【作者】 李磊；

【导师】 王兴鹏； 靖大道； 徐敏； 吴恺；

【作者基本信息】 复旦大学 ， 内科学， 2005， 博士

【摘要】 研究目的: 观察实验性急性胰腺炎(AP)对脂多糖(LPS)的耐受性并从白细胞基因表达谱变化角度探讨其可能机制;观察和探讨氧化磷脂(OXPAPC)对急性坏死型胰腺炎(ANP)的治疗作用及其可能机制。 研究方法: (1)310只C57BL/6J小鼠随机分为正常对照组(n=25)、AP+生理盐水(NS)组(n=25)、NS+LPS组(n=130)和AP+LPS组(n=130),后两组均以不同LPS剂量分为7个亚组。AP模型制备采用间隔1hr共7次腹腔内注射雨蛙肽(50μg/kg),于第1次雨蛙肽注射后5 hr腹腔内注射LPS;AP+NS组以NS代替LPS;NS+LPS组以NS代替雨蛙肽。各LPS亚组随机分出10只观察7天死亡率,其余于第1次雨蛙肽或NS注射后12hr处死,分离出血白细胞,留取血清及肝、肺、胰腺等组织,检测血清淀粉酶(AMS)、乳酸脱氢酶(LDH)水平及各脏器病理学改变。应用含12489条小鼠全长基因的寡核苷酸芯片分别检测正常对照组、AP+NS组、NS+LPS(15mg/kg)组(n=25)和AP+LPS(15mg/kg)组(n=25)小鼠血白细胞的基因表达谱并重复3次,筛选AP+NS组/正常对照组及AP+LPS(15mg/kg)缈NS+LPS(15mg/kg)组间的表达差异基因。(2)另88只C57BL/6J小鼠随机被分为ANP模型组(n=44)和OXPAPC治疗组(n=44),ANP模型制备采用腹腔内注射雨蛙肽和LPS(5mg/kg),OXPAPC治疗组于LPS注射前5min给予OXPAPC(25mg/kg)腹腔注射,每组20只观察生存率,其余于第1次雨蛙肽注射后第9、12和24小时分批处死小鼠,留取血清及胰腺组织。88只SD大鼠随机被分为ANP模型组(n=44)更多还原

【Abstract】 ObjectivesTo investigate the tolerance to lipopolysacchride(LPS) on experimental acute pancreatitis(AP) and explore the possible mechanism concerning gene expression profile changes of leucocytes; To investigate the therapeutic effect and mechanism of oxidized phospholipids, oxidized 1-palmitoyl-2-arachidonoyl-sn- glycero-3-phosphorylcholine (OXPAPC) on acute necrotizing pancreatitis(ANP) in rodent.Methods(1) Three hundred and ten C57BL/6J mice were randomly divided into normal control group(n=25), AP+NS group(n=25), NS+LPS group(n =130)and AP+LPS group (n=130) . The two latter groups were respectively subdivided into seven subgroups according to different dose of LPS. AP model was induced by seven times administration of cerulein (50 ug/kg). LPS was given 5 hours after the first celulein injection. LPS was replaced by NS in AP+ NS group. Cerulein was replaced by NS in NS+LPS group. Ten mice were randomly separated from every subgroup to investigate mortality rate for 7 days. Others were sacrificed at 12 hours after the first celulein or NS injection. Leucocytes were separated for gene chip test. Liver, lung, pancreas and blood serum were collected to investigate the pathological changes and serum levels of amylase (AMS) and lactate dehydrogenase (LDH). Gene expression profiles of leucocytes in control group, AP+NS group, NS +LPS (15mg/kg) group (n =25) and AP +LPS (15mg/kg) group (n =25) were studied with oligonucleotide microarrays of 12479 full length mouse genes respectively forthree times. (2) Eighty-eight C57BL/6J mice were randomly divided into two groups, ANP group(n =44) and ANP treated with OXPAPC group(n =44). ANP model was induced by seven times administration of cerulein (50 ug/kg) challenged by LPS(5 mg/kg)intraperitoneal injection. Treatment with OXPAPC (25mg/kg) was started 5 mins before LPS injection. Twenty mice of each group were separated to investigate survival rate. The others were sacrificed at the 9 h^ 12 hrs and 24 hrs after the first injection of cerulein, and the pancreas and blood serum were harvested. Eighty-eight SD rats were randomly divided into two groups, ANP group(n =44) and ANP treated with OXPAPC group(n =44). ANP model was induced by injecting sodium taurocholate(5%) into pancreatic duct. Treatment with OXPAPC (25mg/kg) was administrated at Ohr> 6 hrs after sodium taurocholate injection. Twenty rats of each group were separated to investigate survival rate. The others were sacrificed at 12 hrs, 24 hrs, 48 hrs and 72 hrs after injection of sodium taurocholate, and the pancreas and blood serum were collected. Serum levels of amylase(AMS) and lactate dehydrogenase (LDH) were measured by enzyme dynamics chemistry. Severity of pancreatitis was evaluated by histological scoring system. The activities of myeloperoxidase (MPO) in pancreas were determined by zymohistochemistry. Intrapancreatic TNF-alpha, IL-1P, ICAM-1 and E-selectin mRNA expressions in pancreas were studied by semi-quantitative RT-PCR. Inhibitor of kB kinase p (IKKp), protein38(p38) and c-Jun-N-terminal kinase 1(JNK1) protein were investigated by western blot. The binding activities of nuclear factor kB (NF-kB) and activated protein-1 (AP-1) to DNA were testifid by electrophoretic mobility shift assay (EMSA). Results(1) Mortality rate in both NS+LPS group and AP +LPS group was increased with the increasing of LPS dose. Mortality rate in AP +LPS group was significantly less than that in NS+LPS group with equal LPS dose(P<0.05). Serum level of LDH in AP +LPS group was significantly lower than that in NS+LPS group with equal LPS dose(P<0.05), but serum level of AMS was significantly higher than that in NS+LPS group with equal LPS dose(P<0.05).更多还原