实验性重症急性胰腺炎对SD大鼠小肠防御素-5基因表达影响.docVIP

实验性重症急性胰腺炎对SD大鼠小肠防御素-5基因表达影响 　 作者：李钢，胡祖健，王凯诚，罗静，赵金锋，刘世宾，严萍，陈海平 【摘要】 目的 检测实验性重症急性胰腺炎时，SD大鼠小肠防御素-5 mRNA表达的变化，从分子水平上探讨胰腺炎时肠黏膜屏障受损的机制。方法 40只SD大鼠随机分成4组，每组10只。模型组（B～E组）改良Ahos法造模，于造模后第12，24和36小时处死，正常对照组（A组）直接处死。取血送淀粉酶检查，胰腺送病理检查。用Quanty One软件分析PCR产物凝胶图像以检测各组防御素-5 mRNA的表达变化。结果 改良Ahos法导致了SD大鼠重症急性胰腺炎，12 h防御素-5 mRNA表达轻微下调，36 h下调最明显。结论 防御素-5的获得性缺陷可能是胰腺炎时发生肠道细菌移位的重要原因之一。 【关键词】 胰腺炎；防御素-5；mRNA表达 Abstact Objecive To detect the changes of intestinal defensin-5 mRNA expression in SD rats with experimental severe acute pancreatitis and explore the mechanisms of bacterial translocation at the molecular level. Methods Forty SD rats were randomly divided into 4 groups with 10 in each group. Experimental groups (from B to D)， pancreatitis induced by modified Ahos method， were killed at 12， 24 and 36 hours after the injection. The control group(A) was killed directly. Amylase and pancreatic pathological alterations were observed. The amplification products of RT-PCR were electrophoresed and the images were analyzed by Quanty One software. Results Severe acute pancreatitis in SD rats were induced by modified Aho’s method. Intestinal defensin-5 mRNA expression was down regulated slightly at 12th hour and most markedly at 36th hours. Conclusion Acquired defensin-5 deficiency may play an important role in the pathogenesis of bacterial translocation in severe acute pancreatitis. Keywords severe acute pancreatitis； defensin-5； mRNA expression重症急性胰腺炎（severe acute pancreatitis， SAP）的发病过程分为急性全身炎性反应期、坏死感染期、感染愈合期。已有的研究资料提示SAP的坏死性感染与肠道的细菌移位相关，而肠源性感染的发生与肠黏膜免疫屏障（天然免疫和获得性免疫）受损相关。近年来发现的肠道天然免疫物质防御素-5（defensin 5，Dn-5）在肠黏膜抗微生物防御中的作用颇受关注，具有抗菌谱广、不易产生耐药、不影响肠道微生态平衡等优点[1]。本实验通过研究SAP发病过程中小肠Dn-5的表达变化，为SAP肠道细菌移位进一步寻找理论依据，同时为寻找能诱导Dn-5表达的新药打下基础。 1 材料和方法 1．1 实验动物分组和试剂 SD大鼠40只，雌雄各半，体质量为(250±10)g，由浙江省医学科学院动物实验中心提供，随机分为A组（正常对照组）、B组（模型12h组）、C组（模型24h组）、D组（模型36h组）四组。牛磺胆酸钠（taurocholic acid）购自Sigma公司。RNA提取试剂盒（Trizol Reagent）购自GibcoBRL公司，RT-PCR试剂盒购自TaKaRa Biotechnology公司。Dn-5引物由上海生物工程公司合成并提供，上游引物：5’-TCGCAGCCATGAAGAAACTT-3’