【作者】 杨林；

【导师】 蔡端；

【作者基本信息】 复旦大学 ， 外科学， 2011， 博士

【摘要】 第一部分骨桥蛋白在不同体外模拟胆汁体系中成核作用的综合研究目的探讨骨桥蛋白(OPN)在Small模拟胆汁和综合模拟胆汁中的成核效应及其可能的相关机制。方法制备过饱和Small模拟胆汁和综合模拟胆汁,分别用成核时间实验、胆固醇结晶生长实验探讨OPN在体外模拟胆汁中的成核效应。制备Small模拟胆汁通过电镜负染技术研究OPN对胆汁中胆固醇-磷脂泡融合聚集过程的动态影响。结果在Small模拟胆汁和综合模拟胆汁中OPN可显著延缓胆固醇结晶的成核时间,且其抑成核效应表现为剂量依赖性。钙离子表现为促成核效应,其效应被OPN抑制。OPN既可增加胆固醇结晶的成核时间指数,又可降低胆固醇结晶的生长指数,而钙离子仅降低胆固醇结晶的成核时间指数,不影响胆固醇结晶生长。电镜研究提示OPN使模拟胆汁中的胆汁泡结构更加稳定,并抑制胆汁泡的融合、聚集和进展。结论OPN在模拟胆汁中具有抑成核活性,其效应和钙离子相关,OPN可能通过抑制胆固醇结晶成核、结晶生长及稳定胆汁泡等作用起到抑制胆囊胆固醇结石形成的作用。第二部分OPN在人胆囊胆汁中的表达及成核作用研究目的研究OPN在人胆囊胆汁中的成核作用,探讨OPN及脂质成分在结石胆汁和对照胆汁中的分布差异。方法利用Holan成核时间实验研究OPN在胆固醇结石患者胆囊胆汁和对照组胆囊胆汁中的成核作用；超速离心技术制备均相胆汁,磷钨酸负染/电镜观察研究OPN对胆囊胆汁中胆汁泡的影响；ELISA检测OPN及胆汁脂质分子在胆汁中的分布差别。结果在结石患者胆囊胆汁和对照组胆汁中OPN均可使胆固醇成核时间显著延长,并呈剂量依赖性,其抑成核效应表现在生理浓度区。钙离子可使成核时间显著缩短,促进成核,但其促成核效应被OPN抑制。通过电镜扫描观察发现OPN可抑制人胆囊胆汁中胆汁泡的出现、融合聚集过程,从而抑制胆固醇结晶析出、沉淀。结石患者胆囊胆汁中OPN和钙离子低于对照组胆汁中含量(P<0.05),而胆固醇、磷脂、胆酸和CSI则高于对照组胆汁(P<0.05)。结论OPN在人胆囊胆汁具有同模拟胆汁相似的抑成核效应,OPN可能通过抑制胆固醇结晶的出现、胆汁泡进展等步骤影响结石形成,为进一步研究其具体机制提供了基础。第三部分OPN及其受体在胆囊胆固醇结石形成中表达机制的初步研究目的研究OPN及受体integriαv、CD44在胆固醇结石胆囊组织中的表达及作用,检测胆汁及外周血中OPN含量变化的一致性。方法免疫组织化学技术验证OPN、integrinαv和CD44在结石胆囊组织(分两组,结石组Ⅰ：胆囊组织相对正常,无胆囊组织纤维化；病变组Ⅱ：胆囊组织内皮有剥落、破坏,胆囊壁有纤维化改变)和正常对照胆囊组织中的差异表达。RT-PCR及Western Blot验证其在mRNA和蛋白质水平的变化。ELISA检测外周血OPN表达。结果免疫组织化学结果显示OPN及其受体integrinαv、CD44的免疫染色在病变组Ⅰ较对照组增高；而在病变组Ⅱ,OPN及integrinαv、CD44的免疫染色明显降低。RT-PCR及Western Blot结果表明OPN及受体integrinαv、CD44在mRNA水平和蛋白质水平的表达与免疫组织化学染色结果一致。ELISA检测发现结石患者外周血中OPN含量低于对照组,与胆汁中OPN含量变化具有一致性。结论免疫组织化学和RT-PCR、Western Blot对OPN及及integrinαv、CD44的检测结果表明OPN在胆囊胆固醇结石过程中是动态变化的,其作用可能是通过与integrinαv、CD44的结合来实现的。OPN可能作为一个潜在的外周血标志因子。第四部分OPN在胆囊胆固醇结石形成中的动态机制研究目的探讨OPN在胆囊胆固醇结石形成中的变化机制,及其他促／抑成核因子在胆汁和血液中的动态改变。方法RT-PCR检测胆囊组织和肝脏组织中OPN mRNA在豚鼠胆囊胆固醇结石形成过程中的动态改变,ELISA检测OPN及其他相关因子在胆汁和血液中的变化。结果成功建立豚鼠胆囊胆固醇结石动物模型,与对照组相比,OPN mRNA在豚鼠胆囊组织和肝脏组织中的表达逐渐增高,至第6周到高峰,随后逐渐下降。而ELISA发现OPN在胆汁中表达也是逐渐增高,第6周达高峰,随后逐渐下降,呈峰型分布,其在血液中的变化与胆汁中表现一致,但OPN峰值改变早于胆汁。抑成核因子载脂蛋白AI在胆汁和血液中的变化与OPN类似。而促成核因子粘蛋白和α1-酸性糖蛋白在胆汁和血液中逐渐升高,并一直维持高水平而不下降。结论肝脏和胆囊的功能参与调节OPN在胆囊胆固醇结石形成中的作用,OPN等抑成核因子的动态变化和促成核因子不同,提示胆汁中促/抑成核平衡的变化在胆结石形成中起重要作用。本课题通过体外胆汁的成核相关实验技术表明OPN具有抑制胆囊胆固醇结石形成的作用,其抑成核效应和钙离子相关,并呈剂量依赖性。通过人体标本试验表明OPN在人胆囊胆固醇结石中同样具有抑成核效应,OPN在结石患者中作用可能是通过与其受体结合实现的。并进一步通过动物模型试验揭示OPN在胆囊胆固醇结石形成过程中的动态变化机制及其他相关成核相关因子的变化机制。初步探讨了促成核因子和抑成核因子在结石胆汁中的变化作用,为进一步阐明促/抑成核平衡在胆结石形成中的机制提供了一定基础。更多还原

【Abstract】 Part one The synthetic study of the nucleation role of osteopontin in different model bile systems in vitroObjective To investigate the nucleation role and the related mechanisms of osteopontin (OPN) in Small and Synthetic model bile systems in vitro. Methods The Small and Synthetic model bile systems have been prepared according the previously described methods, then the nucleation time assay and cholesterol crystal growth assay have been used to determine the nucleation role of the osteopontin in vitro. The effect of OPN on cholesterol-phosphalipids vesicles have been studied by the negative staining under the electron microscopy. Results OPN prolonged the nucleation time in Small and Synthetic model bile in vitro, this anti-nucleation role showed the dose dependent manner. While the calcium showed the pro-nucleation role in model bile, but this role was inhibited by OPN. Cholesterol crystal growth was inhibited by OPN in a dose dependent manner, but not affected by calcium. Furthermore, the formation, aggregation and fusion of vesicles were suppressed by OPN in model bile as demonstrated by transmission electron microscopy, and the vesicles were stabilized by OPN. Conclusions OPN played the anti-nucleation role in model bile systems and this role correlated with the calcium. These findings indicated OPN could inhibit the cholesterol gallstone formation by inhibiting cholesterol crystal nucleation, growth and stabilizing vesicles in model bile.Part two The expression and nucleation role of OPN in human gallbladder bileObjective To study the nucleation role of OPN in human gallbladder bile, and the concentrations of OPN and biliary lipids in lithogenic bile and control bile. Methods Nucleation role was determined in lithogenic bile and control bile in vitro. Effect of OPN on vesicles of bile was investigated via transmission electron microscopy. OPN and biliary lipids compositions in both gallbladder biles were determined via commercial kits. Results OPN prolonged nucleation time both in lithogenic gallbladder bile and control bile in vitro, this anti-nucleation role showed the dose dependent manner and played the effect at the physical concentration level. Calcium ions shortened nucleation time, but this effect was inhibited by OPN. Furthermore, the formation, aggregation and fusion of cholesterol-phospholipid vesicles were inhibited by OPN in human bile. The concentration of cholesterol, phospholipid, bile acids, and CSI were higher in lithogenic gallbladder bile than in normal gallbladder bile (P＜0.05); the contents of OPN and calcium, nevertheless, were found to be lower in lithogenic bile than those in normal controls. Conclusions OPN played the similar anti-nucleation role in human gallbladder bile as in model bile, which might inhibited the onset of the cholesterol crystal and the dynamic change of vesicles in human bile. These data might be a basis for further investigating the exact mechanism of OPN in the formation of cholesterol gallstone.Part three Preliminary research of the expression menchanism of OPN and its receptors in cholesterol gallstone formationObjective To investigate the expression of OPN and its receptors (integrin av, CD44) in cholesterol gallstone formation, and determine the OPN content in peripheral blood. Methods The immunostaining, mRNA and protein of OPN, integrin av and CD44 were analyzed in human lithogenic gallbladder tissue (case group I, with normal epithelia; case groupⅡ, with degenerated epithelia and fibrosis) and normal tissue. OPN content was detected by the ELISA kits. Results The immunostaining for OPN, integrin av and CD44 in human gallbladder tissues showed a higher reactivity in case groupⅠthan the control group, and in case group II, the immunostaining showed a lower reactivity. The mRNA and protein level of OPN, integrin av and CD44 in gallstone gallbladder tissue showed the similar results with the immunostaining assay in human gallbladder tissue. OPN content in blood showed the similar trend with that in gallbladder bile. Conclusions These data demonstrated that OPN, integrin av and CD44 were involved in gallstone formation as the dynamic change, the effect of OPN might be regulated by its receptors, OPN might be a potential blood marker molecule in gallstone formation.Part Four Preliminary investigation of the dynamic changes of OPN in cholesterol gallstone formationObjective To examine the dynamic expression of OPN in cholesterol gallstone formation, and the contents of OPN and other factors in gallbladder bile and blood. Methods Expression of OPN mRNA were detected by RT-PCR in gallbladder and hepatic tissues of cholesterol gallstone formation in guinea pig, contents of OPN and other factors in bile and blood were detected by ELISA kits. Results The lithogenic model have been established successfully in guinea pig. OPN mRNA expression in gallbladder and hepatic tissues were gradually increased and reached to the peak at the sixth week in lithogenic diet fed group, then decreased after six week. The contents of OPN in gallbladder bile and blood showed the similar results, but the peak value of OPN in blood was examined early than that in bile. The other anti-nucleation factor, apoprotein AI showed the similar results with OPN. However, pro-nucleation factors, mucin and AAP showed the increased value in bile and blood, which did not show the descended curve. Conclusions Function of gallbladder and liver might be involved in the anti-nucleation role of OPN, the differential changes of pro-nucleation and anti-nucleation factors in bile might played the important role in cholesterol gallstone formation.In conclusion, the results suggested that OPN played an anti-nucleation role in cholesterol gallstone formation in vitro as the nucleation assays in model bile, and the role of OPN correlated with calcium and showed the dose dependent manner. Then it is found that the OPN showed the similar anti-nucleation role in human bile, and the role of OPN might be regulated by its receptors. Furthermore, the dynamic mechanism of OPN in the process of gallstone formation was determined via the guinea pig lithogenic model. We detected the dynamic role of the pro-nucleation and anti-nucleation factors in gallstone formation, and the possible mechanism of the nucleation equilibrium in cholesterol gallstone formation.更多还原