UDP-GT And β-G MRNA Expression In Hepatocytes Of Patients With Primary Intrahepatic Stone And Animal Experiment

Primary intrahepatic stone (PIS) is a multi-cause disease. Increased concentration of unconjugated bilirubin in bile might be responsible for the initiation of PIS. It has been reported that abnormal glucuronide of bilirubin (UCB) can lead to higher proportion of UCB in bile and result in the PIS formation. β-G and UDP-GT are well-known key factors in maintaining the bilirubin(CB) metabolic balance. To detect the reason of the enzyme changes, the mRNA expressions of UDP-GT and β-G were studied.Materials and Methods1. Sample collection. Thirty-six patients, 20 male and 16 female, who were diagnosed as PIS, involved in this study. Among these patients, HBsAg was positive in 4 patients and one had hepatic cirrhosis. Seven patients whose liver functions were normal, suffered from other diseases, four gallbladder polyps and three gastric cancer, and worked as controls. All of them were biopsied with liver tissue and bile during operation. Each bile sample was divided into two tubes, one for bacteria culture and another was stored in liquid nitrogen.2. Evaluation of UDP-GT activity(1) Standard curve. 0.1mM diphenylamine hydrochloride 0.0ul, 5.0ul, 10ul, 20ul added water to 300ul in total value, then added into sodium nitrite to develop color. The standard curve was drawn based on the OD values detected at 540 run.(2) UDP-GT activities. Biopsied liver tissues were homogenized with 20% sucrose and centrifuged. Reaction solution was prepared as follow: 0.1 ml supernatant of liver tissue homogenate, 0.1ml UDP-glucuronitric acid and 0.1ml diamino phinol dihydrochloride were mixed and incubated for two hours at 37℃. Then sodium nitrite was...