| Intrahepatic cholestasis caused by various liver diseases is an important pathological change, which could promote the liver damage and finally lead to hepatic cirrhosis and liver function failure. Patients usually develop end stage liver disease and present clinical therapies often fail to control it.Composed of transporters, the hepatocyte canalicular surface is characterized by transport activities that carry on the bile secretion and named as the hepatocyte third reaction. Those transporters are ATP dependent proteins involved in the bile excretion of endogenous and exogenous metabolites. Resent studies found that the disorders of bile excretion on hepatocyte canalicular membrane could result in intrahepatic cholestasis. So the molecules associated with bile excretion have been paid attention to in this field. At past decades, some transporters on hepatocyte canalicular membrane were found and etiopathogenisis of some intrahepatic cholestasis diseases was clear, which could be helpful to diagnosis and treatment of these diseases. However, it still could not resolve all problems of intrahepatic cholestasis diseases. Therefore, establishing functional canalicular membrane transport model and finding more molecules associated with transport on hepatocyte canalicular membrane would benefit to further ascertain the hepatic complicate physiological function and etiopathogenisis of intrahepatic cholestasis diseases. Objectives To isolate hepatocyte canalicular membrane vesicles and establish in vitro functional canalicular membrane transport model. To screen and identify the molecules associated with transport on rat hepatocyte canalicular membrane.Materials and methods1. Isolation and identification of rat hepatocyte canalicular membrane vesicles(1) Rat hepatocyte canalicular membrane vesicles (CMV) and sinusoidal membrane vesicles (SMV) were isolated by density gradient centrifugation with percoll and sucrose; (2) CMV and SMV were characterized by one-dimensional gel electrophoresis, specific marker enzyme assays and transmission electron microscopy; (3) With transport of [H3]E217βG in the present ATP, the transport activity of CMV were observed.2. Production and screening of monoclonal antibodies associated with transport on hepatocyte canalicular membrane(1) Using CMV as the immunogen, the preparation of monocolonal antibodies was performed; (2) Monoclonal antibodies associated with transport on hepatocyte canalicular membrane were screened by ELISA, immunohistochemistry and in vitro transport experiment; (3) The production of monocolonal antibodies was prepared by ascites and purified with saturated sulfate ammonium; (4)The isotype of the monoclonal antibody was identified with antibody isotyping kit; (5) The protein band recognized by specific antibodies were analyzed by western blot. 3. Identification of antigen recognized by monoclonal antibodies associated with transport on hepatocyte canalicular membrane(1) The physical characters of antigens recognized by monoclonal antibodies were further analyzed by 2D electrophoresis; (2) Using immunoprecipitation and Western blot, the protein band recognized by monoclonal antibody was obtained; (3) After that, the protein band was identified and analyzed with peptide finger print and database software.Results1. Isolation and character of rat hepatocyte canalicular membrane vesiclesCMV and SMV were successfully isolated. Separated with the SDS-PAGE, the protein bands of CMV or SMV were significantly less than those of hepatocyte homogenate. Meanwhile, many differential protein bands were visualized between CMV and SMV. Their characters were further identified with the morphological and chemical analysis. The transmission electron micrographs of CMV and SMV indicated that vesicles had a similar appearance, displaying the membrane bilayer structure . Alkaline phosphatase and Na+/K+-ATPase were commonly used as maker for CMV and SMV respectively. CMV and SMV were enriched appropriately in the respective canalicular and sinusoidal markers by specific enzyme assay. In the present ATP, CMV could transport [H3]E217βG that is a transport substrate of MRP2.2. Production and screening of monoclonal antibodies associated with transport on hepatocyte canalicular membraneUsing CMV as the immunogen, we performed the preparation of monocolonal antibody. Screened by comparative ELISA, 46 stains of monoclonal antibodies whose expressions on hepatocyte canalicular membrane were more than those on sinusoidal membrane were obtained. Immunohistochemistry showed that a strain of monoclonal antibody named as cm1 was distributed in not only hepatocyte canalicular but also cholangiocell apical membrane. Immunoglobulin subclass determination showed that the isotype of antibody cm1 was confirmed to be IgG2a/κtype. Immunoblotting indicated that the protein band with approximately mass molecular 140kDa was recognized exclusively by cm1 antibody and the expression of isolated CMV was significantly stronger than it of SMV. In addition, two strains of monoclonal antibodies specifically distributed in liver tissue. One displayed a striking heterogeneity among rat liver parenchymal cells and was named as cv1. In intact liver, cv1 was found to localize exclusively within a very small population of the parenchymal cells surrounding the terminal hepatic venules. Interestingly, although in human and rat liver tissue the same protein bands with approximately mass molecular 25kDa were recognized, cv1 localized to the human hepatocyte sinusoidal membrane and certain non-parenchymal cell type. The other distributed specifically to rat hepatocyte nucelus membrane and was named as nm1. Immunoblotting indicated that the protein band with approximately mass molecular 50kDa was recognized exclusively by nm1 antibody. The function screening of these monoclonal antibodies by in vitro transport experiment has been performed now.3. Identification of antigen recognized by monoclonal antibodies associated with transport on hepatocyte canalicular membraneThe antigens of antibody cm1, cv1 and nm1 were first identified. Following immunoprecipitation, further peptide finger print and database software analysis indicated that cm1 might be a novel maker of rat hepatocyte canalicular membrane. More information on physical property of cv1 was obtained. The isoelectric point of cv1 was found to be about 3-4. Following 2D electrophoresis and immunoblotting, further peptide finger print and database software analysis indicated the molecule recognized by nm1 was ATP synthase subunit beta (ATPB).ConclusionsRat hepatocyte canalicular membrane vesicles were successfully isolated by density gradient centrifugation and the hepatocyte in vitro transport experiment model was established, which were firstly performed in our country. It was helpful to further identify the function of known transporters and find the novel transporters. Meanwhile, it also established the bases for the molecular mechanism of bile form and etiopathogenisis of intrahepatic cholestasis diseases. Using CMV as the immunogen, we performed the preparation of monocolonal antibody. Screened by comparative ELISA, 46 stains of monoclonal antibodies whose expressions on hepatocyte canalicular membrane were more than those on sinusoidal membrane were obtained. Of these antibodies, cm1 distributing to hepatocyte canalicular membrane was obtained and identified to probably recognize a novel molecule. It could play an important role on the bile secretion of canalicular membrane. In addition, two strains of monoclonal antibodies that specifically distributed in liver tissue were obtained. Cv1 displayed a striking heterogeneity among rat liver parenchymal cells. In intact liver, cv1 was found to localize exclusively within a very small population of the parenchymal cells surrounding the terminal hepatic venules. Interestingly, cv1 localized to the human hepatocyte sinusoidal membrane. Nm1 distributed specifically to rat hepatocyte nucelus membrane and the molecule recognized by nm1 was identified to be ATP synthase subunit beta.
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