【作者】 赵光；

【导师】 崔乃强； 张淑坤；

【作者基本信息】 天津医科大学 ， 中西医结合临床， 2009， 硕士

【摘要】 目的:建立大鼠重症急性胰腺炎模型,并探讨中药清胰颗粒对重症急性胰腺炎(severe acute pancreatitis,SAP)大鼠肠黏膜屏障功能的影响及其作用机制方法:选取健康雄性Wistar大鼠分为正常组、假手术组和模型A、B、C组,采用牛磺胆酸钠胰胆管逆行注射方法建立SAP动物模型,假手术组和模型A、B、C组大鼠胆胰管插管,分别逆行注射生理盐水和2%、3.5%、5%的牛磺胆酸钠,观察各组死亡率和胰腺损伤程度;在SAP大鼠模型稳定后选取150只健康雄性Wistar大鼠,随机分为3组,分别为假手术组、模型组和治疗组。模型组和治疗组均采用胆胰管逆行注射3.5%牛磺胆酸钠建立SAP模型,治疗组于造模前2h、造模后6h、18h、30h和42h分别给予清胰颗粒灌胃,模型组和假手术组以同样的方法分别给与中药安慰剂和生理盐水灌胃。各组动物分别于造模前、造模后6h、12h、24h、48h取腹主动脉血行淀粉酶和二胺氧化酶的检测,取胰腺组织和末端回肠行HE染色和病理学评分,同时测量肠绒毛高度、黏膜厚度,并应用免疫组化方法检测小肠黏膜紧密连接蛋白Claudin-1的表达。结果:1.假手术组无死亡记录,模型A、B、C组在术后72h死亡率分别为20%、50%、70%;模型B组淀粉酶造模后6h最高达9023.0±1924.5 U/L,以后随时间逐渐下降,在72h降为2245.3±271.6仍高于假手术组和正常组且有统计学意义(p<0.05);镜下所见模型B组胰腺间质水肿;红细胞漏出;炎细胞浸润。腺泡细胞水肿;点片状出血;灶性变性、坏死;部分导管扩张。病理学评分模型B组与假手术组各时点比较9.89±2.11 vs 1.27±1.13、10.25±1.98 vs 1.32±1.28、11.71±2.11 vs 1.03±0.11、13.85±1.05 vs 1.01±0.19、13.89±0.89 vs 0.98±0.07均有显著性差异。2.模型组大鼠精神萎靡、竖毛、少动倦卧;治疗组大鼠精神状态较好、活动较多、排便次数增加、肛周污染明显。假手术组无死亡记录,治疗组SAP大鼠死亡率低。模型和治疗组6h、12h、24h、48h的死亡率分别为0%(0/10)、20%(2/10)、40%(4/10)、50%(5/10)和0%(0/10)、20%(2/10)、30%(3/10)、30%(3/10);治疗组24h死亡率低于模型(p<0.05)3.假手术组淀粉酶在造模后6h一过性升高,随后恢复正常;模型组淀粉酶造模后6h最高,以后随时间逐渐下降,但在48h仍高于假手术组(p<0.05);与模型比较,治疗组造模后12h降低开始有统计学意义(p<0.05),48h与假手术组比较无差异4.假手术组血清DAO水平在造模前后无变化(p>0.05);治疗组血清DAO水平在造模后24 h和48 h较模型组降低有显著性差异(p<0.05)5.假手术组术后6h可见胰腺腺泡细胞轻度水肿、无出血及坏死。模型组可见胰腺间质水肿,红细胞漏出,炎细胞浸润,腺泡细胞水肿,点片状出血,灶性变性、坏死,部分胰腺导管扩张,随时间推移胰腺组织病理学评分逐渐加重。治疗组24h以上改变均减轻病理学评分降低有统计学意义。6.光镜下假手术组肠黏膜绒毛结构完整,上皮呈高柱状,间质无水肿、无中性粒细胞浸润。模型组肠黏膜结构损伤明显,并呈进行性变化,尤以制模24h最明显,可见大鼠回肠间质水肿并有点状出血,血管淋巴管均扩张,还可见大量中性粒细胞浸润伴淋巴滤泡增生,肠绒毛水肿增粗,高度缩短,顶端上皮有片状坏死、脱落,杯状细胞减少,排列紊乱;黏膜厚度变薄,黏膜腺体排列稀疏,腺体减少;中药清胰颗粒治疗组在造模12h时与模型组相比病理损伤有所减轻,表现为炎症细胞浸润减少,绒毛损伤脱落减少,黏膜厚度及绒毛高度均有改善,造模后24 h开始有显著的差异。术后48 h各指标均接近正常值7.各组小肠黏膜claudin-1均分布于肠上皮紧密连接的细胞膜(包括细胞膜顶部),膜内近侧的胞质区少量染色,为棕黄色染色颗粒,胞核及核膜无表达。假手术Claudin-1蛋白在细胞膜表达且为强阳性;与假手术组比较,模型组claudin-1表达明显下降p<0.05);与模型组比较,治疗组造模后各时间点Claudin-1表达升高且有统计学意义(p<0.05)结论:1.应用静脉留置针导管经十二指肠乳头逆行注射3.5%牛磺胆酸钠(0.1ml/100g体重、注射速度为0.15ml/min)诱导的SAP大鼠动物模型是Aho法制备SAP大鼠模型的改良,是进行SAP中药临床研究科研较为理想的动物模型2.早期应用清胰颗粒能够改善SAP大鼠胰腺组织的损伤,减轻肠黏膜和肠绒毛萎缩,提高肠道紧密连接蛋白Claudin-1水平的表达水平,降低肠黏膜通透性保护肠屏障,降低死亡率。更多还原

【Abstract】 Objectives:To create the animal model with severe acute pancreatitis which can adapt to scientific research,and to investigate the effects of Qingyi granules on the intestinal mucosa barrier in rats with severe acute pancreatitis(SAP). Methods:Healthy male Wistar rats were divided into normal group,sham operation group and model A,B,C group,Severe acute pancreatitis model was induced by retrograde injection of sodium deoxycholate into the biliopancreatic duct in rats.,the sham operation group and model A,B,C Group rat,respectively,by retrograde injection of saline and 2%,3.5%,5%sodium deoxycholate,observed mortality and the extent of pancreatic injury;then 150 healthy male Wistar rats were randomly divided into 3 groups:sham-operation group,model group and treatment group.The model group and the treatment group were retrograde biliopancreatic injection of 3.5%sodium deoxycholate to create SAP model,and treatment group were given orally Qingyi granules at 2 hours before induction of SAP,at 6h,18h,30h and 42h after SAP,the model group were given placebo in the same way.The levels of serum amylase and diamine oxidase were determined,the histopathology of pancreatic and terminal ileum tissue were examined by hematoxylin and eosin(HE) staining at 0h,6 h、12 h、24 h and 48h after SAP and histologic assessment was performed.At the same time,we measured intestinal villus height,mucosal thickness and detection of small intestinal mucosal tight junction protein Claudin-1 expression by immunohistochemistry.Results:1.Sham-operated group was no death records,the mortality of models A,B,C groups after 72h were 20%,50%,70%;the level of amylase in model B group after 6 h up to 9023.0±1924.5 U/L and was a gradual decline over time,after 72 h reduced to 2245.3±271.6 U / L,but still higher than the sham-operated group and the normal group and were statistically significant(p <0.05);In model B group,the pancreas showed that interstitial edema,red blood cell leakage,inflammatory cell infiltration, acinar cells edema,point sheet hemorrhage,focal degeneration and necrosis. Pathology score of model B group and sham-operated group in every time point 9.89±2.11 vs 1.27±1.13,10.25±1.98 vs 1.32±1.28,11.71±2.11 vs 1.03±0.11, 13.85±1.05 vs 1.01±0.19,13.89±0.89 vs0.98±0.07 were showed difference significantly.2.Model rats showed apathetic,erect hair,lying tired paucimobilis;In treated group rats showed better mental state,activities of large increase in the number of defecation,perianal obvious pollution.Sham-operated group without death records, the mortality of rats with SAP were lower in treatment group.The mortality of rats with SAP in model group and treatment group were 0%(0/10),20%(2/10)、40% (4/10)、50%(5/10) and 0%(0/10)、20%(2/10)、30%(3/10)、30%(3/10) respectively.The mortality of the treatment group in 24 h was lower than the model group(p <0.05).3.The level of amylase in sham-operated group appeared transient increase after model 6h and then returned to normal;In model group model,the level of amylase was highest after model 6 h,and gradually decreased with time,but in the 48 h was still higher than sham-operated group(p <0.05);Compared with the model group,the level of amylase in treatment group 12 h after began to reduce(p <0.05),in 48 h they showed no differences4.The level of serum DAO in sham-operated group before and after operation was no change (P> 0.05);The levels of serum DAO in treatment group after 24 h and 48 h was lower than the model group(p <0.05).5.After model 6h in sham-operated group acinar cells can be seen with mild edema, no bleeding and necrosis.In model group,the pancreas was Observated that interstitial edema,red blood cell leakage,inflammatory cell infiltration,acinar cells,edema, patchy hemorrhage point,focal degeneration and necrosis,and some glandular dilatation,the pathology score of pancreatic tissues gradually increased.The changes with 24 h and more in treatment group was decreasing,which was significance in statistics.6.Under light microscope,the villi structural integrity of the intestinal mucosa in sham-operated group was showed a high columnar epithelium,interstitial edema,no neutrophil infiltration.In model group,the structure of intestinal mucosa was damaged significantly in a progressive manner,particularly in the most obvious mold 24h,showing interstitial edema in rat ileum and a bit like bleeding,the expansion of lymphatic vessels,but was also showed that a large number of neutrophil infiltration with lymphoid follicular hyperplasia,thickening of intestinal villi edema,high degree of shortening,there was patchy epithelial necrosis of the top,falling,goblet cell decrease with disorder;mucosal thickness were becamed thinner,mucosa with sparse glands,glands reduced;When compared with the model group,the treatment of traditional Chinese medicine Qing Yi granules 12h in the model group reduced the pathological damage,manifested as reduced inflammatory cell infiltration,villous injury to reduce shedding,mucosal thickness and villous height were improved,24h after the beginning of the model with significant differences.48h after the operation, the indicators were close to normal.7.The claudin-1 of Small intestinal mucosa in each group were distributed in intestinal epithelial tight junction of cell membranes(including the cell membrane at the top),cytoplasmic area of membrane proximal appeared a small amount of staining, with staining brown granules,but nucleus and nuclear membrane without expression. The Claudin-1 protein of sham-operation group expression in the cell membrane and was strongly positive;Compared with the sham-operated group,the expression of claudin-1 was low in model group(p <0.05);Compared with the model group,the Claudin -1 of treatment group at different time points after model expression was higher(p < 0.05)Conclusion:1.Application of intravenous catheter system from duodenal catheter by retrograde cholangiopancreatography injection of 3.5%sodium taurocholate to establish SAP model(0.1ml/100g weight,speed of injection 0.15ml/min) is a improved model from preparation Aho SAP method,and is a good research animal model for SAP.2.Early application of Qingyi granules can improve the damage of pancreatic tissue in rats with SAP,reduce the intestinal mucosa and intestinal villus atrophy,improve intestinal tight junction protein Claudin-1 expression levels,reduce the permeability of intestinal mucosa,and decrease mortality.更多还原