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
급성간쇠갈대서모형장도방어소-5、분비형린지매A2화용균매표체변화급기여세균이위적관계
Expression of intestinal defensin-5, soluble phospholipase A2 and lysozyme and the relation to bacterial translocation in rat models of acute liver failure
  • 万方数据
    中华肝脏病杂志 중화간장병잡지
    CHINESE JOURNAL OF HEPATOLOGY
    2014年 12期 932-937 ,共6页

    黄娟君%李小鹏%程晓宇%程娜%刘娟%雷弯%刘碧霞%张伦理

    황연군%리소붕%정효우%정나%류연%뢰만%류벽하%장윤리

    肝功能衰竭,急性%防御素-5%磷脂酶A2%溶菌酶%细菌移位 肝功能衰竭,急性%防禦素-5%燐脂酶A2%溶菌酶%細菌移位
    간공능쇠갈,급성%방어소-5%린지매A2%용균매%세균이위
    Liver failure,acute%Defensin-5%Phospholipase A2%Lysozyme%Bacterial translocation
    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,且不伴有明显肠黏膜损伤的发生.
    목적 탐토급성간쇠갈(ALF)대서모형장도방어소-5(RD-5)、분비형린지매A2(sPLA2)화용균매(Lysozyme)표체변화급여세균이위적관계. 방법 장48지건강웅성SD대서분위대조조8지화급성간쇠갈모형조40지,모형조재안조모후불동시간점분위5개아조:8、16、24、48、72 h조.모형조복강주사D-안기반유당유도ALF.취각조간、비화장계막림파결조직균장진행세균배양;간조직화말단회장조직경HE염색후광경하관찰병리변화;검측각조말단회장RD-5、sPLA2、Lysozyme mRNA급sPLA2화Lysozyme적단백표체.조간균수비교채용단인소방차분석. 결과 용D-안기반유당성공유도ALF모형.정상대조조미출현장기세균배양양성,모형조24、48、72 h장기세균이위솔분별위8.3％、37.5％화58.3％,단72 h소서모형적말단회장결구상완정,미견점막상피세포명현탈락배사정황.모형조대서회장점막반씨세포RD-5、sPLA2 mRNA상대표체량재조기축점승고,16h상승지고봉분별위1.291 ±0.153、1.131±0.128,여대조조적0.725±0.116、0.722±0.112비교,t치분별위69.25、95.71,P치균＜0.01,차이유통계학의의;RD-5、sPLA2mRNA상대표체량수시간연장축점하강,재72 h분별위0.415±0.104、0.425±0.076,명현저우대조조,t치분별위31.55、44.98,P치균＜0.01,차이유통계학의의.대조조Lysozyme mRNA상대표체량위0.853±0.093,모형조조기역승고,8h상승지고봉위1.211±0.107,수후축점하강,72h위0.704±0.103,명현저우대조조,차이유통계학의의(t=9.224,P＜0.01).Western blot검측대조조sPLA2화Lysozyme적단백상대표체량,분별위0.583±0.121화0.650±0.093,고우모형조72 h적0.327±0.086화0.382±0.057,t치분별위12.28、15.83,P치균＜0.01.면역조직화학염색결과여Western blot검사결과상일치. 결론 ALF대서모형적회장점막면역병장공능하강,수착반씨세포RD-5、sPLA2화Lysozyme mRNA급단백표체하강,장기세균이위솔증가,차불반유명현장점막손상적발생.
    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 and 0.425±0.076) was significantly lower than that of the control group (t =31.55 and 44.98,all P ＜ 0.01).Lysozyme mRNA expression in the model group peaked at 8 hours after ALF induction (1.211±0.107),which was higher than that of the control group at this time point (0.853±0.093),and by 72hours after ALF induction it declined to 0.704±0.103,which was significantly lower than that of the control group (t =9.224; all P =0.009).In addition,at 72 hours after ALF induction the protein expression of both lysozyme and sPLA2 was significantly lower in the model group (0.327±0.086 and 0.382±0.057) than in the control group (0.583±0.121 and 0.650±0.093,t =12.28 and 15.83,P =0.004 and 0.001).Similar results were obtained with immunohistochemical staining.Conclusion The function of the ileal mucosal immune barrier in the rat model of acute liver failure decreased,along with decreases in expression of RD-5,sPLA2 and lysozyme in the Paneth cells.At the same time,the rate of organ bacterial translocation increased without obvious injury to the intestinal mucosa structure.
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