急性肝衰竭大鼠模型肠道防御素-5、分泌型磷脂酶A2和溶菌酶表达变化及其与细菌移位的关系  

Expression of intestinal defensin-5, soluble phospholipase A2 and lysozyme and the relation to bacterial translocation in rat models of acute liver failure

作　　者：黄娟君[1] 李小鹏[2] 程晓宇[2] 程娜[2] 刘娟[2] 雷弯 刘碧霞[2] 张伦理[2] 

机构地区：[1]赣州市人民医院感染性疾病科,341000 [2] 南昌大学第一附属医院感染病科

出　　处：《中华肝脏病杂志》2014年第12期932-937,共6页Chinese Journal of Hepatology

基　　金：中国肝炎防治基金会一天晴肝病研究基金（20120055）;江西省教育厅基金（CJJ11322）

摘　　要：目的 探讨急性肝衰竭（ALF）大鼠模型肠道防御素-5（RD-5）、分泌型磷脂酶A2（sPLA2）和溶菌酶（Lysozyme）表达变化及与细菌移位的关系. 方法 将48只健康雄性SD大鼠分为对照组8只和急性肝衰竭模型组40只,模型组再按造模后不同时间点分为5个亚组：8、16、24、48、72 h组.模型组腹腔注射D-氨基半乳糖诱导ALF.取各组肝、脾和肠系膜淋巴结组织匀浆进行细菌培养;肝组织和末段回肠组织经HE染色后光镜下观察病理变化;检测各组末段回肠RD-5、sPLA2、Lysozyme mRNA及sPLA2和Lysozyme的蛋白表达.组间均数比较采用单因素方差分析. 结果 用D-氨基半乳糖成功诱导ALF模型.正常对照组未出现脏器细菌培养阳性,模型组24、48、72 h脏器细菌移位率分别为8.3％、37.5％和58.3％,但72 h小鼠模型的末端回肠结构尚完整,未见黏膜上皮细胞明显脱落坏死情况.模型组大鼠回肠黏膜潘氏细胞RD-5、sPLA2 mRNA相对表达量在早期逐渐升高,16h上升至高峰分别为1.291 ±0.153、1.131±0.128,与对照组的0.725±0.116、0.722±0.112比较,t值分别为69.25、95.71,P值均＜0.01,差异有统计学意义;RD-5、sPLA2mRNA相对表达量随时间延长逐渐下降,在72 h分别为0.415±0.104、0.425±0.076,明显低于对照组,t值分别为31.55、44.98,P值均＜0.01,差异有统计学意义.对照组Lysozyme mRNA相对表达量为0.853±0.093,模型组早期亦升高,8h上升至高峰为1.211±0.107,随后逐渐下降,72h为0.704±0.103,明显低于对照组,差异有统计学意义（t=9.224,P＜0.01）.Western blot检测对照组sPLA2和Lysozyme的蛋白相对表达量,分别为0.583±0.121和0.650±0.093,高于模型组72 h的0.327±0.086和0.382±0.057,t值分别为12.28、15.83,P值均＜0.01.免疫组织化学染色结果与Western blot检查结果相一致. 结论 ALF大鼠模型的回肠黏膜免疫屏障功能下降,随着潘氏细胞RD-5、sPLA2和Lysozyme mRNA及蛋白表达下降,脏器细菌移位率增加,Objective To study the intestinal expression of defensin-5 （RD-5）,soluble phospholipase A2 （sPLA2） and lysozyme in acute liver failure （ALF） using rat models,and to determine the relation of these expressions to intestinal bacterial translocation.Methods Forty-eight healthy male Sprague-Dawley rats were divided into a control group （n =8） and a model group （n =40; intraperitoneal injection of 10% D-galactosamine）.The model group was further divided into five subgroups according to the time lapse after model establishment （8,16,24,48,and 72 hours）.At the end of the experiments,homogenates of mesenteric lymph nodes,liver and spleen were cultured in agar for bacterial outgrowth.Hematoxylin-eosin stained sections of liver and terminal ileum were examined under an optical microscope to assess pathological changes.mRNA expression of RD-5,sPLA2 and lysozyme in the terminal ileum was determined by reverse transcription-polymerase reaction （RT-PCR）,and protein expression of sPLA2 and lysozyme from the same anatomic location was determined by western blotting and immunohistochemistry.Means between groups were compared with one-way analysis of variance.Results ALF was successfully induced in the D-galactosamine injected rats.No bacteria grew in the organ cultures from the control group,while 8.3%,37.5% and 58.3% of the rats in the 24-,48-and 72-hour model groups showed positive cultures.Despite this,the structure of the terminal ileum from the rats in the 72-hour model group was nearly intact,without obvious necrosis of mucosal epithelial cells.Expression of RD-5 and sPLA2 mRNA in the model groups gradually increased at early time points and peaked at 16 hours after induction of ALF （1.291±0.153 and 1.131±0.128）,which was significantly higher than that detected in the control group （0.725±0.116 and 0.722±0.112,t =69.25,95.71,all P 〈 0.01）.After that,the expression of RD-5 and sPLA2 mRNA progressively decreased,and by 72 hours after the induction of ALF,the expression （0.415±0.104

关 键 词：肝功能衰竭 急性 防御素-5 磷脂酶A2 溶菌酶 细菌移位 

分 类 号：R575.3[医药卫生—消化系统] R-332[医药卫生—内科学]