎Ⅰ号治疗后大鼠胃黏膜腺体排列规则,未见明显胃黏膜坏死,可见个别腺体轻度萎缩、壁薄轻度囊性扩张等病变。低剂量胃炎Ⅰ号治疗后大鼠胃黏膜腺体排列不规则,出现较多的胃腺体壁变薄、萎缩,胃腺体腔囊性扩张,未见明显化生及异型增生等病变。高剂量胃炎Ⅰ号治疗后大鼠胃黏膜腺体排列欠规则,出现少许的胃腺体壁变薄、萎缩,胃腺体囊性扩张,可见少许胃黏膜坏死,未见明显肠化生及异型增生等病变。西药维酶素治疗后大鼠胃黏膜腺体排列欠规则,出现轻度胃腺体壁变薄、萎缩,胃腺体轻度囊性扩张,未见明显坏死、肠化生及异型增生等病变。②荧光定量PCR检测发现CAG模型大鼠胃黏膜MMP1mRNA表达低于空白对照组(P＜0.05),经治疗后中剂量胃炎Ⅰ号MMP1mRNA表达明显上升,疗效优于低剂量和西药维酶素(P＜0.05),基本接近空白对照大鼠水平(P＞0.05);同时应用明胶酶谱法检测MMP1酶活性发现CAG模型大鼠胃黏膜MMP1酶活性高于空白对照组(P＜0.01),经治疗后高剂量和中剂量胃炎工号大鼠胃黏膜MMP1酶活性明显降低(P＜0.001),低剂量胃炎Ⅰ号及西药维酶素对降低MMP1酶活性疗效不明显(P＞0.01),其中以中剂量胃炎Ⅰ号疗效最佳,基本接近空白对照大鼠水平(P＞0.01);③荧光定量PCR检测发现CAG模型大鼠与空白对照大鼠胃黏膜TIMP1mRNA表达无明显差异(P＞0.05),治疗后各组间以及与模型大鼠胃黏膜TIMP1mRNA表达亦无明显差异(P＞0.05);同时应用反向明胶酶谱法检测发现CAG模型大鼠胃黏膜TIMP1酶活性低于空白对照组(P＜0.01),经高、中剂量胃炎Ⅰ号及西药治疗后大鼠胃黏膜组织TIMP1酶活性增加(P＜0.01),其中以中剂量胃炎Ⅰ号疗效最佳,基本接近空白对照大鼠胃黏膜水平(P＞0.05);④免疫组织化学定量法检测发现CAG模型大鼠胃黏膜组织整合素β1免疫组化显色平均数目和显色平均密度都增加(P＜0.01),经高、中、低剂量胃炎Ⅰ号治疗后大鼠胃黏膜组织整合素β1免疫组化显色平均数目和平均显色密度都有明显降低(P＜0.01),其中以中剂量胃炎Ⅰ号疗效最佳,基本接近空白对照大鼠整合素β1表达水平(P＞0.05),此外西药维酶素对降低大鼠胃黏膜组织整合素β1免疫组化平均显色密度也有一定效果;⑤对指标相关性进行Spearman统计分析,发现MMP1酶活性与TIMP1酶活性之间呈负相关(r=-0.642,P＜0.01);MMP1酶活性与整合素β1免疫组化显色平均数目及平均光密度均呈正相关性(r=0.753、P＜0.01,r=0.638、P＜0.01)。结论:①本实验采取综合造模法,虽能成功构建大鼠慢性萎缩性胃炎模型,但却有造模试剂对人体及环境有害等缺陷。因此在今后的实验研究中,可探讨更优越的造模方法。②本实验首次同时对MMP1及TIMP1的mRNA表达及酶活性进行了研究,结果表明,MMP1及TIMP1主要在酶活性调节层次相互制约参与大鼠慢性萎缩性胃炎的发生和发展过程。其可能的作用机制是通过调节MMP1和TIMP1酶的活性来调节细胞外基质的修复、再生等生理功能的。③本实验研究表明,整合素β1参与了大鼠慢性萎缩性胃炎形成机制。其作用机制可能是慢性萎缩性胃炎大鼠胃黏膜组织长期反复的损伤与修复过程使整合素β1的表达增加,以加强各种细胞间的黏附作用,并通过细胞信号转导作用,传递各种生长、分化信息,促进胃黏膜组织的再生。④相关性统计分析结果表明,MMP1与整合素β1可能存在协同机制在CAG大鼠胃黏膜损伤修复中起作用,共同维持细胞外环境的稳定及对细胞的凋亡、再生、分化、增殖及衰老等生物学行为起调控作用。⑤本实验研究表明,胃炎Ⅰ号对慢性萎缩性胃炎大鼠胃黏膜损伤具有良好的治疗及修复作用。可通过调节MMP1、TIMP1酶活性及整合素β1表达等机制,促进大鼠胃黏膜的炎症恢复及逆转腺体萎缩等病理变化趋势。其中以中剂量胃炎Ⅰ号疗效为最佳。更多还原

【Abstract】 Objective:Chronic atrophic gastritis(CAG) is a refractory disease of digestive system diseases,which’s main pathological changes including gastric mucosa and gastric glands thinning,lamina muscularis mucosae thickening,intestinal metaplasia,atypical hyperplasia, etc.According to the linkage theory of gastric cancer carcinogenesis,the pathological changes of gastric cancer carcinogenesis follow the step from chronic superficial gastritis (CSG),chronic atrophic gastritis(CAG),intestinal metaplasia,atypical hyperplasia and the end to gastric cancer.CAG as a kind of lesion precancerous has been generally accepted. Modern medicine can cure gastritis by eradicating HP,anti gastric acid secretion and providing nutritional support.But as to the pathological changes of atrophic gastritis, intestinal metaplasia and atypical hyperplasia,it doesn’t have active drug to cure.But Tradition Chinese Medicine(TCM) according to the entirety theory to zheng differentiation-treatment can receive excellent curative effect in curing CAG.It has very important Science significance and social benefit to lucubrate the mechanism of TCM treatment to CAG.My tutor professor Liu Youzhang according to the Clinical symptoms of CAG,treat it as stomach painful abdominal mass of TCM.The basic pathogenesis include weakness of the spleen and stomach,dys-splenism,stomach downward propelling disorders,insufficiency of vital energy and blood,middle energizer disorder of vital energy and ascent-descent disorder,.Therapeutically,professor Liu Youzhang treat CAG by strengthening spleen,benefiting vital energy,nourishing blood,regulating stomach and promoting granulation.GastritisⅠis an effective prescription which was found by Professor Liu Youzhang in his several decades clinical practice.It has the effectiveness of strengthening spleen,benefiting vital energy,nourishing blood,regulating stomach and promoting granulation,and has been confirmed it’s effectiveness in reconditioning gastric mucosa and resisting epithelial metaplasia of CAG.The purpose of our empirical study is to lucubrate the mechanism of gastritisⅠcuring the CAG rats by lucubrating the mechanism of gastritisⅠregulating to MMP1,TIMP1 and integrin-β1 in stomach of the CAG rats,and to provide scientific experimental evidence of the clinical therapeutic effect of gastritisⅠ.Our empirical study for the first time lucubrate the interrelation of mRNA expressing and enzymic activity between MMP1 and TIMP1,and their synergy in the mechanism of the CAG rats.Our empirical study also researches the mechanism of cell signal transduction between extra cellular matrix(ECM) and cell adhesion molecule of the CAG rats.Methods:Our research was divided into overview and empirical study.It doesn’t have the CAG disease name in TCM.So in the overview,the first we unscrambled the corresponding disease name in TCM to the CAG.We expounded the etiological factor, pathogenesis and therapeutic principle of ancient and contemporary doctor of TCM.We also expounded the advancement of modern medicine.In the empirical study,84 healthy SD rats were divided into 6 groups,including model group(24),normal group(12), western medicine control group(12),and 3 GastritisⅠtreatment group including high dose group(12),medium dose group(12),low-dose group(12).the CAG model rats was constructed by freely drinking the MNNG solution(50μg/ml),being intragastric administration and irregular diet.After the CAG model was constructed successfully, giving 5g/(kg·d) GastritisⅠto high dose treatment group,giving 2.5g/(kg·d) GastritisⅠto medium dose group,giving 1.25g/(kg.d) GastritisⅠto low-dose group,giving 0.86 g/ (kg.d) Wei Mei Su Pian to western medicine control group,the normal group was feed normally.We adopted gelatinase zymography to detect MMP1 enzymatic activity,inverted gelatinase zymography to detect TIMP1 enzymatic activity,fluorescent quantitation PCR to detect MMP1 and TIMP1 mRNA expression,adopted immunohistochemistry to detect integrinβ1 expression.Result:①Result of light microscope observe:The changes of gastric mucosa of CAG model rats included structure disorder,comprehensive glandular organ thinningz and atrophy and Saccular ectasia,the intestinal metaplasia and atypical hyperplasia dysplasia hadn’t develop jet.The gastric mucosa of normal rats was normal.The gastric mucosa of the CAG rats treated by medium dose gastritisⅠwas line up in order,and glandular organ thinningz and atrophy and Saccular ectasia,the intestinal metaplasia and atypical hyperplasia dysplasia hadn’t develop jet.The gastric mucosa of the CAG rats treated by low dose gastritisⅠwas structure disorder,major glandular organ thinningz and atrophy and Saccular ectasia.The gastric mucosa of the CAG rats treated by high dose gastritisⅠwas slightly structure disorder,a little glandular organ thinningz and atrophy and Saccular ectasia,and slightly necrosis.The gastric mucosa of the CAG rats treated by Wei Mei Su Pian was slightly structure disorder,a little glandular organ thinningz and atrophy and Saccular ectasia.②The result of fluorescent quantitation PCR found that the MMP1 mRNA expression of the CAG model group rats was lower than normal control group (P＜0.05).After the treatment of gastritisⅠ,the curative effect of medium dose gastritisⅠwas better than low dose and Wei Mei Su Pian(P＜0.05);the result of gelatinase zymography found that the MMP1 enzymatic activity of the CAG model group rats was higher than normal control group(P＜0.01),after the treatment of gastritisⅠ,the MMP1 enzymatic activity of medium dose and high dose gastritisⅠwere lower(P＜0.001),but the MMP1 enzymatic activity of western medicine control group and low-dose group were not obviously change(P＞0.01),the curative effect medium dose gastritisⅠwas best.③Result of fluorescent quantitation PCR found that the TIMP1 mRNA expression between the CAG model group rats and normal control group wasn’t significant deviation(P＞0.05), and it was the same between the groups after the treatment(P＞0.05).Result of inverted gelatinase zymography found that the TIMP1 enzymatic activity of the CAG model group was lower than normal control group(P＜0.01).After the treatment of medium dose gastritisⅠ,the TIMP1 enzymatic activity was higher(P＜0.01),and didn’t have significant deviation with normal dose(P＞0.05).Wei Mei Su Pian also had effect to raise the TIMP1 enzymatic activity(P＜0.05),but the curative effect medium dose gastritisⅠwas best.④Result of quantitative immunohistochemistry found that the integrinβ1 expression average coloration and average coloration density of the CAG model group was higher than normal control group(P＜0.01),after the treatment of medium dose gastritisⅠ,the integrinβ1 expression average coloration and average coloration density were lower obviously(P＜0.01),and didn’t have significant deviation with normal dose(P＞0.05).The high dose and low-dose gastritisⅠalso had effect to raise the integrinβ1 expression average coloration and average coloration density,but the curative effect of medium dose gastritisⅠwas best.Conclusion:①Our study adopted synthetic methods to construct the CAG model rats. Those methods were able to build the CAG model rats successfully,but the reagents of this method was harmful to human body and environment,the time of intragastric administration was too long,that would be increase death rate.Therefore,we can explore better methods of modeling in the future experiment.②In our experiment,we simultaneously studied the interrelation of mRNA expression and enzymatic activity between MMP1 and TIMP1 for the first time.The result found that MMP1 and TIMP1 participates the development of the CAG model rats in the enzymatic activity level.The mechanism of action was adjusting the repair and regenerate of ECM by regulating MMP1 and TIMP1 enzymatic activity.③The result of our study found that integrinβ1 participates the development of the CAG model rats.The mechanism of action was that the long-term damage and reparative process of the CAG rats made the expression of integrinβ1 increase, the adhesions between cells was strengthened,and transfer various kinds of growth and cell differentiation information by signal transduction of cells,and the end to promote the regeneration of stomach mucous membrane.④The result of our study found that gastritisⅠhad satisfactory curative effect and renovation to the damage of the stomach of the CAG rats.It could promote inflammation recovery and deteriorate glandular organ atrophy of the CAG rats,the curative effect of medium dose gastritisⅠwas the best.更多还原