【作者】 阮氏秋河；

【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】 胃癌是消化道最常见的肿瘤之一,对胃癌早发现、早诊断、早治疗可明显提高其生存率。一般认为,胃粘膜上皮细胞在发展为恶性肿瘤之前,常经历多年持续的癌前病变,及早识别和治疗癌前病变是防治胃癌的有效途径。世界卫生组织胃癌专家会议指出慢性萎缩性胃炎(CAG)伴有不典型增生时是癌前病变。中药治疗慢性萎缩性胃炎癌前病变取得了较好的临床疗效,对于慢性萎缩性胃炎发病机理的研究正在逐渐深入到分子和基因水平。本课题运用分子生物学技术探讨中药胃炎Ⅰ号治疗慢性萎缩性胃炎的分子机制。本文分为两个部分,第一部分文献研究主要总结了中医对慢性萎缩性胃炎病因病机的认识以及现代医学对慢性萎缩性胃炎癌前病变病因、发病机制、治疗方面的研究成果;第二部分实验研究,在成功复制慢性萎缩性胃炎动物模型的基础上,运用生化,放射免疫,明胶酶谱法,荧光定量PCR(SYBR Green)方法,研究慢性萎缩性胃炎自由基,炎症因子,金属基质蛋白酶2(MMP2)和金属基质蛋白酶抑制因子2(TIMP2)的活性,和表达的影响。1.研究目的观察胃炎Ⅰ号对慢性萎缩性胃炎癌前病变大鼠胃组织病理变化、自由基损伤、炎症因子,MMP-2及其抑制因子TIMP-2活性和RNA表达的影响。通过观察慢性萎缩性胃炎癌前病变大鼠血清自由基损伤情况、炎症因子表达,胃组织MMP-2及其抑制因子TIMP-2活性和基因表达情况,及胃炎Ⅰ号的干预机制,以期揭示慢性萎缩性胃炎癌前病变的发病机制,并为胃炎Ⅰ号治疗慢性萎缩性胃炎癌前病变寻找新的靶点。2.研究方法60只SD大鼠,随机分为6组:模型组、空白阻、胃炎Ⅰ号方高、中、低剂量组和维酶素组,每组10只。除空白组只给生理盐水灌胃外,其余各组均采用甲基-硝基-亚硝基胍(MNNG)(1 g/L)和盐酸雷尼替丁胶囊(0.03 g/kg)灌胃建立慢性萎缩性胃炎模型,造模后各组均给予胃炎Ⅰ号(2.16g/kg·d;1.08g/kg·d;0.54g/kg·d)治疗90天。疗程结束后取血,分离出血清,使用生化方法测定过氧化物歧化酶(SOD),丙二醛(MDA)和含量,使用放射免疫法测定血清IL-1β、TNF-α含量。应用明胶酶谱法测定胃组织MMP2和TIMP2的活性,荧光定量PCR(SYBR Green)技术检测大鼠胃组织MMP-2,TIMP-2的mRNA表达水平,同时进行形态学观察。3.研究结果3.1.空白组的胃粘膜厚度正常,上皮细胞及腺体排列整齐。模型组出现明显的胃粘膜炎症、腺体萎缩及化生表现,未见明显的异型增生。胃炎Ⅰ号各治疗组与模型组比较有明显差异,表明胃炎Ⅰ号能有效的改善CAG的病理改变。3.2.与模型组比较,胃炎Ⅰ号方3个治疗组血清SOD含量明显升高(P＜0.05),血清MDA的含量明显下降(P＜0.01),胃炎Ⅰ号各治疗组之间及胃炎Ⅰ号各组与维酶素组之间比较无统计学差异(P＞0.05)。3.3.与空白组比较模型组血清IL-1β含量略升高,但没有统计学差异(P＞0.05)。与模型组比较各治疗组血清IL-1β含量略有降低,但没有统计学差异(P＞0.05)。与空白组比较各组大鼠血清TNF-α含量均有不同程度升高,但只有模型组有显著差异(P＜0.05)。与模型组比较,给予胃炎Ⅰ号方中,低剂量组和维酶素组血清TNF-α含量明显降低,而胃炎Ⅰ号高剂量组TNF-α含量降低不明显。3.4.与空白组比较各组大鼠胃粘膜MMP-2活性均有不同程度升高,以模型组最为明显(P＜0.05)。与模型组比较,给予胃炎Ⅰ号各组和维酶素组MMP-2活性明显降低(P＜0.05),而胃炎Ⅰ号中剂量组MMP-2活性降低最明显。与空白组比较各组胃粘膜TIMP-2活性均有不同程度下降但只有模型组有显著差异(P＜0.05)。与模型组比较,给予胃炎Ⅰ号方各组和维酶素组胃组织TIMP-2活性明显升高(P＜0.05),其中胃炎Ⅰ号中剂量组升高最明显,优于维酶素组。3.5.造模后各组大鼠胃粘膜MMP-2 RNA的表达均有不同程度升高。给予胃炎Ⅰ号和维酶素后,治疗组MMP-2RNA的表达明显降低,其中胃炎Ⅰ号中剂量组效果最佳。造模后各组大鼠胃粘膜TIMP-2 RNA的表达均有不同程度降低于空白组。与模型组比较给予胃炎Ⅰ号方各治疗组和维酶素组胃粘膜TIMP-2RNA的表达有升高趋势,但没有统计学差异(P＞0.05)。4.结论4.1胃炎Ⅰ号能改善实验性慢性萎缩性胃炎大鼠的胃粘膜病理改变。4.2胃炎Ⅰ号能通过升高血清SOD含量、降低血清MDA含量来减少慢性萎缩性胃炎大鼠的自由基损伤的作用。4.3胃炎Ⅰ号能减少炎症因子TNF-α含量。4.4慢性萎缩性胃炎大鼠胃组织中MMP2的活性升高而TIMP-2活性降低损伤因素增加而保护因素降低,所以有增加癌前病变损伤。胃炎Ⅰ号各组和维酶素组可降低MMP-2活性,增加TIMP-2活性从而达到降低慢性萎缩性胃炎的癌前病变的作用。更多还原

【Abstract】 Gastric carcinoma is one of the most common tumors.It early detection, diagnosis,and treatment will greatly decrease mortality rate.There is a long time period of precancerous change before the normal mucosa change into carcinoma.Detecting and treating precancerous lesions is a good way to prevent gastric carcinoma.WHO experts indicated that Chronic atrophic gastritis with dysplasia is a precancerous lesion.Traditional chinese medicine has good curative effects on it.My thesis can be divided into two parts.In the part 1,I summarized the cognition of cause and mechanisms of chronic atrophic gastritis in the view of traditional chinese medicine,and review progress of its etiological factors,pathogenesis,treatment of western and traditional medicine.In the part 2,based on replicating animal model of chronic atrophic gastritis,using biochemical,radioimmunoassay,western blott and PCR technique to study effects of Weiyanyihao formula on freeradical,inflammatory factors, activities and expression of MMPZ and TIMP2.1.PurposeTo study effects of Weiyan yihao formula on histopathological changes, freeradical damage,inflammatory factors,activities and expression of MMP2 and its inhibitor TIMP-2 of gastric tissues in chronic atrophic rats with precancerous lesions.Observating serum free radical damage,the expression of inflammatory cytokines,gastric tissue MMP-2 and its inhibitor TIMP-2 activity and gene expression in rats with chronic atrophic gastritis precancerous lesions and intervention mechanism of Weiyanyihao,with a view to reveal pathogenesis of precancerous lesions in chronic atrophic gastritis,as so as treatment effects of Weiyanyihao on chronic atrophic gastritis precancerous lesions to find a new target.2.Method60 SD rats were radomisely devided into 6 groups:(10 animals in each): model group,control group,Weiyanyihao with high-dose,medium-dose and low-dose group and Weimeisu group.Except the control group received intragastrical administration of normal saline,the rest five groups underwent intragastric MNNG and ranitidine hydrochloride capsules administration to be made into CAG models.Afterwards,corresponding medication was administered to each group for 90 days.After the end of treatment,isolating from the serum, using biochemical method to measure SOD and MDA content,using of radioimmunoassay to measure Serum IL-1β,TNF-αcontent;morphological observation at the same time.Using Western blot to measure activity of MMP2 and TIMP2 in gastric tissue,using PCR technic(SYBR Green) to detecte levels of mRNA MMP-2,TIMP-2 expression in rat gastric tissue.3.Results3.1.The model group has significant stomach membrane inflammation,gland atrophy and metaplasia.There is no significant dysplasia.There are significant differences between Weiyanyihao groups and model group.It indicated that Weiyanyihao formula has improvement effect on the pathological changes in CAG。3.2.Compared to the model group,that of SOD level in the three Wei yanyihao groups significantly higher while the contents of MDA is significantly lower,and the difference was statistically significant.But, there was no statistical difference between Weiyanyihao groups and Weimeisu group.3.3.Compared with the control group,serum IL-1βconcentration slightly increased but there is no statistical difference.Compared with the model group,in the treatment group the serum IL-1βcontent is slight decrease but no statistical difference.Compared with control group,serum TNF-αcontent in treatment groups were increased,but only the model group had significantly different.Compared with model group,serum TNF-αin Weiyanyihao with middle and low-dose groups and Weimeisu group were significantly lower,and in Weiyanyihao with high dose group TNF-αwas decreased not obviously. 3.4.Compared with control group,MMP-2 activity in all rats groups were increased in varying degrees but only in model group the increase was most obvious.Compared with model group,treatment with Weiyanyihao and Weimeisu groups MMP-2 activity was significantly lower,and Weiyan yihao with middle dose had the best effect.Compared with the control group,TIMP-2 activity in all groups were decreased in varying degrees but only the model group were significantly different.Compared with the model group,treatment with Weiyanyihao and Weimeisu,TIMP-2 activity was significantly increased,Weiyanyihao with middle dose had the best effect.3.5.In chronic atrophic model rats,MMP-2 RNA expression increased.After treatment with Weiyanyihao and Weimeisu,the expression of MMP-2RNA significantly decreased,Weiyanyihao with middle dose had the best effect.In chronic atrophic model rats,TIMP-2 RNA expression of gastrictissue was reduced.After treatment with Weiyanyihao and Weimeisu,the expression of TIMP-2RNA trend to be increased,but there was no statistical difference.4.Conclusion4.1.Weiyanyihao can improve pathological changes of of experimental chronic atrophic gastritis in rats.4.2.It is suggested that Weiyanyihao can increase the level of serum SOD,decrease the content of serum MDA,which maybe contribute to treatment effect on chronic atrophic gastritis rats.4.3.Weiyanyihao can reduce the inflammatory cytokines TNF-αcontent.4.4.In gastric tissue of rat chronic atrophic gastritis activity of MMP2 is increased and activity of TIMP-2 is reduced,so injury factors are increased and protective factors are reduced,so it increase precancerous lesions.Weiyanyihao and Weimeisu can reduce MMP-2 activity,while increase TIMP-2 activity,so they can reduce the the possibility of gastric precancerous lesions.更多还原