【作者】 丁震；

【导师】 侯晓华；

【作者基本信息】 华中科技大学 ， 内科学， 2007， 博士

【摘要】 第一部分不同胰腺炎模型胰腺病理改变与MCP-1表达的关系目的:验证不同浓度胆酸钠诱导的急性胰腺炎严重程度及胰腺病理评分与MCP-1表达的相关性。方法:分别在胰管内给予浓度为5%和0.5%的胆酸钠诱导急性胰腺炎,测定不同时间段胰腺的病理评分,同时利用免疫组化和RT-PCR两种方法在蛋白及mRNA水平测定MCP-1在胰腺内的表达,并通过相关性分析检测MCP-1表达与胰腺病理改变的相关性。结果:利用5%和0.5%的胆酸钠成功的诱导了SAP和MAP,两者的病理评分有显著性差异(MAP组2.75±0.86,SAP组10.15±1.44,p< 0.001);而免疫组化提示MCP-1在MAP组及SAP组的胰腺中均有明显表达,RT-PCR半定量分析显示SAP组MCP-1胰腺内表达水平明显高于MAP组(MAP组1.37±0.47,SAP组3.55±0.65,p< 0.001),并且MCP-1的表达量与胰腺损伤的严重程度呈正相关(pearson相关系数=0.922,p< 0.001)。结论:0.5%的胆酸钠胰管内注射可以成功的诱导MAP;在急性胰腺炎过程中胰腺MCP-1的表达明显升高,显示MCP-1可能是参与胰腺炎症的重要介质。第二部分急性胰腺炎时血脑屏障通透性的变化规律目的:观察急性胰腺炎时血脑屏障通透性的变化规律。方法:通过不同浓度的胆酸钠构建不同程度的急性胰腺炎模型,并在不同时间点静脉给予伊文兰(Evan’s blue),在生理盐水灌注冲洗后取出大鼠脑组织,匀浆、离心、取上清并用分光光度计读数,通过Evan’s blue的标准曲线得到脑组织中Evan’s blue的含量,而该含量直接反映了BBB的通透性。结果:轻症急性胰腺炎两组的血脑屏障通透性和对照组相比没有明显升高(MAP2h 1.64±0.17 p=0.443; MAP6h 1.69±0.24 p=0.321),而BBB通透性在SAP组中2小时开始升高,持续升高至48小时才有所下降(与对照组相比,SAP2h 1.89±0.12 p=0.013;SAP48h 1.84±0.07 p=0.019;SAP6h 2.66±0.32,SAP12h 2.91±0.29,SAP24h 2.89±0.69, p value均小于0.001)。结论:SAP时存在血脑屏障通透性的升高,并且这种改变呈现一定的时间依赖性。第三部分急性胰腺炎时血脑屏障通透性变化的相关因素分析目的:观察急性胰腺炎时血脑屏障通透性的升高与胰腺病变水平及MCP-1、TLR4表达的关系。方法:通过不同浓度的胆酸钠构建不同程度的急性胰腺炎模型,根据处死时间对大鼠进行分组,通过伊文兰(Evan’s blue)观察不同时间点大鼠BBB的通透性,利用病理评分评估胰腺病理损伤的水平,同时采用免疫组化和RT-PCR的方法在蛋白和RNA水平来测定MCP-1和TLR4的表达,观察其表达水平与BBB通透性的相关性。结果: BBB通透性在轻症急性胰腺炎组没有明显升高,在SAP组中2小时开始升高,持续升高至48小时才有所下降;而重症急性胰腺炎各组的病理评分均较MAP组明显升高(SAP2h=8.50±1.07; SAP6h=9.75±0.71; SAP12h10.25±1.28; SAP24h=11.13±1.25; SAP48h=11.13±1.13)BBB通透性与胰腺病理评分的相关系数为0.626(p<0.01);与此同时,MCP-1和TLR4的表达相一致,均在SAP的各组中有明显表达,而对照组和MAP组表达为阴性,相关性分析提示MCP-1和TLR4的表达水平均与BBB通透性的升高显著相关。(MCP-1 Pearson相关系数: 0.812 p=0.007<0.01;TLR4 Pearson相关系数: 0.208 p<0.05),同时MCP-1的表达与TLR4的表达也相关(相关系数: 0.818 p<0.001)。结论:随着胰腺炎的加重,血脑屏障通透性逐渐升高,而由TLR4导致的MCP-1表达上调可能参与了重症急性胰腺炎时血脑屏障通透性的改变。第四部分急性胰腺炎时CNS的损伤与BBB通透性的关系目的:观察急性胰腺炎时血脑屏障通透性的升高与CNS损伤的关系。方法:在不同的急性胰腺炎模型中利用伊文兰(Evan’s blue)观察不同时间点大鼠BBB的通透性,同时利用利用胶质纤维酸性蛋白(Glial fibillary acidic protein, GFAP)作为神经损伤的标志物,利用免疫组织化学和RP-PCR的方法检测其表达,同时观察其表达水平与BBB通透性的相关性。结果: BBB通透性在轻症急性胰腺炎组没有明显升高,在SAP组中2小时开始升高,持续升高至48小时才有所下降;而重症急性胰腺炎各组的病理评分均较MAP组明显升高;与此同时,GFAP在对照组中呈基础性的低表达,GFAP在SAP2h、SAP6h与SAP12h三组中表达明显上升,12h组时表达最为明显,然而在24h以后的SAP各组中表达量迅速下降至基础水平(对照组1.13±0.33;MAP2h=1.00±0.33; MAP6h=1.10±0.55; SAP2h=1.81±0.27; SAP6h=2.26±0.65; SAP12h 2.49±0.64; SAP24h=1.05±0.64; SAP48h=1.00±0.61);而GFAP的表达水平和血脑屏障通透性之间呈正相关(pearson相关系数=0.383,p=0.007<0.01)。结论:GFAP的一过性表达提示诱导急性胰腺炎后CNS没有出现严重的病理损伤,然而血脑屏障通透性的升高可能是GFAP表达增加的原因。更多还原

【Abstract】 PartⅠThe pathological injury in different acute pancreatitis model and its relationship with pancreatic MCP-1 expressionObjective:To explore the pathological injury after inducing acute pancreatitis by intrapancreatic ductal injection with different concentration choleate sodium and find the relationship between pancreatic damage and MCP-1 expression.Method:After injection of 5% and 0.5%choleate sodium, we evaluated the pancreatic injury by a score system. Then, we may detect MCP-1 expression by immunohistochemistry and RT-PCR and analyze the relationship between MCP-1 expression and pancreatic inflammation.Result:SAP and MAP had been made by intraductal injection with 5% and 0.5% choleate sodium. Pathological score was significantly different between these two groups(MAP group 2.75±0.86,SAP group 10.15±1.44,p< 0.001). In addition, MCP-1 expression could be found in both these two groups in pancreas, but RT-PCR indicated their levels were different: MCP-1 expressed higher in SAP group than that in MAP group (MAP group 1.37±0.47,SAP group 3.55±0.65,p< 0.001). Correlation analyse showed Pearson correlation coefficient was 0.922,p< 0.001.Conclusion:0.5% choleate sodium can induce mild acute pancreatitis simply and cheaply. MCP-1 may be an important cytokine in the pathogenesis of acute pancreatitis. PartⅡThe change of blood brain barrier permeability in acute pancreatitis Objective:To explore the rule of blood brain barrier permeability change after inducing acute pancreatitis.Method : In different models of acute pancreatitis, we injected Evan’s blue intravenously before sacrificing,then we got supernatant fluid after homogenate and centrifuging, then measured its OD value by spectrophotometer. We could get the quantity of Evan’s blue in brain by standard curve which reflect the relationship between the quantity of Evan’s blue and OD’s value.Result:Compared to control group, there was no significant increase of BBB permeability in MAP groups(MAP2h 1.64±0.17 p=0.319; MAP6h 1.69±0.24 p=0.249),However, BBB permeability increased in all SAP groups (SAP2h 1.89±0.12;SAP6h 2.66±0.32;SAP12h 2.91±0.29;SAP242.89±0.69;SAP48h 1.84±0.07,p value are all below 0.05).Conclusion:BBB permeability increase in SAP groups time-dependently.PartⅢThe relationship among pancreatic injury、MCP-1 expression and the change of blood brain barrier permeability in acute pancreatitisObjective:To investigate the relationship among pancreatic injury、MCP-1/TLR4 expression and the change of blood brain barrier permeability in acute pancreatitis.Method:In different models of acute pancreatitis, we used Evan’s blue to detect the BBB permeability. The grade of pancreatic injury was measured by a score system and the expression of MCP-1 and TLR4 were measured by immunohistochemistry and RT-PCR.Result:Compared to control group, there was no significant increase of BBB permeability in MAP groups. However, BBB permeability increase in all SAP groups. In the same time, the pathological score of pancreas were significantly higher in SAP groups than those in MAP groups(SAP2h=8.50±1.07; SAP6h=9.75±0.71; SAP2h=8.50±1.07; SAP6h=9.75±0.71; SAP12h10.25±1.28; SAP24h=11.13±1.25; SAP48h=11.13±1.13). Therefore, the expression of MCP-1 and TLR4 only existed in SAP groups.Conclusion:The increase of BBB permeability is related to the severity of pancreatic inflammation and MCP-1 may contribute to the change of BBB permeability. The activation of TLR4 may be the cause of MCP-1 expression increase.PartⅣThe relationship between blood brain barrier permeability and CNS injury in acute pancreatitisObjective:To investigate the relationship between blood brain barrier permeability and CNS injury in acute pancreatitis.Method:In different models of acute pancreatitis, we used Evan’s blue to detect the BBB permeability. The grade of CNS injury was measured by expression of GFAP which was an indicator of astrocyte activation. The expression of GFAP was measured by immunohistochemistry and RT-PCR.Result:Compared to control group, there was no significant increase of BBB permeability in MAP groups. However, BBB permeability increased in all SAP groups. In the same time, the expression of GFAP was lower in control group and MAP groups(control group 1.13±0.33;MAP2h=1.00±0.33; MAP6h=1.10±0.55). However, in SAP groups, the level of GFAP expression began to increase in 2 hours and reach the peak value in 12h, then it return to normal(*SAP2h=1.81±0.27; *SAP6h=2.26±0.65; *SAP12h 2.49±0.64; SAP24h=1.05±0.64; SAP48h=1.00±0.61, * means p value <0.05). Therefore, the expression of GFAP related to the BBB permeability.Conclusion:GFAP expression increase transiently which suggest there is no severe injury in CNS during acute pancreatitis. However, BBB permeability may be the cause of GFAP expression.更多还原