自噬在大鼠急性坏死性胰腺炎时的变化及其意义  

Changes and its significance of autophagy in rats with acute necrosis pancreatitis

作　　者：刘晓[1] 郭晓榕[1] 张蓓蓓[1] 李洁[1] 吴敏[1] 湛先保[1] 

机构地区：[1]上海长海医院消化内科,200433

出　　处：《中华消化杂志》2015年第1期39-43,共5页Chinese Journal of Digestion

基　　金：国家自然科学基金（81170434）;上海市科委自然科学基金（11ZR1447600）

摘　　要：目的 探讨自噬在实验性急性坏死性胰腺炎（ANP）大鼠发病中的变化及其意义.方法 按随机数字法将18只SD大鼠随机分为对照组、ANP组、ANP＋雷帕霉素组.采用腹腔注射20％ L-精氨酸的方法制作大鼠ANP模型,造模后9h处死.ANP＋雷帕霉素组于造模前30 min按大鼠体质量1.2 mg/kg腹腔注射雷帕霉素.对照组予腹腔注射等量0.9％ NaCl溶液.ELISA方法检测各组大鼠血清胰蛋白酶原活化肽（TAP）、IL-1、IL-6、TNFα水平,进行胰腺组织病理评分,电子显微镜下观察大鼠胰腺腺泡细胞自噬相关结构,实时定量-PCR、Western蛋白印迹法及免疫组织化学检测大鼠胰腺组织自噬标志物微管相关蛋白轻链3（LC3）-Ⅱ及Beclin-1 mRNA和蛋白的表达.组间均数比较采用单因素方差分析.结果 成功建立大鼠ANP模型,胰腺病理评分显示ANP＋雷帕霉素组腺泡细胞坏死评分为2.19±1.38,高于ANP组（0.97±0.68）,差异有统计学意义（F=33.75,P＜0.05）.Western蛋白印迹法显示ANP组LC3-Ⅱ及Beclin-1蛋白表达（分别为35.25±2.68和49.40±5.28）明显高于对照组（分别为1.54±0.16和0.78±0.06）;而ANP＋雷帕霉素组LC3-Ⅱ及Beclin-1蛋白表达量（分别为123.53±3.21和76.41±3.80）比ANP组更高,差异均有统计学意义（F=2 045.54、326.87,P均＜0.01）.免疫组织化学法显示ANP＋雷帕霉素组LC3-Ⅱ及Beclin-1蛋白表达量（分别为7 570.63±4 357.67和3 418.09±2 035.78）明显高于ANP组（分别为1 926.53±1 414.44和536.11±403.10）,差异均有统计学意义（F=39.83、41.58,P均＜0.01）.Beelin-1 mRNA在ANP组的表达（107.12±29.10）较对照组（7.01±3.39）明显升高,差异有统计学意义（F=3.61,P＜0.05）,但在ANP＋雷帕霉素组的表达（97.63±65.38）与ANP组比较差异无统计学意义（P＞0.05）.LC3-Ⅱ mRNA在ANP组的表达（1.76±1.59）与对照组（1.51±0.95）相比差异无统计学意义（P＞0.05）,但在ANP＋雷帕霉素组的表达（4.37±1.Objective To investigate the changes and significance of autophagy in rats with experimental acute necrosis pancreatitis （ANP）.Methods According to method of random number,18 rats were randomly divided into control group,ANP group,ANP＋rapamycin （RAP） group.The ANP rat model was established by intraperitoneal injection of 20% L-arginine.The rats of ANP＋RAP group were intraperitoneal injected with RAP 1.2 mg/kg at 30 minutes before modeling.The rats of control group were intraperitoneal injected with 0.9% NaCl solution.The blood was drawed from the hearts nine hours after modeling for subsequent experiments.Serum levels of trypsinogen activation peptide （TAP）,interleukin （IL-1）,IL-6 and tumor necrosis factor （TNF） α were measured with enzyme-linked immunosorbent assay.The pancreatic tissues were pathologically scored.Autophagy-related structures in rat pancreatic acinar cells were observed by transmition electron microscopy.The expression of autophagy marker microtuble assciated protein 1 light chain 3 （LC3）-Ⅱ and Beclin-1 at mRNA and protein level were measured by quantitative real-time polymerase chain reaction （qRT-PCR）,Western bloting and immunohistochemistry.The single factor analysis of variance was used for mean comparison among groups.Results A rat model of ANP was successfully established.Histopathological score of pancreas acinar cell necrosis of ANP＋RAP group （2.19±1.38） was higher than that of ANP group （0.97±0.68）,and the difference was statistically significant（F=33.75,P〈0.05）.The results of Western blotting indicated that the protein expression of LC3-Ⅱ and Beclin-1 in ANP group （35.25±2.68 and 49.40±5.28）were higher than those in control group （1.54±0.16 and 0.78±0.06）,furthermore the expressions in ANP＋RAP group（123.53±3.21 and 76.41±3.80） were higher than those in ANP group,and the differences were statistically significant（F=2 045.54,326.87,both P〈0.01）.Immunohistochemistry results also indicated that the LC3Ⅱ and

关 键 词：胰腺炎 急性坏死性 自噬 胰蛋白酶原 

分 类 号：R576[医药卫生—消化系统]