维胃方对大鼠胃溃疡愈合作用及细胞生长因子EGF含量影响的研究
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摘要
课题依据:
消化系统是人体最大的免疫器官系统,包含人体最多的免疫器官和组织,如肝、脾和胃肠的集合淋巴组织,因此在机体防御和清除外来抗原中起着重要作用。大量临床资料已证实,消化系统自身免疫性疾病的发生率高,所涉及的器官较多,免疫学发病机制和临床特征较复杂。近年来,消化系统免疫的理论和临床研究进展十分迅速,而作为消化系统免疫结构的主要组成部分——黏膜免疫系统,由于结构和功能的特异性,其相关性研究已逐渐发展成为一门独立的学科。胃肠道免疫系统在机体抵抗和清除外来抗原以及维持自身稳态中发挥极其重要的作用。若免疫系统功能紊乱,即可能导致某些免疫相关性疾病的发生。而在胃肠道免疫系统中,主要细胞生长因子——表皮生长因子(EGF),对胃肠道黏膜免疫系统的维护和稳定起着重要的作用。胃肠道疾病研究以免疫细胞因子为突破口,是现实可行而有重要意义的,近年来也有大量的课题围绕着细胞生长因子类指标进行研究,这些研究也为消化系统疾病的免疫诊断和治疗展现了广阔的应用前景。
胃溃疡病(GU)即消化性溃疡病(PU),是一种多病因疾病。各种与发病有关的因素如胃酸、胃蛋白酶、感染、遗传、体质、环境、饮食、生活习惯、神经精神因素等,通过不同途径或机制,使胃液中胃酸和胃蛋白酶的侵袭作用增强和(或)防御机制减弱,从而促成溃疡发生。GU在全球发病率占人口10%左右。该病属于祖国医学的胃脘痛范畴。中医对该病有着悠久的理论研究历史,积累了大量的临床诊治经验。尤其是金元时期形成雏形的脾胃学说,以李东垣的《脾胃论》为标志。本课题以中医理论为指导,以细胞生长因子EGF为切入点,参照Watanebet等的经典造模方法,并加以改进,建立了乙酸诱导大鼠胃溃疡病模型。该模型是一种慢性溃疡模型,持续时间短则8-10天,长则半年,一般60天左右可自然愈合,肉眼观察和组织形态学观察均与人类的消化性溃疡非常相似,其自然复发率约为30%。本课题通过检测大鼠胃液PH值变化、观察胃体标本,并进一步通过对造模处病理切片进行镜下病理观察;通过放射性免疫分析(RIA)方法检测血清EGF含量,从而观察探讨维胃方对大鼠实验性溃疡的治疗机制。
实验方法:
1.SPF级Wistar雄性大鼠,180～220g/只,随机分为9组,每组8只:①正常组;②模型组;③自愈组;④维胃方高剂量组;⑤维胃方中剂量组;⑥维胃方低剂量组;⑦雷尼替丁组;⑧白及、三七中剂量组;⑨白及、三七高剂量组。正常组予每同正常饮食。模型组在造模三天后处死。自愈组观察胃溃疡自愈情况,在造模3天后正常饮食,与用药组同时处死。其余各用药组均在造模三天后予以相应药物每日每只2ml灌胃,灌胃七天后处死。除正常组外,余各组大鼠造模前禁食不禁水二十四小时后,予10%水合氯醛腹腔注射麻醉,于剑突下纵向逐层切开腹壁约2cm长,暴露胃体,并在胃前壁窦体交界处靠胃窦侧浆膜面,用100%乙酸浸泡过的直径5mm的滤纸片压贴60秒,然后将胃纳入体内,胃于原位,逐层缝合,75%酒精消毒伤口。采集标本胁大鼠禁食不禁水二十四小时,具体步骤如下:①称重,10%水合氯醛腹部麻醉后备毛,固定;②剥离胃,结扎幽门,于十二指肠及食管靠贲门两处剪断取胃,由贲门挤胃液用PH试纸上测值并记录;③10%甲醛溶液10ml由贲门注入胃腔,结扎贲门,放入10%甲醛溶液固定30分钟;④沿胃大弯剪开胃,展平,冲洗胃内壁,于造模处寻找溃疡面,以溃疡瘢痕平行于胃长轴方向的最长径为中心选材,剪取全层胃组织置入10%甲醛溶液内固定,进一步石蜡包埋,制作病理切片。
2.实验对象的选取、分组及造模、给药同前。采集标本前大鼠禁食不禁水二十四小时。动物处死后每只腹主动脉采血4ml于真空管内,倾斜放置30分钟后,放入低速离心机,以3000转/分钟,离心30分钟,取上清2ml,置于EP管内,-20℃冷藏,进一步进行放射性免疫测定(RIA)分析。统计学处理:
本实验数据以(?)±s表示,使用SPSS11.5软件进行单因素方差分析方法(One-way ANOVA)进行多样本均数比较。当方差齐性时,使用Least-significantdifference(LSD)方法检验,P＜0.05为差异显著性标准。
主要结果:
1.从大体标本观察来看,各用药组溃疡面愈合情况与自愈组及模型组有明显区别,尤其是雷尼替丁组及维胃方中、高剂量组,无穿孔、较少有溃疡面炎性渗出现象,甚至很难辨别出溃疡面,浆膜粘连不严重,愈合效果均优于其他组别。从镜下病理观察情况来看,维胃方中剂量组较接近正常组,腺体形态基本修复,炎性细胞较少,黏膜下微循环未见明显病理状态,恢复情况较好;而模型组破坏较严重,缺损偶及肌层,造模处腺体完全破坏,非造模处炎性细胞浸润严重、腺体中度不典型性增生,黏膜下微循环可见有血栓闭锁性动脉内膜炎样病理改变。从PH值的数据统计结果来看,F值为4.202,P值为0.000,各组之间的差异具有显著性意义。其中模型组最高(3.288±0.300),与维胃方三组及雷尼替丁组组间的差异均有显著性意义(P＜0.05),与正常组比较(P＜0.01)。
2.模型组大鼠的EGF水平较正常组明显升高(差异具有显著性意义,P＜0.01)。各用药组血清EGF含量均较模型组下降(各用药组与模型组差异均具显著性意义,P＜0.05,白及、三七高剂量组除外),其中雷尼替丁组和维胃方中、高剂量组下降程度更为明显(P＜0.01),以维胃方中剂量组最低(0.253±0.055ng/ml)。自愈组EGF水平较模型组虽然有所下降,但是差异不显著,而除白及、三七高剂量组外,各用药组与自愈组的差异比较均具显著性意义(P＜0.05)。
结论:
1.通过乙酸直接作用于大鼠胃浆膜表面,形成实验性强刺激的造模方法模型典型,成功率较高,操作性、可行性较强。
2.根据大体标本观察情况,用药组别溃疡面愈合的情况与自愈组有明显区别,尤其是雷尼替丁组及维胃方中、高剂量组,愈合效果均优于其他组别。
3.从镜下病理观察情况来看,维胃方中剂量组更接近正常组,腺体形态基本修复,炎性细胞较少,黏膜下循环未见明显病理状态,恢复情况较好;而模型组破坏较严重,缺损偶及肌层,造模处腺体完全破坏,非造模处炎性细胞浸润严重、腺体变形,黏膜下循环病理改变明显。
4.从胃液PH值的数据统计结果中可以看出溃疡程度越严重,胃泌酸功能越差,胃内PH值越高(模型组与正常组比较P＜0.01)。而维胃方三组的PH值均接近正常组(三组与模型组比较均P＜0.05),与雷尼替丁组的PH值亦很接近。说明经维胃方治疗确实能有效的修复损害的胃组织,恢复胃的正常泌酸功能,其效果与临床常用胃溃疡治疗药物雷尼替丁相似。这也符合大体标本的观察结果。
5.对大鼠血清EGF水平的测定结果反映出:经维胃方治疗,对胃溃疡愈合效果是明显的。数据中雷尼替丁组,维胃方中、高剂量组更接近正常组(0.239±0.432ng/ml)水平(三组与模型组比较均P＜0.01,与自愈组比较P＜0.05),其中维胃方中剂量组(0.253±0.05 ng/ml)最接近。维胃方的治疗效果与GU治疗常用药雷尼替丁相似,差别不具有统计学意义(而且维胃方中剂量组更低)。而白及、三七配伍用药的组别虽然对造模后血清EGF含量有影响,但均不及维胃方三组和雷尼替丁组,其中高剂量组与自愈组差异无显著性,这说明维胃方中各药联合配伍要比其拆方白及、三七配伍用药的疗效明显。
6.维胃方的拆方——白及、三七配伍用药的治疗效果无论从大体观察、怖砭迪鹿鄄臁H值测定(拆方两组的均值都高于维胃方三组和雷尼替丁组,其中白及、三七高剂量组与维胃方低剂量组比较差异显著,P＜0.05),还是血清EGF水平测定结果(拆方两组的均值分别为0.3050、0.3288,都高于均值皆小于0.2800的维胃方中、高剂量组和雷尼替丁组)来看,均不及维胃方原方和雷尼替丁组别的效果明显。
综上所述,本研究的实验造模是成功的。维胃方能有效治疗胃溃疡,修复胃的生理结构,从而达到恢复正常的胃泌酸功能和维持正常的血清EGF浓度的效果。这表明维胃方具有间接的免疫调节功能,其中维胃方的临床标准剂量——中剂量组的效果最佳,与胃溃疡病临床常用抑酸剂雷尼替丁疗效相当。而其拆方白及、三七配伍组的疗效不如原方。
Background:
Digestive system is the largest immune organ system in human body,which includes most of the human body's immune organs and tissues,such as liver,spleen and aggregate lymphoid tissues of gastrointestinal mucosa,therefore plays an important role in the body defense and the removal of foreign antigen.Lots of clinical data have confirmed that the autoimmune diseases(AID) of digestive system have high incidence and involves more organs.In additional,the immune diseases have more complex pathogenesis and clinical features.In recent years,the basic and clinical research on the immune diseases of digestive system progress very rapidly. And the research on mucosal immune system(MIS)—the major component of digestive system immune structure,has developed to be an independent discipline based on the specific structure and function of the MIS.Gastrointestinal mucosa is the place for food digestion and absorption.Meantime,the immune system of gastrointestinal mucosa also plays an important role in the body defense,the removal of foreign antigen and the maintenance of body's steady state.Once the mucosal immune system dysfunction occurs,which may lead to certain immune-related diseases.However epidermal growth factor(EGF),the major cell growth factor of gastrointestinal mucosal immune system,plays an important role in the maintenance of mucosa and the stability of gastrointestinal mucosal immune system.Therefore, the study of gastrointestinal diseases based on immune cell factors is practical and significant.In recent years,the studies of many topics have been focused on the cell growth factor indicators.We can see a broad prospect of the immunodiagnosis and immunotherapy on the digestive system diseases through these studies.
Gastric ulcer(GU),i.e.peptic ulcer(PU),is a multi-causal disease.This disease has various incidence causes,such as gastric acid,pepsin,infection,inheritance, physique,environment,diet,life habits,neuropsychiatric factors and etc.These causes for disease ineidence enhance the invasion of gastric acid and the pepsin in gastric juice and/or weaken the defense mechanism through different ways or mechanisms,and cause to ulcer.The people suffering from gastric ulcer account for about 10 percent of the total global population.This disease was included in the areas of epigastralgia in the Chinese Traditional Medicine.The Chinese medicine practitioners have experienced a long history of theoretical research on this disease, accumulated a great deal of clinical treating experience for it and established the embryonic form of spleen-stomach theory in Jin and Yuan Dynasties,which landmark literature is Li Dongyuan's Spleen-Stomach Theory.In this study,we took the Chinese medicine theory as the guide,set the Epidermal Growth Factor(EGF) as the breakthrough point,referred to and improved some typical modeling methods,such as WATANABET,and then established the model for acetic acid induced gastric ulcer in rats.It's a chronic ulcer model,which short duration is eight to ten days,while the long duration is six months.In general,it can heal normally in about 60 days.This chronic ulcer is very similar to peptic ulcer through the naked eye observation and the histomorphological observation,which normal recurrence rate is about 30%.By detecting the changes of PH value of gastric juice in rats,observation of the gastric body samples,further microscopic pathological observation of the mucosal pathological sections at the model position,detecting the blood serum EGF through radioimmunoassay(RIA),we observed and discussed the treatment mechanism of WWF on the experimental ulcer in rats.
Experimental Methods:
1.SPF Wistar Male Rats:180～220g/rat,divided into nine groups randomly, eight rats for each group;①Normal Group②Model Group③Control Experiment Group④WWF High Dose Group⑤WWF Medium Dose Group⑥WWF Low Dose Group⑦Ranitidine Group⑧Baiji Sanqi Medium Dose Group⑨Baiji Sanqi High Dose Group.The rats in the Normal Group were given normal daily diet.The rats in the Model Group were killed after establishing model.The rats in the Control Experiment Group were observed for the healing of gastric ulcer,given normal diet after three days of establishing model,and killed at the same time with those in the drug groups.The rats in the remaining drug groups were given gastric perfusion with the corresponding drugs for 2ml/day/rat after three days of establishing model and killed after gastric perfusion for seven days.Except of the Normal Group,the rats in the remaining groups were fasted for 24 hours but water was given.They were given intraperitoneal injection of 10%chloral hydrate for anesthesia;vertically opened the abdominal walls for about 2cm layer by layer for the exposure of gastric body;pressured for 60 seconds with the filter paper with 5ram diameter and immerged by 100%acetic acid at the side serosal surface of gastric antrum nearby the juncture of anterior gastric wall and gastric antrum;placed the stomach in the body situ,sutured layer by layer and then disinfected the wounds with 75%alcohol.The rats were fasted for 24 hours but water was given before collecting the samples.The concrete steps are as follows:①Weighting,giving abdominal anesthesia with 10%chloral hydrate,carrying through hair preparation and then fixing the rats.②Stripping stomach,ligating pylorus,snipping cardiac and taking out the stomach from the cardiac nearby duodenum and esophagus,crowing gastric juice onto the PH indicator paper for measured value.③Injecting 10ml 10% formaldehyde solution from cardiac into gastric cavity,ligating pylorus,placing into 10%formaldehyde solution for 30 minutes.④Opening stomach along the greater curvature,flattening,washing the inner gastric wall and searching ulcer surface at the model position;Selecting gastric tissue at the longest track of ulcer scar in longitudinal direction,obtaining and placing full-thickness gastric tissue into 10% formaldehyde solution and then further carrying through paraffin embedding and producing pathological sections.
2.The selection,grouping,modeling,administration of experimental objects were the same as the above.Before collecting samples,the rats were fasted for 24 hours but water was given.4ml blood were sampled from each abdominal aorta after the rats were killed,the blood were transferred into vacuum tube and slant placed for 30 minutes.The tube was placed into low speed centrifuge,centrifuged for 30 minutes with the speed rate of 3000 r/min.2ml supernatant were transferred into EP tube,kept at the cold storage of-20℃and further for radioimmunoassay(RIA) analysis.
Statistically:
Lots of experimental data were showed with~((?)±s).SPSS11.5 software was applied into One-way ANOVA for mean comparison based on multiple samples. When the variance was homogeneous,Least-significant difference(LSD) was applied for test and the value of P＜0.05 would mean significant difference.
Major findings:
1.Based on the observation of gross sample,healing of ulcer surface in the rats of drug groups showed a clear distinction with that of the Control Experiment Group and the Model Group.In particular,there was no perforation,less inflammatory exudation on ulcer surface which caused difficulty to identify ulcer surface,and no serious serous adhesion in the rats of the Ranitidine Group,the WWF Medium Dose Group and the WWF High Dose Group.Therefore thc healing effects of these three groups are better than that of the other groups.Based on the pathological observation by microscope,the WWF Medium Dose Group is closer to the Normal Group,which had basically repaired glands morphology,less inflammatory cells and no obvious pathological sub-mucous circulation.Therefore these two groups were in better recovery condition.However,the Model Group had more serious mucosa damage, mucosa defect involving muscular layer,and completely destroyed glands at the model position,serious infiltration of inflammatory cells at the non-model position, and moderate atypical glands hyperplasia.In this group,the arterioles of serous layer showed an inflammatory pathological changes on the thrombus closed arterial intima. Based on the statistical results of PH value,these groups had F value of 4.202 and P value of 0.000 and showed significant difference.Of which,the Model Group had the highest value(3.288±0.300),which showed a significant difference with the three WWF groups and the Ranitidine Group(P＜0.05),and with the Normal Group(P＜0.01).
2.The EGF in the rats of the Model Group were significantly higher than that of the Normal Group(the difference is significant,P＜0.01).The serum EGF concentrations of the drug groups were significantly lower than that of the Model Group(the difference between the drug groups and the Model Group is significant,P＜0.05,except of the BaiJi Sanqi High Dose Group).Of which,the Ranitidine Group, the WWF Medium Dose Group and the WWF High Dose Group dropped more obviously(P＜0.01),especially the WWF Medium Dose Group dropped to the lowest concentrations(0.253±0.055 ng/ml).Compared with the Model Group,the EGF of the Control Experiment Group also dropped,but the difference was not significant. Except of the BaiJi SanQi High Dose Group,the difference between the drug groups and the Control Experiment Group was significant(P＜0.05).
Conclusion:
1.The typical experimental model with strong stimulation established through treating acetic acid on the gastric serosa surface of rats has higher success rate, stronger feasibility and is easier to operate.
2.Based on the observation of gross samples,healing of ulcer surface in the rats of drug groups had a clear distinction with that of the Control Experiment Group.In particular,the healing effects of the Ranitidine Group,the WWF Medium Dose Group and the WWF High Dose Group were better than that of the other groups.
3.Based on the pathological observation by microscope,the WWF Medium Dose Group was closer to the Normal Group,which had basically repaired glands morphology,less inflammatory cells and no obvious pathological sub-mucous circulation;therefore these two groups were in better recovery condition.However, the Model Group had more serious mucosa damage,mucosa defect involving muscular layer,and completely destroyed glands at the model position,serious infiltration of inflammatory cells at the non-model position,glands deformation and obvious pathological sub-mucous circulation.
4.The statistical results of gastric juice PH value showed that more serious ulcer, worse gastric acid secretion and higher gastric PH value(compared the Model Group with the Normal Group,P＜0.01).However the PH values of the three WWF groups were close to that of the Normal Group(compared the three WWF groups with the Model Group,P＜0.05) and also very close to that of the Ranitidine Group.It showed that WWF is effective in repairing the damaged gastric mucosa tissues and recovering the normal acid secretion of gastric mucosa.The efficacy of WWF is similar to that of Ranitidine,the clinical drug commonly used for gastric ulcer by the Western mcdicine. This is also in line with the results of observation on the gross samples.
5.The measured results of serum EGF in rats showed that WWF has obvious effect for the healing of gastric ulcer.The data of the Ranitidine Group,the WWF Medium Group and the WWF High Group were closer to that of the Normal Group (0.239±0.432ng/ml)(compared the three groups with the Model Group,P＜0.01; compared the three groups with the Control Experiment Group,P＜0.05).Of which, the data of the WWF Medium Dose Group(0.253±0.055 ng/ml) was closest to that of the Normal Group.The efficacy of WWF was similar to that of Ranitidine,the routine drug for gastric ulcer.There was no statistical difference between them(the WWF Medium Dose Group has less difference).The BaiJi SanQi Group affected the serum EGF after modeling,but it was less effective than that of the three WWF groups and the Ranitidine Group.Of which,the effect between its high dose group and the Control Experiment Group had no significant difference,which showed that the efficacy of the combined detections of components in WWF is better than the efficacy of combination of BaiJi and SanQi.
6.Combination of BaiJi and SanQi is the decomposed recipes of WWF.Based on the general observation,the pathological observation by microscope,the PH measurement and the serum EGF measurement,the mean PH value of the two decomposed recipes groups were higher than that of the three WWF groups and the Ranitidine Group.Of which,the difference between the BaiJi SanQi High Dose Group and the WWF Low Dose Group is significant(P＜0.05).In additional,the mean serum EGF value of the two decomposed recipes groups are 0.3050 and 0.3288 respectively,which were higher than the mean serum EGF of less than 0.2800 of the WWF Medium Dose Group,the WWF High Dose Group and the Ranitidine Group. Therefore,we can see that the combination of BaiJi and SanQi is less effective than WWF original formula and Ranitidine.
In conclusion,the experimental model of this study is successful.WWF is effective for the treatment of gastric ulcer and the restoration of the normal physiological structure of gastric mucosa,so as to restore normal gastric acid secretion and maintain normal serum EGF concentrations.It showed that WWF has indirect immunity regulation function.The clinical usual dose of WWF is medium and the medium dose group has best efficacy which is equivalent to the efficacy of Ranitidine,the clinical usual acid inhibitor for gastric ulcer.However the decomposed recipes of WWF,i.e.combination of BaiJi and SanQi,are less effective than the original formula of WWF.
消化系统是人体最大的免疫器官系统,包含人体最多的免疫器官和组织,如肝、脾和胃肠的集合淋巴组织,因此在机体防御和清除外来抗原中起着重要作用。大量临床资料已证实,消化系统自身免疫性疾病的发生率高,所涉及的器官较多,免疫学发病机制和临床特征较复杂。近年来,消化系统免疫的理论和临床研究进展十分迅速,而作为消化系统免疫结构的主要组成部分——黏膜免疫系统,由于结构和功能的特异性,其相关性研究已逐渐发展成为一门独立的学科。胃肠道免疫系统在机体抵抗和清除外来抗原以及维持自身稳态中发挥极其重要的作用。若免疫系统功能紊乱,即可能导致某些免疫相关性疾病的发生。而在胃肠道免疫系统中,主要细胞生长因子——表皮生长因子(EGF),对胃肠道黏膜免疫系统的维护和稳定起着重要的作用。胃肠道疾病研究以免疫细胞因子为突破口,是现实可行而有重要意义的,近年来也有大量的课题围绕着细胞生长因子类指标进行研究,这些研究也为消化系统疾病的免疫诊断和治疗展现了广阔的应用前景。
胃溃疡病(GU)即消化性溃疡病(PU),是一种多病因疾病。各种与发病有关的因素如胃酸、胃蛋白酶、感染、遗传、体质、环境、饮食、生活习惯、神经精神因素等,通过不同途径或机制,使胃液中胃酸和胃蛋白酶的侵袭作用增强和(或)防御机制减弱,从而促成溃疡发生。GU在全球发病率占人口10%左右。该病属于祖国医学的胃脘痛范畴。中医对该病有着悠久的理论研究历史,积累了大量的临床诊治经验。尤其是金元时期形成雏形的脾胃学说,以李东垣的《脾胃论》为标志。本课题以中医理论为指导,以细胞生长因子EGF为切入点,参照Watanebet等的经典造模方法,并加以改进,建立了乙酸诱导大鼠胃溃疡病模型。该模型是一种慢性溃疡模型,持续时间短则8-10天,长则半年,一般60天左右可自然愈合,肉眼观察和组织形态学观察均与人类的消化性溃疡非常相似,其自然复发率约为30%。本课题通过检测大鼠胃液PH值变化、观察胃体标本,并进一步通过对造模处病理切片进行镜下病理观察;通过放射性免疫分析(RIA)方法检测血清EGF含量,从而观察探讨维胃方对大鼠实验性溃疡的治疗机制。
实验方法:
1.SPF级Wistar雄性大鼠,180～220g/只,随机分为9组,每组8只:①正常组;②模型组;③自愈组;④维胃方高剂量组;⑤维胃方中剂量组;⑥维胃方低剂量组;⑦雷尼替丁组;⑧白及、三七中剂量组;⑨白及、三七高剂量组。正常组予每同正常饮食。模型组在造模三天后处死。自愈组观察胃溃疡自愈情况,在造模3天后正常饮食,与用药组同时处死。其余各用药组均在造模三天后予以相应药物每日每只2ml灌胃,灌胃七天后处死。除正常组外,余各组大鼠造模前禁食不禁水二十四小时后,予10%水合氯醛腹腔注射麻醉,于剑突下纵向逐层切开腹壁约2cm长,暴露胃体,并在胃前壁窦体交界处靠胃窦侧浆膜面,用100%乙酸浸泡过的直径5mm的滤纸片压贴60秒,然后将胃纳入体内,胃于原位,逐层缝合,75%酒精消毒伤口。采集标本胁大鼠禁食不禁水二十四小时,具体步骤如下:①称重,10%水合氯醛腹部麻醉后备毛,固定;②剥离胃,结扎幽门,于十二指肠及食管靠贲门两处剪断取胃,由贲门挤胃液用PH试纸上测值并记录;③10%甲醛溶液10ml由贲门注入胃腔,结扎贲门,放入10%甲醛溶液固定30分钟;④沿胃大弯剪开胃,展平,冲洗胃内壁,于造模处寻找溃疡面,以溃疡瘢痕平行于胃长轴方向的最长径为中心选材,剪取全层胃组织置入10%甲醛溶液内固定,进一步石蜡包埋,制作病理切片。
2.实验对象的选取、分组及造模、给药同前。采集标本前大鼠禁食不禁水二十四小时。动物处死后每只腹主动脉采血4ml于真空管内,倾斜放置30分钟后,放入低速离心机,以3000转/分钟,离心30分钟,取上清2ml,置于EP管内,-20℃冷藏,进一步进行放射性免疫测定(RIA)分析。统计学处理:
本实验数据以(?)±s表示,使用SPSS11.5软件进行单因素方差分析方法(One-way ANOVA)进行多样本均数比较。当方差齐性时,使用Least-significantdifference(LSD)方法检验,P＜0.05为差异显著性标准。
主要结果:
1.从大体标本观察来看,各用药组溃疡面愈合情况与自愈组及模型组有明显区别,尤其是雷尼替丁组及维胃方中、高剂量组,无穿孔、较少有溃疡面炎性渗出现象,甚至很难辨别出溃疡面,浆膜粘连不严重,愈合效果均优于其他组别。从镜下病理观察情况来看,维胃方中剂量组较接近正常组,腺体形态基本修复,炎性细胞较少,黏膜下微循环未见明显病理状态,恢复情况较好;而模型组破坏较严重,缺损偶及肌层,造模处腺体完全破坏,非造模处炎性细胞浸润严重、腺体中度不典型性增生,黏膜下微循环可见有血栓闭锁性动脉内膜炎样病理改变。从PH值的数据统计结果来看,F值为4.202,P值为0.000,各组之间的差异具有显著性意义。其中模型组最高(3.288±0.300),与维胃方三组及雷尼替丁组组间的差异均有显著性意义(P＜0.05),与正常组比较(P＜0.01)。
2.模型组大鼠的EGF水平较正常组明显升高(差异具有显著性意义,P＜0.01)。各用药组血清EGF含量均较模型组下降(各用药组与模型组差异均具显著性意义,P＜0.05,白及、三七高剂量组除外),其中雷尼替丁组和维胃方中、高剂量组下降程度更为明显(P＜0.01),以维胃方中剂量组最低(0.253±0.055ng/ml)。自愈组EGF水平较模型组虽然有所下降,但是差异不显著,而除白及、三七高剂量组外,各用药组与自愈组的差异比较均具显著性意义(P＜0.05)。
结论:
1.通过乙酸直接作用于大鼠胃浆膜表面,形成实验性强刺激的造模方法模型典型,成功率较高,操作性、可行性较强。
2.根据大体标本观察情况,用药组别溃疡面愈合的情况与自愈组有明显区别,尤其是雷尼替丁组及维胃方中、高剂量组,愈合效果均优于其他组别。
3.从镜下病理观察情况来看,维胃方中剂量组更接近正常组,腺体形态基本修复,炎性细胞较少,黏膜下循环未见明显病理状态,恢复情况较好;而模型组破坏较严重,缺损偶及肌层,造模处腺体完全破坏,非造模处炎性细胞浸润严重、腺体变形,黏膜下循环病理改变明显。
4.从胃液PH值的数据统计结果中可以看出溃疡程度越严重,胃泌酸功能越差,胃内PH值越高(模型组与正常组比较P＜0.01)。而维胃方三组的PH值均接近正常组(三组与模型组比较均P＜0.05),与雷尼替丁组的PH值亦很接近。说明经维胃方治疗确实能有效的修复损害的胃组织,恢复胃的正常泌酸功能,其效果与临床常用胃溃疡治疗药物雷尼替丁相似。这也符合大体标本的观察结果。
5.对大鼠血清EGF水平的测定结果反映出:经维胃方治疗,对胃溃疡愈合效果是明显的。数据中雷尼替丁组,维胃方中、高剂量组更接近正常组(0.239±0.432ng/ml)水平(三组与模型组比较均P＜0.01,与自愈组比较P＜0.05),其中维胃方中剂量组(0.253±0.05 ng/ml)最接近。维胃方的治疗效果与GU治疗常用药雷尼替丁相似,差别不具有统计学意义(而且维胃方中剂量组更低)。而白及、三七配伍用药的组别虽然对造模后血清EGF含量有影响,但均不及维胃方三组和雷尼替丁组,其中高剂量组与自愈组差异无显著性,这说明维胃方中各药联合配伍要比其拆方白及、三七配伍用药的疗效明显。
6.维胃方的拆方——白及、三七配伍用药的治疗效果无论从大体观察、怖砭迪鹿鄄臁H值测定(拆方两组的均值都高于维胃方三组和雷尼替丁组,其中白及、三七高剂量组与维胃方低剂量组比较差异显著,P＜0.05),还是血清EGF水平测定结果(拆方两组的均值分别为0.3050、0.3288,都高于均值皆小于0.2800的维胃方中、高剂量组和雷尼替丁组)来看,均不及维胃方原方和雷尼替丁组别的效果明显。
综上所述,本研究的实验造模是成功的。维胃方能有效治疗胃溃疡,修复胃的生理结构,从而达到恢复正常的胃泌酸功能和维持正常的血清EGF浓度的效果。这表明维胃方具有间接的免疫调节功能,其中维胃方的临床标准剂量——中剂量组的效果最佳,与胃溃疡病临床常用抑酸剂雷尼替丁疗效相当。而其拆方白及、三七配伍组的疗效不如原方。
Background:
Digestive system is the largest immune organ system in human body,which includes most of the human body's immune organs and tissues,such as liver,spleen and aggregate lymphoid tissues of gastrointestinal mucosa,therefore plays an important role in the body defense and the removal of foreign antigen.Lots of clinical data have confirmed that the autoimmune diseases(AID) of digestive system have high incidence and involves more organs.In additional,the immune diseases have more complex pathogenesis and clinical features.In recent years,the basic and clinical research on the immune diseases of digestive system progress very rapidly. And the research on mucosal immune system(MIS)—the major component of digestive system immune structure,has developed to be an independent discipline based on the specific structure and function of the MIS.Gastrointestinal mucosa is the place for food digestion and absorption.Meantime,the immune system of gastrointestinal mucosa also plays an important role in the body defense,the removal of foreign antigen and the maintenance of body's steady state.Once the mucosal immune system dysfunction occurs,which may lead to certain immune-related diseases.However epidermal growth factor(EGF),the major cell growth factor of gastrointestinal mucosal immune system,plays an important role in the maintenance of mucosa and the stability of gastrointestinal mucosal immune system.Therefore, the study of gastrointestinal diseases based on immune cell factors is practical and significant.In recent years,the studies of many topics have been focused on the cell growth factor indicators.We can see a broad prospect of the immunodiagnosis and immunotherapy on the digestive system diseases through these studies.
Gastric ulcer(GU),i.e.peptic ulcer(PU),is a multi-causal disease.This disease has various incidence causes,such as gastric acid,pepsin,infection,inheritance, physique,environment,diet,life habits,neuropsychiatric factors and etc.These causes for disease ineidence enhance the invasion of gastric acid and the pepsin in gastric juice and/or weaken the defense mechanism through different ways or mechanisms,and cause to ulcer.The people suffering from gastric ulcer account for about 10 percent of the total global population.This disease was included in the areas of epigastralgia in the Chinese Traditional Medicine.The Chinese medicine practitioners have experienced a long history of theoretical research on this disease, accumulated a great deal of clinical treating experience for it and established the embryonic form of spleen-stomach theory in Jin and Yuan Dynasties,which landmark literature is Li Dongyuan's Spleen-Stomach Theory.In this study,we took the Chinese medicine theory as the guide,set the Epidermal Growth Factor(EGF) as the breakthrough point,referred to and improved some typical modeling methods,such as WATANABET,and then established the model for acetic acid induced gastric ulcer in rats.It's a chronic ulcer model,which short duration is eight to ten days,while the long duration is six months.In general,it can heal normally in about 60 days.This chronic ulcer is very similar to peptic ulcer through the naked eye observation and the histomorphological observation,which normal recurrence rate is about 30%.By detecting the changes of PH value of gastric juice in rats,observation of the gastric body samples,further microscopic pathological observation of the mucosal pathological sections at the model position,detecting the blood serum EGF through radioimmunoassay(RIA),we observed and discussed the treatment mechanism of WWF on the experimental ulcer in rats.
Experimental Methods:
1.SPF Wistar Male Rats:180～220g/rat,divided into nine groups randomly, eight rats for each group;①Normal Group②Model Group③Control Experiment Group④WWF High Dose Group⑤WWF Medium Dose Group⑥WWF Low Dose Group⑦Ranitidine Group⑧Baiji Sanqi Medium Dose Group⑨Baiji Sanqi High Dose Group.The rats in the Normal Group were given normal daily diet.The rats in the Model Group were killed after establishing model.The rats in the Control Experiment Group were observed for the healing of gastric ulcer,given normal diet after three days of establishing model,and killed at the same time with those in the drug groups.The rats in the remaining drug groups were given gastric perfusion with the corresponding drugs for 2ml/day/rat after three days of establishing model and killed after gastric perfusion for seven days.Except of the Normal Group,the rats in the remaining groups were fasted for 24 hours but water was given.They were given intraperitoneal injection of 10%chloral hydrate for anesthesia;vertically opened the abdominal walls for about 2cm layer by layer for the exposure of gastric body;pressured for 60 seconds with the filter paper with 5ram diameter and immerged by 100%acetic acid at the side serosal surface of gastric antrum nearby the juncture of anterior gastric wall and gastric antrum;placed the stomach in the body situ,sutured layer by layer and then disinfected the wounds with 75%alcohol.The rats were fasted for 24 hours but water was given before collecting the samples.The concrete steps are as follows:①Weighting,giving abdominal anesthesia with 10%chloral hydrate,carrying through hair preparation and then fixing the rats.②Stripping stomach,ligating pylorus,snipping cardiac and taking out the stomach from the cardiac nearby duodenum and esophagus,crowing gastric juice onto the PH indicator paper for measured value.③Injecting 10ml 10% formaldehyde solution from cardiac into gastric cavity,ligating pylorus,placing into 10%formaldehyde solution for 30 minutes.④Opening stomach along the greater curvature,flattening,washing the inner gastric wall and searching ulcer surface at the model position;Selecting gastric tissue at the longest track of ulcer scar in longitudinal direction,obtaining and placing full-thickness gastric tissue into 10% formaldehyde solution and then further carrying through paraffin embedding and producing pathological sections.
2.The selection,grouping,modeling,administration of experimental objects were the same as the above.Before collecting samples,the rats were fasted for 24 hours but water was given.4ml blood were sampled from each abdominal aorta after the rats were killed,the blood were transferred into vacuum tube and slant placed for 30 minutes.The tube was placed into low speed centrifuge,centrifuged for 30 minutes with the speed rate of 3000 r/min.2ml supernatant were transferred into EP tube,kept at the cold storage of-20℃and further for radioimmunoassay(RIA) analysis.
Statistically:
Lots of experimental data were showed with~((?)±s).SPSS11.5 software was applied into One-way ANOVA for mean comparison based on multiple samples. When the variance was homogeneous,Least-significant difference(LSD) was applied for test and the value of P＜0.05 would mean significant difference.
Major findings:
1.Based on the observation of gross sample,healing of ulcer surface in the rats of drug groups showed a clear distinction with that of the Control Experiment Group and the Model Group.In particular,there was no perforation,less inflammatory exudation on ulcer surface which caused difficulty to identify ulcer surface,and no serious serous adhesion in the rats of the Ranitidine Group,the WWF Medium Dose Group and the WWF High Dose Group.Therefore thc healing effects of these three groups are better than that of the other groups.Based on the pathological observation by microscope,the WWF Medium Dose Group is closer to the Normal Group,which had basically repaired glands morphology,less inflammatory cells and no obvious pathological sub-mucous circulation.Therefore these two groups were in better recovery condition.However,the Model Group had more serious mucosa damage, mucosa defect involving muscular layer,and completely destroyed glands at the model position,serious infiltration of inflammatory cells at the non-model position, and moderate atypical glands hyperplasia.In this group,the arterioles of serous layer showed an inflammatory pathological changes on the thrombus closed arterial intima. Based on the statistical results of PH value,these groups had F value of 4.202 and P value of 0.000 and showed significant difference.Of which,the Model Group had the highest value(3.288±0.300),which showed a significant difference with the three WWF groups and the Ranitidine Group(P＜0.05),and with the Normal Group(P＜0.01).
2.The EGF in the rats of the Model Group were significantly higher than that of the Normal Group(the difference is significant,P＜0.01).The serum EGF concentrations of the drug groups were significantly lower than that of the Model Group(the difference between the drug groups and the Model Group is significant,P＜0.05,except of the BaiJi Sanqi High Dose Group).Of which,the Ranitidine Group, the WWF Medium Dose Group and the WWF High Dose Group dropped more obviously(P＜0.01),especially the WWF Medium Dose Group dropped to the lowest concentrations(0.253±0.055 ng/ml).Compared with the Model Group,the EGF of the Control Experiment Group also dropped,but the difference was not significant. Except of the BaiJi SanQi High Dose Group,the difference between the drug groups and the Control Experiment Group was significant(P＜0.05).
Conclusion:
1.The typical experimental model with strong stimulation established through treating acetic acid on the gastric serosa surface of rats has higher success rate, stronger feasibility and is easier to operate.
2.Based on the observation of gross samples,healing of ulcer surface in the rats of drug groups had a clear distinction with that of the Control Experiment Group.In particular,the healing effects of the Ranitidine Group,the WWF Medium Dose Group and the WWF High Dose Group were better than that of the other groups.
3.Based on the pathological observation by microscope,the WWF Medium Dose Group was closer to the Normal Group,which had basically repaired glands morphology,less inflammatory cells and no obvious pathological sub-mucous circulation;therefore these two groups were in better recovery condition.However, the Model Group had more serious mucosa damage,mucosa defect involving muscular layer,and completely destroyed glands at the model position,serious infiltration of inflammatory cells at the non-model position,glands deformation and obvious pathological sub-mucous circulation.
4.The statistical results of gastric juice PH value showed that more serious ulcer, worse gastric acid secretion and higher gastric PH value(compared the Model Group with the Normal Group,P＜0.01).However the PH values of the three WWF groups were close to that of the Normal Group(compared the three WWF groups with the Model Group,P＜0.05) and also very close to that of the Ranitidine Group.It showed that WWF is effective in repairing the damaged gastric mucosa tissues and recovering the normal acid secretion of gastric mucosa.The efficacy of WWF is similar to that of Ranitidine,the clinical drug commonly used for gastric ulcer by the Western mcdicine. This is also in line with the results of observation on the gross samples.
5.The measured results of serum EGF in rats showed that WWF has obvious effect for the healing of gastric ulcer.The data of the Ranitidine Group,the WWF Medium Group and the WWF High Group were closer to that of the Normal Group (0.239±0.432ng/ml)(compared the three groups with the Model Group,P＜0.01; compared the three groups with the Control Experiment Group,P＜0.05).Of which, the data of the WWF Medium Dose Group(0.253±0.055 ng/ml) was closest to that of the Normal Group.The efficacy of WWF was similar to that of Ranitidine,the routine drug for gastric ulcer.There was no statistical difference between them(the WWF Medium Dose Group has less difference).The BaiJi SanQi Group affected the serum EGF after modeling,but it was less effective than that of the three WWF groups and the Ranitidine Group.Of which,the effect between its high dose group and the Control Experiment Group had no significant difference,which showed that the efficacy of the combined detections of components in WWF is better than the efficacy of combination of BaiJi and SanQi.
6.Combination of BaiJi and SanQi is the decomposed recipes of WWF.Based on the general observation,the pathological observation by microscope,the PH measurement and the serum EGF measurement,the mean PH value of the two decomposed recipes groups were higher than that of the three WWF groups and the Ranitidine Group.Of which,the difference between the BaiJi SanQi High Dose Group and the WWF Low Dose Group is significant(P＜0.05).In additional,the mean serum EGF value of the two decomposed recipes groups are 0.3050 and 0.3288 respectively,which were higher than the mean serum EGF of less than 0.2800 of the WWF Medium Dose Group,the WWF High Dose Group and the Ranitidine Group. Therefore,we can see that the combination of BaiJi and SanQi is less effective than WWF original formula and Ranitidine.
In conclusion,the experimental model of this study is successful.WWF is effective for the treatment of gastric ulcer and the restoration of the normal physiological structure of gastric mucosa,so as to restore normal gastric acid secretion and maintain normal serum EGF concentrations.It showed that WWF has indirect immunity regulation function.The clinical usual dose of WWF is medium and the medium dose group has best efficacy which is equivalent to the efficacy of Ranitidine,the clinical usual acid inhibitor for gastric ulcer.However the decomposed recipes of WWF,i.e.combination of BaiJi and SanQi,are less effective than the original formula of WWF.
引文
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[5]Go MF.Review article:natural history and epidemiology of Hilicobater pylori infection.Aliment Pharmacol Ther.2002,16 suppll:3
[6]Andre J.P.M.Smout Louis M.A Akkarmans.MOTILITY OF THE GASTRO INAL TRACT.Wrightson Biomedical Publishing Ltd.1992:6-9
[7]Versalovic J.Helicobacter pylori.Pathology and diagnostic strategies.Am J Chin Pathol.2003,119:403
[8]王继山,陈俭红。实用小儿胃肠病学[M]。北京:北京医科大学中国协和医科大学联合出版社,1997:9
[9]Konturek PC,Konturek SJ,Brzozowski T,et al.Epidermal growth factor and transforming growth factor-α:role in protection and healing of gastric mucosal lesions[J].Eur J Gastroenterol Hepatol,1995,7(10):933-938
[10]Szabo I,Tarnawski AS.Apoptosis in the gastric mucosa:molecular mechanism,basic and clinical implications.J Physiol Pharmacol,2000,51:3-15
[11]崔忠敏,李兆申,湛先保,许国铭。应急状态下胃耐受性细胞保护中TGF α,EGFR的免疫组化研究。第二军医大学学报,2000,21(1):4-6
[12]王永炎主编。中医内科学(第六版)。上海科学技术出版社,1997.6: 163-165
[13]雷载权主编。中药学(第六版) 上海科学技术出版社,1999.6
[14]耿志国。白及对盐酸引起的大鼠胃损伤的保护作用[J]。中草药,1990,21(2):24
[15]Tarnawski A,Stachura J,Krause W J,etal.Quality of ulcer healing:a new emerging concept[J].J Clin Gastroenterol,1991,13(suppl1):42
[16]Arakawa T.Quality of ulcer healing-a new concept to rank healed peptic ulcer[J].Gastroenterol Jpn.1993,28(suppl5):158-162
[17]邓兰琼,王国荣。柴胡桂枝汤对乙酸胃溃疡大鼠再生黏膜的影响[J]。中国中西医结合消化杂志,2005,13(4):243-244
[18]林宇,王勇,孟文芳,等。灯盏花素对乙酸所致胃溃疡的保护作用[J]。齐齐哈尔医学院学报,2001,22(1):1
[19]赵琰,李宇航,王庆国,等。半夏泻心汤不同性味拆方对胃溃疡大鼠血清胃泌素的影响[J]。上海中医药杂志,2004,38(10):45-47
[20]Yanaka A,Suzuki H,Shibahara,etal.EGF promotes gastric mucosal restitution by activating Na~+/H~+ exchange of epithelial cells[J].Am J Physiol Gastrointest Liver Physiol,2002,282(5):866-876
[21]Milani S,Calabro A.Role of growth factors and the irreceptors in gastric ulcer healing[J].Microsc Res Tech,2001,53(5):360-371
[22]Lidong W,Elizabeth J W,Jon O,etal.Gastroenterology 1996;110(2):469-477
[23]Tepperman B L,Soper B D.Dig Dis Sci,1990;35(8):943-949
[24]Wright NA,Pike C M,Ella G.Digestion,1990;46(2):125-133
[25]WrightNA,PikeC,EliaG.Nature,1990;343(6235):82-85
[26]Yi S X,Yang R D,Chang X R,etal.World J Gastroenterol,2006;12:1761-1765
[27]唐志鹏,许鑫梅,叶柳忠,等。健中愈疡片对乙酸诱导胃溃疡大鼠表皮生长因子及其受体表达的影响[J]。中国中西医结合消化杂志,2006,4(2):99-101
[28]Playford R J,Hanby AM,Goodlad RA,etal.Gastroenterology,1995;108(4):A747
[29]Tunio AM,Hobsley M.Gut,1995;37(3):335-339
[30]巨卫平,朱振江。胃溃疡患者治疗前后血清TNF、EGF水平的观察。放射免疫学杂志。2002,15(1):35
[31]曹剑凡,马云宝,张晓懿。消化性溃疡患者血清多肽生长因子及相关激素水平测定[J]。放射免疫学杂志。2005,(02)
[2]徐叔云,卞如濂,陈修。药理学实验方法[M]。第3版。北京:人民卫生出版社,2002:1331-1332
[3]李仪奎。中药药理实验方法学[M]。第2版。上海:上海科学技术出版社,2006:493-497
[4]陈灏珠主编。实用内科学(第12版)。人民卫生出版社,2005.5.12:1867,1868
[5]Go MF.Review article:natural history and epidemiology of Hilicobater pylori infection.Aliment Pharmacol Ther.2002,16 suppll:3
[6]Andre J.P.M.Smout Louis M.A Akkarmans.MOTILITY OF THE GASTRO INAL TRACT.Wrightson Biomedical Publishing Ltd.1992:6-9
[7]Versalovic J.Helicobacter pylori.Pathology and diagnostic strategies.Am J Chin Pathol.2003,119:403
[8]王继山,陈俭红。实用小儿胃肠病学[M]。北京:北京医科大学中国协和医科大学联合出版社,1997:9
[9]Konturek PC,Konturek SJ,Brzozowski T,et al.Epidermal growth factor and transforming growth factor-α:role in protection and healing of gastric mucosal lesions[J].Eur J Gastroenterol Hepatol,1995,7(10):933-938
[10]Szabo I,Tarnawski AS.Apoptosis in the gastric mucosa:molecular mechanism,basic and clinical implications.J Physiol Pharmacol,2000,51:3-15
[11]崔忠敏,李兆申,湛先保,许国铭。应急状态下胃耐受性细胞保护中TGF α,EGFR的免疫组化研究。第二军医大学学报,2000,21(1):4-6
[12]王永炎主编。中医内科学(第六版)。上海科学技术出版社,1997.6: 163-165
[13]雷载权主编。中药学(第六版) 上海科学技术出版社,1999.6
[14]耿志国。白及对盐酸引起的大鼠胃损伤的保护作用[J]。中草药,1990,21(2):24
[15]Tarnawski A,Stachura J,Krause W J,etal.Quality of ulcer healing:a new emerging concept[J].J Clin Gastroenterol,1991,13(suppl1):42
[16]Arakawa T.Quality of ulcer healing-a new concept to rank healed peptic ulcer[J].Gastroenterol Jpn.1993,28(suppl5):158-162
[17]邓兰琼,王国荣。柴胡桂枝汤对乙酸胃溃疡大鼠再生黏膜的影响[J]。中国中西医结合消化杂志,2005,13(4):243-244
[18]林宇,王勇,孟文芳,等。灯盏花素对乙酸所致胃溃疡的保护作用[J]。齐齐哈尔医学院学报,2001,22(1):1
[19]赵琰,李宇航,王庆国,等。半夏泻心汤不同性味拆方对胃溃疡大鼠血清胃泌素的影响[J]。上海中医药杂志,2004,38(10):45-47
[20]Yanaka A,Suzuki H,Shibahara,etal.EGF promotes gastric mucosal restitution by activating Na~+/H~+ exchange of epithelial cells[J].Am J Physiol Gastrointest Liver Physiol,2002,282(5):866-876
[21]Milani S,Calabro A.Role of growth factors and the irreceptors in gastric ulcer healing[J].Microsc Res Tech,2001,53(5):360-371
[22]Lidong W,Elizabeth J W,Jon O,etal.Gastroenterology 1996;110(2):469-477
[23]Tepperman B L,Soper B D.Dig Dis Sci,1990;35(8):943-949
[24]Wright NA,Pike C M,Ella G.Digestion,1990;46(2):125-133
[25]WrightNA,PikeC,EliaG.Nature,1990;343(6235):82-85
[26]Yi S X,Yang R D,Chang X R,etal.World J Gastroenterol,2006;12:1761-1765
[27]唐志鹏,许鑫梅,叶柳忠,等。健中愈疡片对乙酸诱导胃溃疡大鼠表皮生长因子及其受体表达的影响[J]。中国中西医结合消化杂志,2006,4(2):99-101
[28]Playford R J,Hanby AM,Goodlad RA,etal.Gastroenterology,1995;108(4):A747
[29]Tunio AM,Hobsley M.Gut,1995;37(3):335-339
[30]巨卫平,朱振江。胃溃疡患者治疗前后血清TNF、EGF水平的观察。放射免疫学杂志。2002,15(1):35
[31]曹剑凡,马云宝,张晓懿。消化性溃疡患者血清多肽生长因子及相关激素水平测定[J]。放射免疫学杂志。2005,(02)