Expression And Signification Of MUC2 And MUC5AC In The Primary Intrahepatic Stones

Objective(s):Intrahepatic bile duct stones are caused by infection,cholestasis,abnormal bile duct anatomy,and gene mutations or expression abnormalities,among which the endogenous and exogenous β-Glucuronidase(β-G1 ucuronidase,β-G)produced by bile duct infections and the mucin secreted by goblet cell and biliary epithelium after metaplasia are considered to be an important factor in the formation of primary intrahepatic stones(PIS).Surgical treatment for type I or localized primary intrahepatic bile duct stones is the best treatment method,but even after surgery,there is still a high recurrence rate of intrahepatic bile duct stones.In this study,we will use Western blot(WB)to detect the expression of MUC5 AC and MUC2 of intrahepatic bile duct epithelial cells in patients with primary intrahepatic bile duct stones and patients without stones,clarify the relationship between primary intrahepatic bile duct stones and mucin MUC5 AC and MUC2,and preliminarily explore the formation and recurrence mechanism of primary intrahepatic bile duct stones,thereby find more effective clinical treatment and prevention methods.Methods:1.Five patients with primary intrahepatic stones who were treated by regular partial hepatectomy in the First Affiliated Hospital of Kunming Medical University between May 2022 and December 2022 were selected,and all the patients with stones are intrahepatic bile duct stones type I according to the Nakayama classification in Japan or the biliary surgery group of the Surgical Credit Association of the Chinese Medical Association.Chose 5 patients who underwent partial hepatectomy due to hepatic hemangioma,FNH or other non-calculous benign liver diseases during the same period.Three sets of bile duct epithelial specimens containing stones were taken from each patient according to the sampling distance.Collected the general information,clinical data,auxiliary examination and pathologic examination results of all surgical patients.There was no statistical difference in age,sex and infection indicators of patients(p<0.05).All patients were diagnosed clearly before operation by relevant test and imaging results,and were further confirmed by surgical treatment.The age distribution of patients is between 26 and 77.2.Strictly follow the principle of sterility during operation.After surgical resection,bile duct specimens were immediately collected from patients with primary intrahepatic bile duct stones.Samples were taken from intrahepatic bile ducts containing stones,and a total length of 0.6cm of bile duct epithelial specimens were peeled from the cutting edge.The specimens were divided into three groups based on their distance from the cutting edge,labeled as the cutting-edge group,0.2cm group(0.2cm from the cutting edge)and 0.4cm group(0.4cm from the cutting edge),all the specimens were placed in a specimen tube and labeled.For non-stone patients,selected the intrahepatic bile duct with the same standard to obtain the samples,and placed them in the sample tubes.The samples were subsequently used for Western blot to analysis the expression of mucin MUC2 and MUC5 AC.After being taken out and placed in a sample tube,the specimens were quickly stored in liquid nitrogen and immediately transported to the laboratory for storage in a-80 ℃ refrigerator.3.Using Western blotting to detect MUC2 and MUC5 AC in intrahepatic bile duct specimens.Compared the expression levels of MUC5 AC and MUC2 in bile duct epithelial cells of patients with and without intrahepatic bile duct stones,and compared the expression levels of MUC5 AC and MUC2 in bile duct epithelial cells of the same patient at different distances from the cutting edge to infer the relationship between primary intrahepatic bile duct epithelium and mucin MUC5 AC and MUC2 to explore the role of mucin MUC5 AC and MUC2 in PIS.Result:1.Western blotting was performed on samples of intrahepatic bile ducts from non-stone patients and stone patients,and detected the expression levels of mucin MUC5 AC and MUC2.The results showed that mucin expression was detected in 30 out of 60 samples,with a positive rate of 50%(30/60).Among them,the positive rate of MUC in patients with stones is 100%(30/30),and the positive rate of MUC in non-stone patients is 20%(6/30).2.In the stone patients’ group,the MUC5 AC expression were found in a total of15 samples,with a positive rate of 100%(15/15).Among non-stone patients,3samples had MUC5 AC expression,with a positive rate of 20%(3/15),indicating significant statistical significance between two groups.3.In the stone patients’ group,the MUC2 expression were found in a total of 9samples,with a positive rate of 60%(9/15).Among non-stone patients,3 samples had MUC2 expression,with a positive rate of 20%(3/15).There was no significant statistical significance between two groups.4.4.In the stone group,the expression level of MUC2 in each sample in showed an overall upward trend as the distance increased,but there was no significant statistical significance.And there was no significant trend and statistical significance in the comparison of expression level of MUC5 AC in each sample.Conclusion(s):1.This study used Western blot to detect the expression levels of mucin MUC5 AC and MUC2 in the intrahepatic bile ducts of patients with primary intrahepatic stones and benign liver diseases,in order to discover the association and speculate the relationship between mucin MUC5 AC and MUC2 and intrahepatic bile duct stones.It was also the first attempt to segment the intrahepatic bile duct to obtain more samples and more accurate data.The experimental method is mature and stable,and with strong repeatability.2.The results revealed a significant statistical difference in the expression of MUC5 AC between stone patients and non-stone patients,while there was no significant statistical difference in the expression of MUC2 between stone patients and non-stone patients.This suggests that MUC5 AC may be one of the main mucins leading to bile stasis and the formation of PIS,and MUC2 may not be the main mucin leading to the formation of PIS.3.All patients with stones who detected MUC expression had mucin expression in their incisal margin group,indicating that there may still be high expression of MUC in the residual bile ducts of patients after surgery,which may be one of the reasons for stone recurrence.