消痈生肌法治疗消化性溃疡的实验与临床研究

【作者】 周天羽；

【导师】 周学文；

【作者基本信息】 辽宁中医药大学 ， 中医内科学， 2009， 博士

【摘要】 目的:本课题旨在探求中药消痈生肌法治疗消化性溃疡的疗效,通过对胃溃疡大鼠治疗后在病理组织学、分子生物学、血清学等方面研究对比,以及本方在消化性溃疡患者中的疗效观察,探索本方治疗消化性溃疡的作用机制及对临床应用的价值。材料与方法:1材料1.1研究对象动物实验:SPF级SD雄性大鼠70只,体重(200±10)g,随机将大鼠分为7组,即正常对照组、假手术组、模型组、消痈溃得康组、中药汤剂组、溃疡胶囊组、奥美拉唑组,每组10只。临床实验:待检胃粘膜标本来自辽宁中医药大学附属医院胃镜室,胃镜活检组织保存于液氮中待用,其中胃溃疡10例,慢性糜烂性胃炎10例,慢性浅表性胃炎9例。临床观察:病例均为2007年2月至2008年10月符合入选标准的本院门诊患者,共60例,按随机数字表法分为2组。治疗组30例,对照组30例。1.2药物与试剂消痈生肌方,消痈溃得康,溃疡胶囊,奥美拉唑镁肠溶片,三叶因子2上下游引物,西咪替丁片。100%冰乙酸,10%水合氯醛溶液,10%甲醛溶液,IL-1β,IL-2 ELIASA酶免疫试剂盒;抑肽酶;NO,NOS试剂盒;焦碳酸二乙酯(DEPC);琼脂糖;嗅化乙锭(EB);TRIZOL液;异丙醇;氯仿;DNA-marker DL2000;RT-PCR试剂盒;100%乙醇。1.3主要实验仪器电热恒温水温箱,恒温震荡器,TDL-5A台式离心机,721B分光光度计,LeicaRM2135切片机;Olympus光学显微镜;1601紫外分光光度计;INFINITE M200酶标仪;SN-682型放射免疫r计数器;游标卡尺;Milli-Q超纯水机;DY89-Ⅰ电动玻璃匀浆机;JY92-Ⅱ超声波细胞粉碎机;DU-600蛋白核酸分析仪;BIOFUGE28RS低温高速离心机;Indeslt低温冰箱;PE9600 PCR仪;EPS-300电泳仪;DYCP-33A电泳槽。2方法2.1动物实验部分将传统冰乙酸烧灼法加以改良,制备大鼠胃溃疡模型。普通人按体重60kg计算,人与200g体重大鼠折算比为6.25:1,计算出每种药物的对应浓度:消痈生肌方1.28g/kg,消痈溃得康0.2 g/kg,溃疡胶囊0.018 g/kg,奥美拉唑肠溶片0.2 mg/kg。各组实验大鼠从造模后第2天开始灌胃,早晚各一次,共12天。正常组,正常饲养,不进行灌胃;假手术组、模型组,每日灌胃蒸馏水2ml,早晚各一次;汤药组,每日灌胃1.28g/kg浓度的消痈生肌方溶液2ml,早晚各一次;溃得康组,每日灌胃0.2g/kg浓度的消痈溃得康溶液2ml,早晚各一次;胶囊组,每日灌胃0.018g/kg浓度的溃疡胶囊溶液2ml,早晚各一次;奥美组,每日早晨灌胃1.28g/kg浓度的奥美拉唑肠溶片溶液2ml,晚上灌胃蒸馏水2ml。灌胃12天,禁食一晚后取材。各组大鼠用10%水合氯醛麻醉,开腹,找到胃,在幽门、贲门两端剪断,将胃取出,取胃液;沿胃大弯剪开,用生理盐水清洗,在洁净工作台将胃壁展平,观察溃疡情况,然后沿溃疡边缘3mm切下胃壁,制作HE染色的病理切片。拨开肠管、网膜找到腹主动脉,用头皮针刺入,取血约5ml,每ml血中加入抑肽酶10μl,混匀,凉置2小时;再以3000转/分钟离心10分钟,取上部血清,装入EP管中,按组别1-70编号标记,冷冻备用。观测指标:胃黏膜大体形态观察、溃疡面积的测定、胃液PH值测定、胃黏膜病理组织检查,ELISA法检测大鼠血清IL-1β,IL-2,硝酸还原法测定NO含量,比色法测定血清中NOS水平。2.2临床部分将各组患者胃粘膜标本加入1ml TRIZOL试剂,用组织剪剪碎,用细胞粉碎机破碎,做总RNA的提取,反转录反应,PCR反应,琼脂糖电泳,得出TFF2的反应条带。治疗组予消痈生肌方治疗。每天1剂,水煎,取汁300mL,分3次口服。对照组予胃热清胶囊治疗。每次4粒,每日3次。两组均治疗2月为1疗程,同时加服西咪替丁200mg,每日三次口服。治疗期间停用其他药物,并避免进食粗糙、辛辣等刺激性食物,戒烟酒。主要观察临床症状及纤维胃镜改变、复发率及不良反应。结果:1.模型组溃疡面积明显大于正常组和假手术组(P＜0.01),但各组与正常组间仍有差异(P＜0.01);胶囊组、奥美组与模型组比较有显著性差异(P＜0.01),汤药组和溃得康组的溃疡面积明显小于奥美组和胶囊组,汤药组与其它各对照组存在着差异(P＜0.05)。汤药组与奥美组的胃酸值比较无显著性差异(P＞0.05)2.汤药组中IL-1β含量高于正常组、假手术组,与之比较有非常显著性差异(P＜0.01);汤药组中IL-1β含量低于溃得康组、胶囊组、奥美组,与之比较有非常显著差异(P＜0.01);模型组含量最高,各组与之比较均有非常显著性差异(P＜0.01);溃得康组与奥美组比较,无显著差异(P＞0.05)。汤药组中IL-2含量低于正常组、假手术组,与之比较有非常显著性差异(P＜0.01);汤药组中IL-2含量高于溃得康组、胶囊组、奥美组,与之比较有非常显著差异(P＜0.01);模型组含量最低,各组与之比较均有非常显著性差异(P＜0.01);溃得康组与奥美组比较,无统计学意义(P＞0.05)。3.本实验结果显示,汤药组NO含量明显高于模型组,也高于胶囊组,低于正常组和假手术组;溃得康组与汤药组比较,略高于后者;奥美组NO含量高于各治疗组,甚至高于正常组和假手术组;胶囊组的NO含量仅高于模型组,低于其它各组。汤药组的NOS活力均高于各对照组,溃得康组次之,且明显高于模型组;正常组和假手术组NOS活性最高。4.每个病例胃黏膜TFF2均有表达,浅表性胃炎组表达较强,糜烂性胃炎组次之,而胃溃疡组表达最弱。实验结果显示,溃疡组TFF2表达低于另两组,与糜烂组比较有非常显著性差异(P＜0.01),与浅表组比较也有非常显著性差异(P＜0.01);糜烂组与浅表组比较有显著性差异(P＜0.05),均有统计学意义。5.本观察表明,消痈生肌方治疗胃毒热证消化性溃疡疗效较好,优于对照组的疗效,且复发率低,半年仅复发一例,在用药过程中未发现有心、肝、肾等重要器官及造血系统的毒性损害。结论:1.消痈生肌方能使大鼠实验性溃疡面积缩小,促使胃黏膜上皮细胞增殖,使肉芽组织的迅速生成,促进溃疡愈合,能够通过降低胃液酸度,减轻攻击因素对胃黏膜的损伤,由此我们认为消痈生肌方也具有较强的抑酸能力。2.消痈生肌方能够通过降低和提高大鼠血清中IL-1β,IL-2水平,缓解炎症介质释放、降低炎症反应、增强胃粘膜的防御功能,以此来促进溃疡的愈合。3.消痈生肌方能有效增强NOS活性,调节局部血流,适度提高NO的生成,使之接近正常水平,调节胃酸分泌,维护胃黏膜的完整性和防御功能,调节胃肠黏膜血流量,从而达到促进溃疡愈合的目的。4.三叶因子2在胃溃疡黏膜组织中表达最低,其表达程度与胃粘膜损害程度称负相关,由此我们也可以推断其表达量应远低于正常人。我们认为三叶因子2具有保护胃黏膜屏障,防止攻击因子侵袭的作用。5.我们认为消痈生肌法是一种在临床上疗效确切治疗消化性溃疡的方法,并且有望在抗溃疡复发方面取得一定突破。更多还原

【Abstract】 Purpose:This topic is for the purpose of seeking the traditional Chinese medicine to disappearing carbuncle and growing muscle method to treat peptic ulcer’s curative effect.Through we study and contract in aspects of histopathology, molecular biology,serology and so on,after gastric ulcer rat treatment,as well as in the peptic ulcer patient’s curative effect observation,explore to treat the peptic ulcer’s functional mechanism and to the clinical application value.Material and method:1 Material1.1 Object of studyAnimal experimentation:SPF SD male rat 70,weight(200±10) g,divided randomly into 7 groups,named the normal control group,the sham-operation group, the model group,kui de kang group,the decoction group,ulcer capsule group, Omeprazole group,each group of 10.Clinical experiment:The gastric mucosa specimen got from the affiliated hospital gastro-endoscope department,the specimen preserved in the liquid nitrogen,include the gastric ulcer 10 examples,the chronic erosive gastritis 10 examples,chronic superficial gastritis 9 examples.Clinical observation:60 outpatients serviced from February 2007 to October 2008 which are conformed to the selected standard,divide into 2 groups randomly. Treatment group are 30 examples,control group are 30.1.2 Medicines and reagentDisappearing carbuncle and growing muscle decoction;Kui de kang granule, Ulcer capsule;Omeprazole tablete;TFF2 upstream and downstream primers; Cimetidine tablete.100%glacial acetic acid,10%solution of chloral hydrate,10%formal dehyde solution,IL-1β,IL-2 ELIASA enzyme immunoassay kit;aprotinin;NO,NOS kit; DEPC;agarose;sniff of bromide(EB);TRIZOL solution;isopropanol;chloroform; DNA-marker DL2000;RT-PCR kit;100%ethanol.1.3 Main experiment instrumentElectric thermostat temperature box,constant temperature oscillator, TDL-5A desktop centrifuge,721B spectrophotometer,Leica RM2135 slicer;Olympus optical microscope;UV-1601 spectrophotometer;INFINITE M200 microplate reader; SN-682 type r Radioimmunoassay Counter;vernier caliper;Milli-Qultrapure water machine;DY89-Ⅰelectric machine glass homogenate;JY92-Ⅱultrasonic cell pulverizer;DU-600 protein,nucleic acid analyzer;BIOFUGE28RS low-temperature high-speed centrifuge;Indeslt low-temperature refrigerator;PE9600 PCR instrument;EPS-300 Bio-Rad;DYCP-33A electrophoresis tank.2 Method2.1 Animal experimentation partImproves the traditional glacial acetic acid burning method,prepares the rat gastric ulcer model.60kg calculated according to the weight of ordinary people,people with 200g of rat body weight conversion ratio of 6.25:1,calculate the corresponding concentration of each drug:Disappearing carbuncle and growing muscle decoction 1.28g/kg,Kui de kang granule 0.2 g/kg,ulcer capsule 0.018 g/kg,Omeprazole tablete 0.2 mg/kg.Each group of experiment rat after making the mold the 2nd day starts to garage,two times every day,altogether 12 days.The normal group,normal raising, does not carry on garage;The sham-operation group,the model group,gavage distilled water 2ml two times every day;The decoction group,garage 1.28g/kg decoction solution 2ml two times every day;Kui de Kang group,garage 0.2g/kg density to Kui de Kang solution 2ml two times every day;The capsule group,garage 0.018g/kg every day the density ulcer capsule solution 2ml two times every day; Omeprazole group,garage 1.28g/kg density Omeprazole solution 2ml in morning, garage distilled water 2ml in everning.Garage for 12 days,after a night of fasting subjects.Rats in each group with 10%chloral hydrate anesthesia,laparotomy,the stomach found in the pylorus, cardia cut at both ends to remove the stomach,gastric juice check;cut along the greater curvature of stomach with a saline wash to clean bench in parietal flattening observed ulcer,and then along the edge of 3mm cut ulcer gastric wall, the production of HE staining of the biopsy.Far from clearing the bowel,omentum find abdominal aorta with a needle into the scalp,blood about 5ml,per ml blood of aprotinin added to 10μl,mixing,cool home two hours;again to 3000 r/m centrifugation for 10 minutes,from the upper levels,into the EP tube,according to group numbers 1-70 mark,frozen reserve.Observation:the general morphology of gastric mucosa,the determination of ulcer area,gastric PH value of the determination,histopathological examination of gastric mucosa,ELISA assay of serum IL-1β,IL-2,nitrate reduction measured NO content was detected NOS levels in serum.2.2 clinical partsGastric mucosa of patients with each of the groups by adding 1ml TRIZOL reagent samples,with shear organization,with cells broken grinder,so the total RNA extraction,reverse transcription reaction,PCR reaction,agarose gel electrophoresis,then receive the reaction of TFF2 drawn strip.The treatment group gives Disappearing carbuncle and growing muscle decoction to treat.Every day one decoction,the water fries,takes juice 300mL, at 3 times the oral.The control group gives the stomach hot clear capsule treatment.Each time 4 grains,daily 3 times.Two groups be treated in for two monthes as one treatment courses,simultaneously take Cimetidine tablete 200mg, three times a day orally.Disable other drugs during treatment,and avoid eating rough,irritating,such as spicy foods,alcohol quit smoking.The main observation include clinical symptoms,changes in fiber endoscopy,relapse rate and adverse reactions.Results:1.Ulcer area of the model group than normal group and sham operation group (P＜0.01),but the group and there are still differences between the normal group (P＜0.01);Capsule Group,Omeprazole Group compared with the model group were significantly different(P＜0.01),the ulcer area of kui de kang group and the decoction group was significantly less than the ulcer area of Omeprazole Group and the capsule groups,the decoction group there are differences in the control group(P＜0.05).Between the acid value of the decoction group and Omeprazole Group are no significant difference(P＞0.05).2.the IL-1βin decoction group was higher than the normal group, sham-operated group,compared with a very significant difference(P＜0.01); decoction group IL-1βcontent was lower than Kui de kang Group,Capsule Group, Omeprazole group,compared with a very significant difference(P＜0.01);the highest content of model group,compared with the other groups there was a significant difference(P＜0.01);Between decoction group and Omeprazole Group are no significant difference(P＞0.05).decoction group IL-2 content lower than the normal group,sham-operated group,compared with a very significant difference(P＜0.01);the IL-2 in decoction group was higher than Kui de kang Group,Capsule Group,Omeprazole Group,compared with a very significant difference(P＜0.01);the minimum content of the model group,compared with the group there was a significant difference(P＜0.01);Between decoction group and Omeprazole Group are no significant difference(P＞0.05).3.The results showed,NO content in decoction group was significantly higher than the of the model group,but also higher than the capsule group,lower than the normal group and sham-operation group;kui de kang group compare with decoction,slightly higher than the latter;NO content of Omeprazole Group higher than the treatment group,and even higher than the normal group and sham-operated group;capsule group is higher than the NO content in the model group only,which is lower than other groups.Decoction of the NOS group were higher than the vitality of the control group,and was significantly higher than the model group;normal group and sham-operation group dynamic highest NOS.4.TFF2 each case are the expression of gastric mucosa,superficial gastritis group to express strong,followed by erosive gastritis group,gastric ulcer group and the weakest expression.Experimental results show that the expression of TFF2 ulcer group than the other two groups,and rotten to the core group have a very significant difference(P＜0.01),with the superficial group also has a very significant difference(P＜0.01);erosive group and superficial group were significantly different(P＜0.05),have statistical significance.5.This observation shows that disappearing carbuncle and growing muscle method treat toxic-heat type peptic have a better efficacy than the control group, low recurrence for six months only one example,in the course of medication did not find heart,liver,kidney and other vital organs and the toxicity of hematopoietic system damage.Conclusion:1.The experimental result may show that disappearing carbuncle and growing muscle decoction can reduce the size of ulcer in rat,promote gastric epithelial cell proliferation,so that the rapid generation of granulation tissue,and promote ulcer healing,and we also believe that disappearing carbuncle and growing muscle decoction also has a ability of inhibit acid.2.Disappearing carbuncle and growing muscle decoction was able to reduce and improve the serum IL-1β,IL-2 level,to ease the release of inflammatory mediators,reduce inflammation,and enhance the defensive function of gastric mucosa in order to promote the healing of ulcers.3.Disappearing carbuncle and growing muscle decoction will effectively enhance the NOS vitality,regulate local blood flow,moderate to increase the generation of NO to near normal levels,regulating gastric acid secretion and maintaining gastric mucosal integrity and defense functions,regulation of gastrointestinal mucosal blood flow,so as to achieve the promotion of ulcer healing.4.TFF2 in gastric ulcer mucosal tissue expressed the lowest level,its expression in gastric mucosal is relate to damage that negative correlation. We can infer its expression should be far lower than normal people.We believe that the TFF2 can protect gastric mucosal barrier to prevent attacks on the role of the invasion factor. 5.We thought that Disappearing carbuncle and growing muscle method is one kind method of curative effect accurate to treat peptic ulcer,and makes certain breakthrough hopefully in the anti-ulcer recurrence aspect.更多还原