急性胰腺炎大鼠腸道緊密連接損傷及其與αSNAP表達的關(guān)系
發(fā)布時間：2019-05-19 09:54
【摘要】：研究背景與目的： 急性胰腺炎是臨床常見的急腹癥，重癥患者胰腺局部損傷的同時往往伴發(fā)肺、肝臟、心臟、腸道等多器官功能障礙綜合征（MODS）。腸道是疾病進展過程中最易受累的胰外器官之一，，亦可能是MODS發(fā)生的啟動者。目前，腸屏障損傷在重癥急性胰腺炎中所發(fā)揮的作用受到廣泛關(guān)注。腸黏膜屏障一般由免疫屏障、機械屏障、生物屏障和化學屏障四部分組成。腸機械屏障的主要構(gòu)成蛋白之一（occludin）受多種因素調(diào)節(jié)。ɑSNAP是一種新型的膜融合蛋白，在腸上皮細胞中表達豐富，參與調(diào)節(jié)腸屏障的形成與重塑過程。本的研究目的在于檢測急性胰腺炎大鼠模型中腸屏障損傷情況，以及occludin表達的變化，并探討其與ɑSNAP表達的關(guān)系。 方法： 1．動物實驗 SD大鼠72只，隨機分為三組，分別逆行胰膽管注射生理鹽水、0.5%牛黃膽酸鈉溶液、3.8%牛黃膽酸鈉溶液后制作成SO、MAP、SAP組模型。在1d、2d、3d后通過HE染色觀察大鼠胰腺以及回腸上皮的病理變化。采用酶化學方法檢測血清淀粉酶，ELISA方法檢測血清TNF-ɑ、內(nèi)毒素水平變化，。通過檢測漏出腸道的FITC-dextran量間接反映腸道通透性的變化。occludin和ɑSNAP的分布及表達變化采用免疫熒光和免疫印跡（western blot）方法檢測。 2．細胞實驗 構(gòu)建ɑSNAP shRNA慢病毒載體、轉(zhuǎn)染IEC-6細胞。采用流式細胞術(shù)檢測轉(zhuǎn)染前后IEC-6細胞凋亡；western blot和免疫熒光檢測轉(zhuǎn)染前后IEC-6細胞occludin的表達和分布變化。 結(jié)果： 1、動物實驗 1）SAP組大鼠血清淀粉酶、TNF-α、內(nèi)毒素水平明顯升高。在1d、2d、3d時血清淀粉酶水平：SO組（1243.75±157.88,1182.5±153.09,1008.13±73.29）；MAP組（1662.5±104.68,2326.25±300.03,2325±200.48）； SAP組（7897.5±1006.29,5947.5±554.69,5104.38±1256.31）。在1d、2d、3d時血清TNF-ɑ水平：SO組（11.59±5.55,17.64±5.75,14.11±4.83）；MAP組（55.43±9.62,86.77±22.31,38.26±10.93）；SAP組（174.76±35.50,203.96±60.54,119.02±29.20）。在1d、2d、3d時血清內(nèi)毒素水平：SO組（9.53±7.92,8.87±4.05,8.65±5.03）； MAP組（54.36±11.43,67.98±8.86,88.97±9.06）；SAP組（208.15±24.47,197.51±22.99,137.18±31.70）。各組間比較p＜0.05。 2）胰腺組織病理：SO組胰腺組織無明顯變化；MAP組胰腺組織可見輕度水腫，少量炎性細胞浸潤；SAP組胰腺組織大片壞死、大量炎癥細胞浸潤。在1d、2d、3d時胰腺病理評分分別為：SO組（1.88±0.34,2.09±0.26,2.00±0.68）； MAP組（4.86±0.61;5.06±0.59;5.35±0.62）； SAP組（10.63±0.51;12.95±0.39;13.91±0.45）。各組間比較p＜0.05。 3）空腸上皮組織病理：SO組空腸組織絨毛形態(tài)正常；MAP組腸絨毛輕微縮短，其他未見明明改變；SAP組腸絨毛出現(xiàn)明顯水腫、倒伏、萎縮，腸上皮細胞壞死、脫落。在1d、2d、3d時空腸上皮組織病理評分分別為：SO組（1；1；1）； MAP組（1.500±0.54；1.88±0.35；2.13±0.64）； SAP組（3.38±0.52；4.13±0.84；5.13±0.84）。各組間比較p＜0.05。 4）急性胰腺炎大鼠腸道通透性明顯升高，漏出腸道的FITC-dextran的熒光值：SO組（1d,11.08±4.22;2d,8.31±1.74;3d,16.50±8.83），MAP組（1d,75.39±34.40;2d,123.50±5.09;3d,66.11±9.48）,SAP組（1d,379.34±25.38;2d,586.52±57.35;3d,489.76±105.36）。各組間比較p＜0.05。 5）SO、MAP組大鼠腸上皮細胞occludin和αSNAP蛋白表達無明顯變化；SAP組大鼠腸上皮細胞occludin和αSNAP表達明顯下調(diào)。 2、細胞實驗 ɑSNAP shRNA慢病毒轉(zhuǎn)染后IEC-6細胞凋亡明顯增加；occludin表達明顯下調(diào)。 結(jié)論：急性胰腺炎大鼠腸道通透性增加。急性胰腺炎時腸上皮細胞αSNAP蛋白表達下調(diào)導致occludin蛋白表達減少、腸上皮細胞凋亡增加，進而導致腸屏障通透性增加。
[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鹵25.38;2d,586.52鹵 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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R576
本文編號：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鹵25.38;2d,586.52鹵 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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R576
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