The Investigation Of Nucleation Role And Related Mechanisms Of Osteopontin In Cholesterol Gallstone Formation

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 group?, 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 group?than 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.