【作者】 弓淑珍；

【导师】 刘友章；

【作者基本信息】 广州中医药大学 ， 中医内科学， 2009， 博士

【摘要】 研究背景:胃癌是消化系统常见的恶性肿瘤之一,位居全球肿瘤发病率和癌症病死率的第二位,严重威胁着人类的健康。然而其病因病机尚不明确,无法进行针对病因的一级预防,但胃粘膜的癌变往往经历较长的癌前病变阶段,呈现慢性浅表性胃炎一慢性萎缩性胃炎—肠上皮化生—不典型增生—癌变的趋势。其中,不典型增生和肠上皮化生被称为癌前病变。因此,针对癌前病变的二级预防是防治胃癌的关键。如果能在胃癌前病变阶段及早诊断,及早治疗,则能极大的降低胃癌的死亡率。中医药在防治胃癌前病变方面展示出独特的优势。胃炎Ⅰ号是导师刘友章教授根据中医脾胃为后天之本,气血生化之源的理论,结合临床,创制的纯中药复方。在临床应用中取得了很好的疗效。前期研究已初步证实了胃炎Ⅰ号对脾胃虚弱型慢性萎缩性(CAG)胃炎病人和CAG模型大鼠胃粘膜修复有很好的疗效,并有一定的抗肠上皮化生的作用。本课题拟研究胃炎Ⅰ号治疗胃癌前病变可能的作用机制。近年来许多研究证实胃癌中基质金属蛋白酶(MMPs)出现过度表达和活性提高。MMPs是内源性蛋白水解酶,它主要通过降解细胞外基质、调节细胞间黏附、以及促进新生血管形成等参与组织重构、细胞增殖、恶性细胞浸润、转移等。MMP-7、9是MMPs家族中重要成员,它们的过度表达或活性的提高与肿瘤的生长、浸润和转移等有密切关系。此外,一些研究已证明,MMP-7、9在癌前病变组织中的表达较慢性胃炎组织中的表达升高。那么,胃炎Ⅰ号方治疗胃癌前病变的机制是否与MMP-7、9有关?这将是本课题研究的重点。研究目的:本课题通过临床研究对胃炎Ⅰ号方临床疗效进行较为客观的评价,并使用分子生物学技术以及免疫组织化学技术等,观察大鼠胃组织中MMP-7、9蛋白的表达、酶的活性,以及MMP-7mRNA、MMP-9mRNA的表达,从而初步探索胃炎Ⅰ号治疗胃癌前病变可能的作用机制,为临床更好的使用该方提供理论依据。研究方法:1.动物实验:将84只大鼠,雌雄各半,按体重随机分为6组,模型组24只,空白组、胃炎Ⅰ号大、中、小剂量组及维酶素组各12只。造模期间,空白组给予正常饮食以及等体积生理盐水灌胃。其余各组给予以下处理措施:①50μg/mL N-甲基-N’-硝基-亚硝基胍(MNNG)溶液自由饮用,造模期间不再给予其他饮水;②盐酸雷尼替丁水溶液按每天0.03g/kg灌胃,每日1次;③饥饱失常:连续2d喂食,保证足量,1d停食。造模共20周。造模成功后,胃炎Ⅰ号大、中、小剂量组分别使用2.16g/(kg·d)、1.08g/(kg·d)、0.54g/(kg·d)的胃炎Ⅰ号水煎液灌胃。维酶素组用0.86g/(kg·d)的维酶素水溶液灌胃。空白组和模型组均用0.18g/(kg·d)的生理盐水灌胃。给药3个月。实验结束后观察大鼠一般情况,取胃组织做病理检查。使用免疫组化技术观察MMP-7、MMP-9蛋白的表达。使用明胶酶谱法观察MMP-7、MMP-9酶的活性。使用聚合酶链反应(PCR)技术观察MMP-7mRNA、MMP-9mRNA的表达。2.临床观察:符合纳入标准的胃癌前病变患者30例,按就诊时间随机分为胃炎Ⅰ号治疗组和维酶素对照组,每组各15例。采用中医症候量表进行观察,比较两组治疗前后中医症状评分、两组患者的中医疗效,以及治疗后药物安全性观察。从而对胃炎Ⅰ号方治疗胃癌前病变的临床疗效做出客观的评价。研究结果:1.动物试验:病理结果显示大鼠胃粘膜腺体萎缩,伴有肠上皮化生和不典型增生,提示造模成功。经胃炎Ⅰ号和维酶素治疗后,上述病理改变减轻或消失。大鼠胃组织MMP-7的表达:空白组与模型组比较有统计学意义(P=0.000)。大、中、小剂量组及维酶素组与模型组比较有统计学意义(P=0.000,P=0.000,P=0.005,P=0.032)。大、中、小剂量组之间无统计学意义,中剂量组与维酶素组之间有统计学意义(P=0.032)。大、小剂量组与维酶素组之间无统计学意义(P=0.093,P=0.466)。大鼠胃组织MMP-9的表达:空白组与模型组比较有统计学意义(P=0.001)。大、中、小剂量组及维酶素组与模型组比较有统计学意义(P=0.002,P=0.000,P=0.002,P=0.012)。大、中、小剂量组及维酶素组之间无统计学意义。大鼠胃组织MMP-7活性:χ~2=26.533,df=5,P=0.000,各组间差异有统计学意义。从平均秩次的变化趋势看模型组活性最高,空白组活性最低。中剂量组较大、小剂量组、维酶素组MMP-7活性低。大鼠胃组织MMP-9活性:χ~2=24.386,df=5,P=0.000,各组间差异有统计学意义。从平均秩次的变化趋势看模型组MMP-9活性最高,空白组活性最低。中剂量组较大、小剂量组、维酶素组MMP-9活性低。大鼠胃组织MMP-7mRNA表达:χ~2=11.730,df=5,P=0.039,各组间差异有统计学意义。从平均秩次的变化趋势看模型组MMP-9mRNA表达水平较空白组上调。胃炎Ⅰ号大、中、小剂量组和维酶素组较模型组表达水平下调。大鼠胃组织MMP-9mRNA表达:χ~2=11.385,df=5,P=0.044,各组间差异有统计学意义。从平均秩次的变化趋势看模型组MMP-9mRNA表达水平较空白组上调,胃炎Ⅰ号大、中、小及维酶素组表达水平较模型组下调。2.临床研究:两组在性别、年龄段、病程段等方面比较无统计学意义(P均＞0.05),提示两组具有可比性。中医证候量表调查显示,治疗前两组各症状比较无统计学意义(P均＞0.05)。治疗后两组各症状比较,胃炎Ⅰ号方对于改善痞满胀闷、胃脘疼痛、口干口苦、嗳气反酸、大便稀溏方面与维酶素有差异(P均＜0.05)。二者在改善纳呆乏力症状方面无统计学意义;治疗组和对照组各症状治疗前后比较,胃炎Ⅰ号方和维酶素均能改善各临床症状(P均＜0.05)。两者在改善胃脘疼痛、嗳气反酸、口干口苦、大便箱绶矫嫖秆注窈欧接庞谖杆?在痞满胀闷、纳呆乏力方面两者无差别。两组中医疗效比较有统计学意义(P=0.045),胃炎Ⅰ号方疗效优于维酶素。结论:1.胃炎Ⅰ号方对改善胃癌前病变大鼠一般情况,以及胃粘膜慢性炎症,逆转腺体萎缩、肠上皮化生等方面均有一定疗效。胃炎Ⅰ号方对改善大鼠胃癌前病变组织异常的超微结构有一定的作用。2.胃炎Ⅰ号方治疗胃癌前病变的机制可能是降低MMP-7mRNA、MMP-9mRNA的表达,使大鼠胃组织MMP-7、MMP-9蛋白的表达下调,以及降低MMP-7、MMP-9酶的活性,多个环节共同起作用所致。3.胃炎Ⅰ号方和维酶素均能改善患者临床症状,两者中医疗效比较,胃炎Ⅰ号方疗效优于维酶素。更多还原

【Abstract】 Background:Gastric cancer is a common cancer in the alimentary system.Its incidence rate and cancer mortality is the second place among the world’s incidence rate and cancer mortality,so it has became a serious threat to human health.Because we don’ t know its pathogen and pathogenesis,we could not prevent the disease according to the pathogenesis.Gastric mucosa cancerization always go though long precancerous phase.The pathological changes had shown the general trend that from chronic superficial gastritis to chronic atrophia gastritis,then intestines epidermis vegetates and atypical hyperplasia.The intestines epidermis vegetate and atypical hyperplasia were named as gastric precancerous lesion.So preventing and treating gastric precancerous lesions is the key of the secondary prevention. If we can diagnose and treat the disease in the gastric precancerous stage, cancer mortality should be decline greatly.Chinese medcine demonstrates advantages in preventing and treating precancerous lesions.According to the theory of Traditional Chinese Medicine(TCM) which spleen and stomach are the postnatal base and the sources for the production and transformation of qi and blood,Professor Liu Youzhang created the TCM compound formula named as WeiYanIHao(WYIH) after the long-term clinical practice.Preliminary clinical researches have shown that WYIH have significant curative effect on the chronic atrophic gastritis patients with weakness of the spleen and the stomach. Preliminary animal experiment have shown that WYIH have significant curative effect on the CAG model rats,as well as reverse intestines epidermis vegetate to some extent.The purpose of this research is to study the possible mechanism.In recent years,several studies have shown that Matrix metalloproteinase(MMPs) can be expressed excessively in gastric cancer.MMPs are endogenic proteolytic enzyme,Which participate in tissue reconstruct,cell proliferation,malignant cell infiltration,and metastasis by means of degrading extracellular matrix,regulating cell adhesion,and promoting vascularization,et al.As the important members of MMPs famility,MMP-7 and MMP-9 had been proved that their excessive expression and increasing activity are connected closely with tumorous growing,immersion,and transferring. Previous researches have shown that the expressions of MMP-7,9 in gastric precancerous lesions are higher than those expressions in the chronic gestritis tissue.Are the control mechanisms that the WYIH formula treat the gastric precancerous lesion related to MMP-7,9? These will be the focus of the experiment.Objective:According to the clinical research,we will draw out an impersonal and fair conclusion on WYIH clinical effects.The animal experiment which provide theoretical basis for clinical working is to explore the possible mechanism of WYIH formula on treatment of gastric precancerous lesions by means of studying the expression of MMPs with the molecular biology techniques and immunohistochemistry technique.Methods:1.Animal experiment:84 rats were randomly divided into six groups:model group,normal group,large dose WYIH group,medium dose WYIH group,small dose WYIH group,and WeiMeiSu(WMS) group.Each groups had 12 rats,but model group had 24 rats.Normal group was given normal water,diet and the same volume of 0.9%saline.Other groups freely drank 50μg/mL N-methyl-N’-nitro-nitrosoguanidine (MNNG) solution,without other water during the produced animal model phase;and were drunk with ranitidine hydrochloride at 0.03g/kg one time a day;and were given hunger-satiety disorders(animals were fed with adequate food for 2 days and not given food for 1 day).A total of 20 weeks were spent for produced animal model.After the animal model was successfully produced,WYIH large,medium and small dose group were given WYIH separately in the dose of 2.16g/(kg·d),1.08g/(kg·d),0.54g/(kg·d).WMS group was given WMS decoction in the dose of 0.86g/(kg·d).Normal group and model group were given 0.9%saline in the dose of 0.18g/(kg·d).At the end of the 3 months experiment,the general state of rats’ health and histopathological examination of stomach were observed,as well as the expression and activity of MMP-7,MMP-9 of gastric tissue were observed by the three methods of immunohistochemistry,gelatin zymography and polymerase chain reaction technique.2.Clinical observations:30 cases were selected and randomly divided into two groups:WYIH group and WMS group.Before and after the treatment,the clinical symptoms and drug safety were compared to know the effect of WYIH by the table of TCM symptom schedule.Results:1.Animal experiment:The abnormalities of the rat’ s gastric mucosa included gland atrophy, intestinal metaplasia,and atypical hyperplasia,which shown the animal model was successfully produced.After the treatment of WYIH and WMS,these histological abnormalities were lessened.Compared with model group,on the expression of MMP-7 in gastric tissue, normal group was statistically significant(P=0.000);large dose WYIH group, medium dose WYIH group,small dose WYIH group and WMS group were statistically significant(P=0.000,P=0.000,P=0.005,P=0.032).There were not significant differences between large dose WYIH group,medium dose WYIH group and small dose WYIH group.There were significant differences between medium dose WYIH group and WMS group(P=0.032);but there were no differences between large dose WYIH group,small dose WYIH group and WMS group(P=0.093,P=0.466).Compared with model group,normal group were statistically significant on the expression of MMP-9 in gastric tissue(P=0.001);large dose WYIH group, medium dose WYIH group,small dose WYIH group and WMS group were statistically significant(P=0.002,P=0.000,P=0.002,P=0.012).There were not significant differences between large dose WYIH group,medium dose WYIH group,small dose WYIH group and WMS group.There were significant differences among each group for the activity of MMP-7 in gastric tissue(P=0.000).From the mean rank trend,the activity of MMP-7 of model group was higher than others groups,and the activity of MMP-7 of normal group was lowest in the groups.The activity of MMP-7 of medium dose WYIH group was lower than large dose WYIH group,small dose WYIH group and WMS group.There were significant differences among each group for the activity of MMP-9 in gastric tissue(P=-0.000).From the mean rank trend,the activity of MMP-9 of model group was higher than others groups,and the activity of MMP-9 of normal group was lowest in the groups.The activity of MMP-9 of medium dose WYIH group was lower than large dose WYIH group,small dose WYIH group and WMS group. There are significant differences among each of group for the expression of MMP-7mRNA in gastric tissue(P<0.05).From the mean rank trend,the expression of MMP-7 of model group was higher than normal group,and large dose WYIH group,medium dose WYIH group,small dose WYIH group and WMS group were lower than model group on the expression of MMP-7.There are significant differences among each of group for the expression of MMP-9mRNA in gastric tissue(P<0.05).From the mean rank trend,the expression of MMP-9 mRNA of model group was higher than normal group,and large dose WYIH group,medium dose WYIH group,small dose WYIH group and WMS group were lower than model group on the expression of MMP-9mRNA.2.Clinical observations:The differences among sex,age and course of disease were not significant between WYIH group and WMS group(P>0.05).So these two groups are comparable. There were no differences of clinical symptoms by estimating table of TCM symptom schedule before treatment(P>0.05).The differences of stuffiness and fullness,stomachpain,dry mouth and thirst,belching and acid regurgitation,sloppy stool between the WYIH group and WMS group after treatment were significant(P<0.05).No difference of poor appetite and lack of strength between WYIH and WMS were found.The Differences of the items of TCM symptom schedule table were significant before and after treating with WYIH or WMS(P<0.05).WYIH and WMS can improve TCM symptoms such as stomachpain, belching and acid regurgitation,dry mouth and thirst,sloppy stool.The effect of WYIH was better than WMS.There was no difference in stuffiness and fullness, torpid intake and lack of strength between WYIH and WMS.The clinical curative effect of TCM between WYIH group and WMS group is significant(P=0.045),WYIH can improve symptom of TCM better than WMS.Conclusion:1.WYIH can improve general situation of rats,and treat and reverse the chronic inflammation of gastric mucosa,glands atrophy,and intestinal metaplasia. WYIH can improve the abnormal ultrastructure of gastric precancerous lesions rats.2.WYIH can decrease the expression and activity of MMP-7 and MMP-9 in gastric tissue of gastric precancerous lesions rats,as well as WYIH can decrease the expression of MMP-7mRNA and MMP-9mRNk.These maybe the possible mechanism of WYIH formula on treatment of gastric precancerous lesions 3.Both WYIH and WMS can improve the clinical symptoms of patient.The clinical curative effect of TCM between WYIH group and WMS group is significant,WYIH can improve symptom of TCM better than WMS.更多还原