清胰颗粒对胰性脑病大鼠血脑屏障和脑组织AQP-4影响的研究

发布时间：2018-03-05 11:34

  本文选题：胰性脑病　切入点：脑损伤　出处：《广东药学院》2014年硕士论文　论文类型：学位论文

【摘要】：【研究背景】胰性脑病（pancreatic encephalopathy, PE）是重症急性胰腺炎（Severeacute pancreatitis, SAP）早期并发症之一，是SAP病人出现中枢神经系统（centralnervous system, CNS）症状，其发病率为18.2%，预后差，病死率高达67%，其主要临床表现为定向力障碍、抑郁、意识模糊、烦躁、反应迟钝、表情淡漠等CNS症状。临床上中药制剂清胰汤用于治疗SAP，使得SAP病人的症状明显缓解，病程缩短，且减少并发症。清胰颗粒（qingyi grains, QYG）即清胰汤方剂的固化制剂，进一步探讨QYG对SAP中脑损伤的作用机制及疗效，有利于进一步探索PE发病机制和研发QYG。 【目的】建立PE大鼠模型，探讨在PE大鼠血脑屏障（blood brain barrier, BBB）通透性变化和脑组织中水通道蛋白-4（Aquaporin-4, AQP-4）的表达情况及相互关系，探讨QYG治疗后PE大鼠血脑屏障通透性变化和脑组织中水通道蛋白-4表达的影响，并探讨QYG治疗胰性脑病的相关保护机制，以其为QYG的临床治疗PE提供实验依据。 【方法】90只健康成年的SD(Sprague-Dawley）大鼠，雌雄各半，随机分成3组：假手术组(sham-operation, SO)组（30只）；PE组（30只）；QYG治疗组(30只)。分别按12、24、48小时时间点处死老鼠10只。SO组逆行胰胆管行0.9%NaCl注射；PE组逆行胰胆管注射5%的牛黄胆酸钠（sodium taurocholate, STC）(0.1ml/100g)建立PE模型组；QYG组建模后2H，QYG溶解以后灌胃，以后12h/次给药，其它组则用0.9%NaCl代替QYG。（1）HE染色观察胰腺病理变化；（2）伊文思蓝测定血脑屏障通透性；（3）ELISA测定脑组织中TNF-α和AQP-4含量；（4）用免疫组化法来检测脑组织中AQP-4蛋白表达情况；（5）定量逆转录聚合酶反应（reverse transcription PCR, RT-PCR）测定脑组织中的AQP-4-mRNA表达的情况。 【结果】（1）PE组胰腺出现大量炎症细胞浸润，可观察到钙化灶，不同程度的出血、坏死，部分细胞出现核溶解或者消失，残留的腺泡结构出现肿胀，小叶间质水肿，细胞极性消失；QYG组可观察到腺泡结构轻度肿胀，散发少量的充血及坏死，病变程度较PE组减轻，与PE组相比病理评分显著降低（P0.05），PE组、QYG组与SO组相比均有病理评分均显著升高（P0.05）。（2）QYG可能具有保护PE模型大鼠胰腺的作用。（3）PE组中BBB通透性从12h起开始升高，于24h达到峰值，，48h仍处于较高水平。与SO组相比，PE组、QYG组BBB通透性显著升高（P0.05）；与PE组相比，QYG组BBB通透性显著降低（P0.05）。（4）PE组和QYG组中脑组织TNF-α水平均高于SO组，PE组升高更明显，在PE组中呈现时间递增的关系，而QYG治疗后TNF-α水平显著降低（P0.05），QYG疗效呈现时间依赖性。（5）大鼠脑组织中各组AQP-4表达水平不同，PE组AQP-412h开始升高，到48h达到峰值，QYG治疗后脑组织AQP-4蛋白表达显著降低，ELISA、免疫组化和QPCR结果变化趋势相似。（6）经统计相关分析脑组织中TNF-α与AQP-4存在正相关（r=0.932,P0.05），而BBB通透性与AQP-4呈现正相关（r=0.757,P0.05）。BBB通透性与脑组织中TNF-α呈现正相关（r=0.804,P0.05） 【结论】（1）大鼠PE模型建立成功，PE模型组中BBB通透显著升高，至24h时达到顶峰，QYG治疗后BBB通透性水平显著降低。（2）QYG可能具有保护PE模型胰腺的作用。（3）PE模型组中脑组织的肿瘤坏死因子-α表达显著升高，QYG治疗后TNF-α水平显著下降，说明QYG具有抗炎作用。（4）PE模型组中脑组织AQP-4蛋白表达显著升高，而QYG治疗后AQP-4表达水平下降，说明QYG对AQP-4介导的脑损伤具有保护作用。（5）PE模型组、QYG治疗组中脑组织中TNF-α、AQP-4水平呈现逐渐升高的趋势，与BBB通透性具有显著相关，且存在正相关，说明TNF-α、AQP-4的升高可能导致BBB通透性增加，致使脑组织损伤。
[Abstract]:[background] pancreatic encephalopathy (pancreatic encephalopathy PE) is a severe acute pancreatitis (Severeacute pancreatitis SAP) is one of the early complications, SAP patients with central nervous system (centralnervous system CNS) symptoms, the incidence rate was 18.2%, poor prognosis and high fatality rate of 67%, the main clinical manifestations of orientation disorder, depression, confusion, irritability, unresponsive, indifferent expression and clinical symptoms of CNS. Traditional Chinese medicine Qingyi Decoction for the treatment of SAP, the SAP patient's symptoms, shorten the course of the disease, and reduce complications. Qingyi granule (Qingyi grains, QYG) - curable formulation of Qingyi Decoction prescription, further study the mechanism of QYG SAP brain injury and curative effect, help to further explore the pathogenesis and development of QYG. PE
[Objective] to establish a rat model of PE in PE rat blood brain barrier (blood brain, barrier, BBB) and the permeability of brain water channel protein -4 (Aquaporin-4, AQP-4) and the expression of the relationship, to explore the effects of QYG after treatment of water channel protein PE in rat blood brain barrier permeability and brain tissue changes the expression of -4, and to investigate the protective mechanism of QYG treatment of pancreatic encephalopathy, to provide experimental evidence for the clinical treatment of the PE QYG.
[Methods] 90 healthy adult SD rats (Sprague-Dawley), male and female, were randomly divided into 3 groups: sham operation group (sham-operation, SO) group (30 rats); group PE (30); QYG group (30). According to the time point of 12,24,48 mice were killed 10 hours in.SO group retrograde pancreatic duct injection of 0.9%NaCl; group PE by retrograde pancreatic duct injection of 5% sodium taurocholate (sodium taurocholate, STC) (0.1ml/100g) to establish PE model group; QYG after the establishment of model 2H, QYG dissolved after gavage, after 12h/ administration, the other group was replaced by 0.9%NaCl QYG. (1) to observe the pancreatic pathology the changes of HE staining; (2) blood brain barrier permeability by Evans blue; (3) determination of TNF- alpha and AQP-4 content in brain tissue of ELISA; (4) to detect the expression of AQP-4 protein in brain tissue by immunohistochemical method; (5) quantitative reverse transcription polymerase chain reaction (reverse transcription PCR, RT-PCR) in brain tissue in AQP- The expression of 4-mRNA.
銆愮粨鏋溿

本文编号：1570106