【作者】 夏时海；

【导师】 房殿春；

【作者基本信息】 第三军医大学 ， 内科学， 2008， 博士

【摘要】 目的探讨血小板活化因子受体(PAF-R)拮抗剂BN52021在大鼠重症急性胰腺炎(SAP)早期病程中的作用和机制。方法1.用免疫组织化学染色方法研究PAF-R在Wistar大鼠胰腺的定位和分布。2.用胰胆管插入逆行注射牛磺胆酸钠制备SAP大鼠模型;用药代动力学方法研究BN52021治疗大鼠SAP的最佳量效关系。3.用RT-PCR和Western blot方法分别研究PAF-R和核因子κB单体(NF-κBp65) mRNA和蛋白质在SAP大鼠胰腺组织的动态变化以及BN52021的影响。结果1.冰冻切片中显示在Wistar大鼠正常胰腺组织微血管内皮细胞有PAF-R表达;石蜡切片中显示胰岛细胞有PAF-R表达,主要分布于细胞浆和细胞核,核膜着色较深,但是血管内皮细胞着色较浅。2.血清淀粉酶、PLA2、腹水、胰腺组织病理学和胰腺组织PAF-R mRNA表达的结果显示在2.5 mg/kg、5.0 mg/kg、10.0 mg/kg BN52021三个剂量中,5.0mg/kg BN52021静脉注射给Wistar大鼠治疗SAP具有最佳的量效关系(P<0.05);这些结果也显示SAP模型制备是成功的。3.PAF-R mRNA结果显示,SAP模型(SAP组)和BN52021治疗后(BN组)均是动态变化的,早期逐渐上升,在3h达到高峰,以后逐渐降低,除1h和2h外其余各时相点与假手术动物(SO组)相比有显著性差异(P<0.05),除6h外BN组与SAP组各时相点之间无显著性差异;PAF-R蛋白质结果显示,SAP组和BN组PAF-R蛋白质与PAF-R mRNA的变化趋势基本是一致的,先升后降,除1h外其余各时相点与SO组有显著性差异(P<0.05),BN组与SAP组各时相点之间无显著性差异。4.NF-κBp65mRNA结果显示,SAP组和BN组均是动态变化的,呈双峰改变,SAP组双峰顶分别在1h和24h,BN组双峰顶分别在1h和12h,于6h降至最低,SAP组在2h、3h、12h和24h较SO组显著增加(P<0.05),BN组在1h较SAP组显著增加(P<0.05),BN组各时相点较SO组显著升高(P<0.05);NF-?Bp65蛋白质结果显示,SAP组在1h,3h和6h较SO组显著升高(P<0.01),在2h,12h和24h较SO组也显著升高(P<0.05),BN组各时相点较SO组显著升高(P<0.05),在1h,3h和6h较SAP组显著降低(P<0.05)。结论1.研究发现PAF-R不仅定位分布于Wistar大鼠胰腺微血管内皮细胞,也定位分布于其胰腺胰岛细胞胞浆和细胞核内。2.经胰胆管插入逆行注射牛磺胆酸钠制备SAP大鼠模型是可靠的;用5.0mg/kg BN52021静脉注射治疗Wistar大鼠SAP具有最佳的量效关系。3.PAF-R在SAP大鼠胰腺组织表达是动态变化的,PAF-R可能参与了SAP的发病机制,对SAP病情的发生发展起一定的作用;BN52021可降低SAP血清淀粉酶和改善其病理变化,但对胰腺组织PAF-R的表达无显著影响。分析认为,BN52021治疗SAP大鼠并不是通过调控胰腺组织PAF-R的表达量,而是通过竞争性抑制上升的PAF与同时表达量上升的PAF-R结合,从而影响PAF-R的信号转导,实现它的治疗作用。4.NF-kB是PAF-R信号转导通路下游的关键成分。NF-kBp65在SAP大鼠胰腺组织表达是动态变化的,NF-kBp65可能参与了SAP的发病机制,对SAP病情的发生发展起一定的作用;BN52021在早期可降低NF-κBp65蛋白质的表达,从而发挥一定的治疗作用。更多还原

【Abstract】 AIMThis research is to investigate the role and mechanism of platelet activating factor receptor (PAF-R) antagonist (BN52021, Ginkgolide B) in early stage of severe acute pancreatitis (SAP) in rats.METHOD1.The method of immunity hisochemistry staining was employed to study the location and distribution of PAF-R in normal pancreas tissues of Wistar rats.2.The sodium aurocholatet was injected through pancreaticobiliary duct in retrograde direction to establish SAP model and the method of pharmacokinetics was used to explore the optimal dosage of BN52021 on rats with SAP.3.The methods of PT-PCR and Western blot were adopted respectively to investigate the changes of PAF-R and NF-κBp65 mRNA and proteins expression in pancreatic tissues of rats and the effect of BN52021.RESULTS1.In frozen section PAF-R located in pancreatic microvascular endothelial cells. In paraffin section PAF-R located in Pancreas islet cytoplasm and cell nucleus; there was little colouration in pancreatic microvascular endothelial cells.2.The results of the serum amylase, PLA2, ascites, pathologic score and PAF-RmRNA expression showed that, compared with 2.5 mg.kg-1 and 10.0 mg.kg-1, 5.0 mg.kg-1 of BN52021 by vein injection was the optimal dosage in treating rats with SAP(P<0.05). The results also showed that SAP model was successful.3.PAF-mRNA showed dynamic changes in SAP and BN groups. It increased gradually in early stage, reached a peak at 3h, and decreased gradually later. There were significant differences between PAF-mRNA in SAP and BN groups and that in SO groups at each time point except at 1h and 2h (P<0.05), whereas there was no significant difference between PAF-mRNA in BN groups and that in SAP groups at each time point except at 6h. The changes of PAF-R protein in SAP and BN groups shared the similar pattern with those of PAF-R mRNA. There were significant differences between PAF-R protein in SAP and BN groups and that in SO groups at each time point except at 1h(P<0.05), whereas there was no significant difference between PAF-R protein in BN groups and that in SAP groups.4.The expression of NF-κBp65 mRNA dynamically changed in both SAP and BN groups. The mRNA level was higher in SAP groups than that in SO groups at 2h, 3h, 12h, and 24h after operation (P<0.05). The mRNA level was higher in BN groups than that in SO groups at each time point (P<0.05), and also higher than that in SAP groups at 1 h (P<0.05). The NF-κBp65 protein level was higher in SAP groups than that in SO groups at 1h, 3h, and 6h (P<0.01), and 2h, 12h, and 24h (P<0.05). The NF-κBp65 protein level was higher in BN groups than that in SO groups at all time points (P<0.05), and lower in BN groups than that in SAP groups at 1h, 3h, and 6h (P<0.05).CONCLUSIONS1. It is found that PAF-R is distributed not only in pancreatic microvascular endothelial cells but also in pancreatic islet cells of Wistar rats.2. It is found that 5.0 mg.kg-1 of BN52021 by vein injection is the optimal dosage in treating rats with SAP.3. PAF-R is found to play an important role in occurrence and development of SAP. BN52021 exerts biological effects through the combination the increase of PAF expression achieved through competitive inhibition and the simultaneous increase of PAF-R expression rather than through regulating PAF-R expression in pancreatic tissues.4. The NF-κB is a key factor in the lower reaches of signal transduction pathways of PAF-R. The expression of NF-κBp65 in pancreatic tissues dynamically changed and the changing might have played a role in pathogenesis of SAP. BN52021 exerted therapeutic effect by reducing the expression level of NF-κBp65 protein in the early stage of SAP.更多还原