急性胰腺炎大鼠肠道紧密连接损伤及其与αSNAP表达的关系
发布时间：2019-05-19 09:54
【摘要】：研究背景与目的： 急性胰腺炎是临床常见的急腹症，重症患者胰腺局部损伤的同时往往伴发肺、肝脏、心脏、肠道等多器官功能障碍综合征（MODS）。肠道是疾病进展过程中最易受累的胰外器官之一，，亦可能是MODS发生的启动者。目前，肠屏障损伤在重症急性胰腺炎中所发挥的作用受到广泛关注。肠黏膜屏障一般由免疫屏障、机械屏障、生物屏障和化学屏障四部分组成。肠机械屏障的主要构成蛋白之一（occludin）受多种因素调节。ɑSNAP是一种新型的膜融合蛋白，在肠上皮细胞中表达丰富，参与调节肠屏障的形成与重塑过程。本的研究目的在于检测急性胰腺炎大鼠模型中肠屏障损伤情况，以及occludin表达的变化，并探讨其与ɑSNAP表达的关系。 方法： 1．动物实验 SD大鼠72只，随机分为三组，分别逆行胰胆管注射生理盐水、0.5%牛黄胆酸钠溶液、3.8%牛黄胆酸钠溶液后制作成SO、MAP、SAP组模型。在1d、2d、3d后通过HE染色观察大鼠胰腺以及回肠上皮的病理变化。采用酶化学方法检测血清淀粉酶，ELISA方法检测血清TNF-ɑ、内毒素水平变化，。通过检测漏出肠道的FITC-dextran量间接反映肠道通透性的变化。occludin和ɑSNAP的分布及表达变化采用免疫荧光和免疫印迹（western blot）方法检测。 2．细胞实验 构建ɑSNAP shRNA慢病毒载体、转染IEC-6细胞。采用流式细胞术检测转染前后IEC-6细胞凋亡；western blot和免疫荧光检测转染前后IEC-6细胞occludin的表达和分布变化。 结果： 1、动物实验 1）SAP组大鼠血清淀粉酶、TNF-α、内毒素水平明显升高。在1d、2d、3d时血清淀粉酶水平：SO组（1243.75±157.88,1182.5±153.09,1008.13±73.29）；MAP组（1662.5±104.68,2326.25±300.03,2325±200.48）； SAP组（7897.5±1006.29,5947.5±554.69,5104.38±1256.31）。在1d、2d、3d时血清TNF-ɑ水平：SO组（11.59±5.55,17.64±5.75,14.11±4.83）；MAP组（55.43±9.62,86.77±22.31,38.26±10.93）；SAP组（174.76±35.50,203.96±60.54,119.02±29.20）。在1d、2d、3d时血清内毒素水平：SO组（9.53±7.92,8.87±4.05,8.65±5.03）； MAP组（54.36±11.43,67.98±8.86,88.97±9.06）；SAP组（208.15±24.47,197.51±22.99,137.18±31.70）。各组间比较p＜0.05。 2）胰腺组织病理：SO组胰腺组织无明显变化；MAP组胰腺组织可见轻度水肿，少量炎性细胞浸润；SAP组胰腺组织大片坏死、大量炎症细胞浸润。在1d、2d、3d时胰腺病理评分分别为：SO组（1.88±0.34,2.09±0.26,2.00±0.68）； MAP组（4.86±0.61;5.06±0.59;5.35±0.62）； SAP组（10.63±0.51;12.95±0.39;13.91±0.45）。各组间比较p＜0.05。 3）空肠上皮组织病理：SO组空肠组织绒毛形态正常；MAP组肠绒毛轻微缩短，其他未见明明改变；SAP组肠绒毛出现明显水肿、倒伏、萎缩，肠上皮细胞坏死、脱落。在1d、2d、3d时空肠上皮组织病理评分分别为：SO组（1；1；1）； MAP组（1.500±0.54；1.88±0.35；2.13±0.64）； SAP组（3.38±0.52；4.13±0.84；5.13±0.84）。各组间比较p＜0.05。 4）急性胰腺炎大鼠肠道通透性明显升高，漏出肠道的FITC-dextran的荧光值：SO组（1d,11.08±4.22;2d,8.31±1.74;3d,16.50±8.83），MAP组（1d,75.39±34.40;2d,123.50±5.09;3d,66.11±9.48）,SAP组（1d,379.34±25.38;2d,586.52±57.35;3d,489.76±105.36）。各组间比较p＜0.05。 5）SO、MAP组大鼠肠上皮细胞occludin和αSNAP蛋白表达无明显变化；SAP组大鼠肠上皮细胞occludin和αSNAP表达明显下调。 2、细胞实验 ɑSNAP shRNA慢病毒转染后IEC-6细胞凋亡明显增加；occludin表达明显下调。 结论：急性胰腺炎大鼠肠道通透性增加。急性胰腺炎时肠上皮细胞αSNAP蛋白表达下调导致occludin蛋白表达减少、肠上皮细胞凋亡增加，进而导致肠屏障通透性增加。
[Abstract]:Background and purpose of the study: Acute pancreatitis is a common acute abdomen, and the local damage of the pancreas in the severe patients is often associated with multiple organ dysfunction syndrome (MODS) such as lung, liver, heart and intestine. ). The intestinal tract is one of the most affected external organs of the pancreas in the course of disease progression, or may be the start-up of the MODS. At present, the role of intestinal barrier injury in severe acute pancreatitis is widely Note. The intestinal mucosal barrier is generally composed of four groups of immunobarrier, mechanical barrier, biological barrier, and chemical barrier one of the major constituent proteins of the intestinal mechanical barrier is regulated by a number of factors The SNAP is a new type of membrane fusion protein, which is rich in intestinal epithelial cells and is involved in the formation and remodeling of the intestinal barrier. Cheng. The purpose of this study is to detect the intestinal barrier damage in the rat model of acute pancreatitis, and to investigate the changes of the expression of octredin and to explore the correlation with the expression of SNAP. Department. Method: 1. The animal experiment SD rats were randomly divided into three groups: retrograde cholangiopancreatography, normal saline, 0.5% sodium taurocholate solution, 3.8% bezoar cholate solution, and making into SO, MAP. The pancreas of the rat and the ileum were observed by HE staining after 1d, 2d, and 3d. The pathological changes of the epithelium were detected by enzyme-chemical method. The serum TNF-1 was detected by enzyme-linked immunosorbent assay (ELISA). The level of the toxin changes, and the intestine was indirectly reflected by the detection of the amount of FITC-dextran from the intestinal tract. The change of channel permeability. The distribution and expression of occlusin and BSNAP were immunofluorescent and immunoblotting (western bl). ot) Method check test.2. Cell experiment construction of SNAP shRNA lentiviral load The IEC-6 cells before and after transfection were detected by flow cytometry. The cells of IEC-6 were detected by western blot and immunofluorescence. din Expression and distribution Changes. Results:1, Animal Experiment 1) Serum starch in the SAP group Serum amylase levels at 1d, 2d, and 3d: SO (1243.75-157.88, 1182.5-153.09, 1008.13-73.29); MAP group (1662.5-104.68, 2326.25-300.03,2325-200.48); SAP group (7897.5-1006.29, 5947.5-300.03,2325-200.48); SAP group (7897.5-1006.29, 5947.5-554.69 , 5104.38 (1256.31). Serum TNF-1 levels at 1d, 2d, and 3d: SO (11.59, 5.55, 17.64, 5.75, 14.11, 4.83); MAP group (55.43, 9.62, 86.77, 22.31, 38.26, 10.93); SAP group (174.76, 35.50, 203.96)60 .54, 119.02 (29.20). Serum endotoxin levels at 1d, 2d, and 3d: SO (9.53, 7.92, 8.87, 4.05, 8.65, 5.03); MAP group (54.36, 11.43, 67.98, 8.86, 88.97, 9.06); SAP group (208.15) 24.47, 197.51-22 .99, 137.18-3 1.70). There was no obvious change in the pancreatic tissue of the group (p <0.05). The pancreatic tissue of the group had no obvious change; the pancreatic tissue of the MAP group had mild edema and a small amount of inflammatory cell infiltration; SA The pathological scores of pancreatic tissue in P group were: SO (1.88, 0.34, 2.09, 0.26, 2.00, 0.68), MAP (4.86, 0.61, 5.06, 0.59, 5.35, 0.62), and SAP (10.63, 0.51;12, respectively). 95% 0.39; 13.9 1 (0.45). The pathology of the jejunum of the jejunum of the jejunum of the jejunum of the jejunum of the jejunum of the jejunum of the group (p <0.05) in the group was normal; the villi of the group in the group was slightly shortened, and the other was not obviously changed; the intestinal villus in the SAP group appeared. Obvious edema, lodging, atrophy, necrosis and shedding of intestinal epithelial cells, the pathological scores of the jejunum of jejunum at 1d, 2d and 3d were: SO (1;1;1); MAP group (1.500, 0.54; 1.88; 0.35; 2.13; 0.64); and SAP group (3.38-0.52). ; 4.13-0.84;5 .13 (0.84). The intestinal permeability of rats with acute pancreatitis increased significantly between groups (p <0.05.4), and the fluorescence of FITC-dextran from the intestinal tract: the SO group (1d, 11.08, 4.22; 2d, 8.31, 1.74; 3d, 16.50, 8.83), the MAP group (1d, 75.39, 34.40; 2d, 123.50, 5.09; 3d, 66.11, 9.48); and the SAP group (1d, 379.3). 4卤25.38;2d,586.52卤 57.35;3d,489 (.76-105.36). There was no significant change in the expression of occlusin and CRESNAP in the intestinal epithelial cells of the group (p <0.05) in the group (p <0.05). intestinal epithelial cell o The expression of ccludin and SNAP was down-regulated.2. The cell experiment was transfected with SNAP shRNA lentivirus. Post-IEC-6 cell apoptosis increased significantly; occ Conclusion: The decrease of the expression of SNAP protein in the intestinal epithelial cells in acute pancreatitis led to the increase of the intestinal permeability in rats with acute pancreatitis.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R576
本文编号：2480627
[Abstract]:Background and purpose of the study: Acute pancreatitis is a common acute abdomen, and the local damage of the pancreas in the severe patients is often associated with multiple organ dysfunction syndrome (MODS) such as lung, liver, heart and intestine. ). The intestinal tract is one of the most affected external organs of the pancreas in the course of disease progression, or may be the start-up of the MODS. At present, the role of intestinal barrier injury in severe acute pancreatitis is widely Note. The intestinal mucosal barrier is generally composed of four groups of immunobarrier, mechanical barrier, biological barrier, and chemical barrier one of the major constituent proteins of the intestinal mechanical barrier is regulated by a number of factors The SNAP is a new type of membrane fusion protein, which is rich in intestinal epithelial cells and is involved in the formation and remodeling of the intestinal barrier. Cheng. The purpose of this study is to detect the intestinal barrier damage in the rat model of acute pancreatitis, and to investigate the changes of the expression of octredin and to explore the correlation with the expression of SNAP. Department. Method: 1. The animal experiment SD rats were randomly divided into three groups: retrograde cholangiopancreatography, normal saline, 0.5% sodium taurocholate solution, 3.8% bezoar cholate solution, and making into SO, MAP. The pancreas of the rat and the ileum were observed by HE staining after 1d, 2d, and 3d. The pathological changes of the epithelium were detected by enzyme-chemical method. The serum TNF-1 was detected by enzyme-linked immunosorbent assay (ELISA). The level of the toxin changes, and the intestine was indirectly reflected by the detection of the amount of FITC-dextran from the intestinal tract. The change of channel permeability. The distribution and expression of occlusin and BSNAP were immunofluorescent and immunoblotting (western bl). ot) Method check test.2. Cell experiment construction of SNAP shRNA lentiviral load The IEC-6 cells before and after transfection were detected by flow cytometry. The cells of IEC-6 were detected by western blot and immunofluorescence. din Expression and distribution Changes. Results:1, Animal Experiment 1) Serum starch in the SAP group Serum amylase levels at 1d, 2d, and 3d: SO (1243.75-157.88, 1182.5-153.09, 1008.13-73.29); MAP group (1662.5-104.68, 2326.25-300.03,2325-200.48); SAP group (7897.5-1006.29, 5947.5-300.03,2325-200.48); SAP group (7897.5-1006.29, 5947.5-554.69 , 5104.38 (1256.31). Serum TNF-1 levels at 1d, 2d, and 3d: SO (11.59, 5.55, 17.64, 5.75, 14.11, 4.83); MAP group (55.43, 9.62, 86.77, 22.31, 38.26, 10.93); SAP group (174.76, 35.50, 203.96)60 .54, 119.02 (29.20). Serum endotoxin levels at 1d, 2d, and 3d: SO (9.53, 7.92, 8.87, 4.05, 8.65, 5.03); MAP group (54.36, 11.43, 67.98, 8.86, 88.97, 9.06); SAP group (208.15) 24.47, 197.51-22 .99, 137.18-3 1.70). There was no obvious change in the pancreatic tissue of the group (p <0.05). The pancreatic tissue of the group had no obvious change; the pancreatic tissue of the MAP group had mild edema and a small amount of inflammatory cell infiltration; SA The pathological scores of pancreatic tissue in P group were: SO (1.88, 0.34, 2.09, 0.26, 2.00, 0.68), MAP (4.86, 0.61, 5.06, 0.59, 5.35, 0.62), and SAP (10.63, 0.51;12, respectively). 95% 0.39; 13.9 1 (0.45). The pathology of the jejunum of the jejunum of the jejunum of the jejunum of the jejunum of the jejunum of the jejunum of the group (p <0.05) in the group was normal; the villi of the group in the group was slightly shortened, and the other was not obviously changed; the intestinal villus in the SAP group appeared. Obvious edema, lodging, atrophy, necrosis and shedding of intestinal epithelial cells, the pathological scores of the jejunum of jejunum at 1d, 2d and 3d were: SO (1;1;1); MAP group (1.500, 0.54; 1.88; 0.35; 2.13; 0.64); and SAP group (3.38-0.52). ; 4.13-0.84;5 .13 (0.84). The intestinal permeability of rats with acute pancreatitis increased significantly between groups (p <0.05.4), and the fluorescence of FITC-dextran from the intestinal tract: the SO group (1d, 11.08, 4.22; 2d, 8.31, 1.74; 3d, 16.50, 8.83), the MAP group (1d, 75.39, 34.40; 2d, 123.50, 5.09; 3d, 66.11, 9.48); and the SAP group (1d, 379.3). 4卤25.38;2d,586.52卤 57.35;3d,489 (.76-105.36). There was no significant change in the expression of occlusin and CRESNAP in the intestinal epithelial cells of the group (p <0.05) in the group (p <0.05). intestinal epithelial cell o The expression of ccludin and SNAP was down-regulated.2. The cell experiment was transfected with SNAP shRNA lentivirus. Post-IEC-6 cell apoptosis increased significantly; occ Conclusion: The decrease of the expression of SNAP protein in the intestinal epithelial cells in acute pancreatitis led to the increase of the intestinal permeability in rats with acute pancreatitis.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R576
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