早期肠内营养在大鼠急性重症胰腺炎肠道免疫屏障中的作用及其机制的实验研究
发布时间：2018-03-04 23:34
本文选题：SPA 切入点：EEN 出处：《泸州医学院》2013年硕士论文 论文类型：学位论文
【摘要】：目的：通过检测早期肠内营养对牛黄胆酸钠诱导大鼠重症急性胰腺炎模型的血清淀粉酶(AMY)、血糖(GLU)、静脉血中内毒素的含量，末段回肠和结肠peyer结MAdcAM-1分子(mucosal addressin celladhesion molecule-1)CD4+、CD8+含量。和大鼠门静脉血、胰腺、肺脏、肠系膜淋巴结的细菌培养。以及大鼠肠道的TLR4表达，观察早期肠内营养在大鼠重症急性胰腺炎肠道免疫屏障里的功能和研究EEN功效原理。方法：把成年身体状况良好的60只雄性大鼠体重约为300克左右，随机分成三组，每组20只大鼠。即假手术组(SO组)：开腹后仅轻轻翻动十二指肠及胰腺组织后关腹，不作处理。将3.8％牛磺胆酸钠lml从大鼠胰腺被膜下各点徐徐匀速注入胰腺从而完成动物模型建立。造模成功后按组分别给予肠外营养和肠内营养。肠外营养组(TPN组)，造模后12小时，经颈外静脉置管输注肠外营养液。肠内营养组(EEN组)，造模12小时后，经胃，空肠做空肠造瘘管，予以肠内营养。各组于造模后一天，经SD大鼠颈静脉取血，检测血清淀粉酶(AMY)、血糖(GLU)水平。肠外营养、肠内营养持续给予五天，第六天两组分别统计SD大鼠死亡率，随即处死大鼠并收集标本，胰腺组织HE染色后行光镜病理学检查。空肠、回肠、结肠HE染色后观察肠粘膜形态学。免疫组化法测定末端回肠和Peyer结肠道的MAdcAM-1、CD4+、CD8+的含量。Westen blot法测定SD大鼠肠道组织TLR4的表达。SD大鼠静脉血检测内毒素含量。取SD大鼠门静脉血、胰腺、肺脏、肠系膜淋巴结做细菌培养，检测细菌移位率。此课题为四川省绵阳市中心医院赵平武教授申请的四川省应用基础研究的课题。结果：(1)各组SD大鼠5天的生存率：假手术组(SO组)SD大鼠全部存活生存率是100％，死亡率为零；肠外营养组(TPN组)5天死亡率为50%，早期肠内营养组(EEN组)5天死亡率为25%，S组和其它实验组的死亡率的对照存在明显差异(P0.01)。TPN和EEN组的死亡率对照无明显差异无统计学意义(P=0.0847)。证明EEN是安全可行的。(2)SAP下SD大鼠血清淀粉酶改变：TPN、EEN组血淀粉酶含量在建模24小时后都明显上升，与SO组对照有明显差异(P0.01)。在建模24小时后，EEN组和TPN对照，血清淀粉酶含量降低，有统计学意义(P0.01)。(3)血糖变化：造模后TPN、EEN组血糖水平在建模24小时后均显著升高，与SO组比较有显著性统计学差异(P0.01)；在建模24小时后，EEN组与TPN组比，血糖含量无明显变化，但有显著统计学差异(P0.01)。(5)胰腺组织学评分：TPN、EEN组建模5天后所采集标本组织学评分与SO组相比，均明显升高。与S组比较有显著差异性(P0.01)。而TPN、EEN组相比，TPN组评分高于EEN组而且有显著性统计学差异(P0.01)。(6)各组SD大鼠静脉血内毒素含量测定：本实验中EEN、TPN两组动物在建立SAP模型5天后血内毒素水平明显比SO组升高，说明EEN、TPN两组均有不同程度的肠道屏障损伤，而EEN组血浆内毒素含量与TPN组对比明显偏低，具有统计学意义，(P0.05)。说明SAP早期肠内营养有效降低了SD大鼠SAP时肠粘膜的通透性，有效保护了其肠粘膜屏障。(7)胰腺、肺脏、肠系膜淋巴结细菌培养和细菌易位率测定：细菌类型主要为革兰阴性菌，其中大肠杆菌占85.7％(12/14)。各种细菌易位率EEN组远低于TPN组，具有统计学意义(P0.01)。而EEN、TPN组与SO组相比细菌移位率远远高于后者，具有明显统计学意义P0.01.(8)末端回肠黏膜和Peyer结CD4+、CD8+、MadcAM-1，免疫组化测定：CD4+、CD8+、MadcAM-1在EEN、TPN组表达均低于SO组(P0.05)，其中TPN组低于EEN组(p0.05）(9)回肠黏膜光镜下改变：在第5d时，TPN组肠粘膜萎缩程度包括黏膜厚度、绒毛长度与EEN组对照，肠粘膜萎缩程度明显加重，有显著性差异(p0.01)。(10)Westen blot法测定SD大鼠肠道组织TLR4的表达：光密度扫描分析显示，EEN、TPN组与SO组相比肠组织TLR4表达增强(P0.01）。EEN组与TPN组相比，，肠组织TLR4表达减弱(P0.01)。结论：(1)将3．8％牛磺胆酸钠lml缓慢匀速注入SD大鼠胰腺被膜下，能建立SAP动物模型。它具有重复性好，操作简便，价格便宜的优点。而且SD大鼠SAP动物模型病情呈渐进性发展。和患者SAP的疾病演变过程十分相像。5d死亡率稳定。完全能够满足SAP的早期肠内营养治疗研究。(2)早期肠内营养不加重胰腺炎病情，是安全、可行的，而且早期肠内营养在维护重症胰腺炎SD大鼠肠道屏障方面的作用效果显著，对肠道屏障的化学、免疫、生物、机械的几方面的功能提高几乎均有效。尤其是EEN能增加MAdcAM-1、CD4+、CD8+表达，提高SAP时肠免疫屏障，减少茵群易位，减少局部及全身感染。可以提高急性重症胰腺炎SD大鼠5d生存率。(3)研究成果预测，TLR4通过和它主要识别G—细菌细胞壁成分脂多糖（LPS）相结合后其复合物在SAP时导致的肠道屏障受损中起到关键作用，TLR4表达强度与SAP时炎症的强弱有一定的正相关性。
[Abstract]:Objective: through the detection of serum amylase of early enteral nutrition on yellow taurocholate induced severe acute pancreatitis model in rats (AMY), blood glucose (GLU), the content of endotoxin in blood, the terminal ileum and colon Peyer MAdcAM-1 (mucosal addressin celladhesion molecule-1) molecules CD4+, CD8+ content. And the rat portal vein blood. The pancreas, lung, bacterial culture of mesenteric lymph nodes. And rat intestinal TLR4 expression, observation function and study of EEN effect principle of early enteral nutrition on intestinal barrier in rats with severe acute pancreatitis. Methods: 60 male adult rats in good physical condition is about 300 grams, random divided into three groups, 20 rats in each group. Sham operation group (SO group): abdominal laparotomy only gently flip the duodenum and pancreas after no treatment. 3.8% sodium taurocholate LML from rat pancreatic capsule each The point slowly injected into the pancreas to complete the animal model. After the success of the model group were given parenteral nutrition and enteral nutrition Parenteral Nutrition Group (TPN group), 12 hours after modeling, through external jugular venous catheter infusion of parenteral nutrition. Enteral nutrition group (EEN group). Modeling after 12 hours, the stomach, jejunum do jejunal fistula, given enteral nutrition. Each day after modeling, jugular vein in SD rats blood serum amylase (AMY), blood glucose (GLU) levels. Parenteral nutrition, enteral nutrition for five days, sixth the two day mortality statistics were SD rats, then the rats were sacrificed and samples were collected, pancreatic tissue HE staining under light microscope. The pathological examination of jejunum, ileum, colon mucosal morphology were observed after HE staining. Immunohistochemical determination of terminal ileum and colon Peyer by MAdcAM-1, CD4+, TLR4 determination of intestinal tissue SD the content of.Westen blot CD8+ in the To detect the expression of endotoxin in blood of.SD rats. SD rats of portal vein blood, pancreas, lung, mesenteric lymph nodes were cultured for bacteria, the detection rate of bacterial translocation. Subject to the basic application research of Sichuan Province, Sichuan province Mianyang Central Hospital professor Zhao Pingwu for the study. Results: (1) the 5 day survival rate in each group SD rats: sham operation group (group SO) SD rats survived survival rate was 100%, the mortality rate is zero; parenteral nutrition group (TPN group) the 5 day mortality rate was 50%, the early enteral nutrition group (EEN group) the 5 day mortality rate was 25%, there are obvious differences in S control group and the other groups the mortality rate (P0.01) and.TPN EEN group were no significant difference between the mortality rate was not statistically significant (P=0.0847). EEN is safe and feasible. (2) serum amylase in SD rats under SAP change: TPN, amylase content of blood EEN 24 hours after modeling were significantly increased, with control group and SO group 鏄庢樉宸紓(P0.01).鍦ㄥ缓妯
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