Identification 1F9 Antigen On Hepatocyte Canalicular Membrane And Observation Its Pathophysiological Value In PBC

【Background】Primary biliary cirrhosis (PBC) is an intrahepatic cholestatic liver disease characterized by the injury of intrahepatic bile ducts that may eventually lead to liver fibrosis and cirrhosis. The annual incidence of this disease increases and can amount to 940 per million when people know this disease and detect autobodies more and more. It has seriously affected the people's health as one of non-viral liver diseases.Genetic factors and environmental factors have an impact on PBC pathogenesis and the pathogenesis of PBC is not completely explained. Most patients diagnosed today with PBC have been at the middle or late stage for no special clinical presentations. For PBC is a cholestatic disease, study on polarized molecules on hepatocyte canalicular membrane will help to reveal the pathogenesis of PBC and develop the diagnostic reagents.The hepatocyte is a polarized epithelial cell with sinusoidal and canalicular membrane. There are many transporters and proteins specially expressed on hepatocyte canalicular membrane. All these polarized molecules participate in the process of bile secretion. Previous study showed that these molecules abnormal expression or dysfunction had a close relationship with PBC. In PBC, the expression of BSEP and MRP2 were down-regulated and these down-regulations might be regulated by HNF1αand RXRα/RARαin transcription level. Besides, it was found that the expression and distributions of these transporters had changed and these changes might be associated with Ezrin misdistributions. Besides, single nucleotide polymorphisms Site change might be the pathogenesis of PBC. Pauli-Magnus and his colleagues also found that G620D and A1228V missense mutation of BSEP gene had a relation with PBC. UDCA is currently the only effective drug treatment for PBC and part of patients could have a normal life expectancy after regular treatment. But the exact mechanism is not clear. Animal experiments revealed that it might promote bile excretion through up-regulation the expression of BSEP and MRP2.However, polarized molecule on hepatocyte canalicular membrane could not completely explain the mechanism of bile excretion obstruction and the finding of new molecules will help to explain the process.【Aims】1. To prepare and screen polarized molecules on hepatocyte canalicular membrane.2. To explore its pathophysiological value in PBC.3. To identify the antigen recognized by polarized molecules.【Methods】1. To isolate PBC liver canalicular vesicles and preparation and screening the polarized molecules on hepatocyte canalicular membrane according to standard hybridoma technique.2. To observe 1F9 subcellular localization by immuno-electron microscopy and confocal microscopy.3. To collect different stages of PBC liver tissues and other liver disease tissues, observe 1F9 expression by immunohistochemistry and explore its pathophysiological value in PBC.4. To identify the antigen recognized by 1F9 with cDNA library screening and immunoprecipitation followed by LC–MS/MS.【Results】1. We have obtained four monoclonal antibodies named 1F2,1F3,1F5 and 1F9.2. Confocal microscopy and immuno-electron microscopy revealed that 1F9 antigen located on endosome capsule on hepatocyte canalicular membrane.3. We collected different stages of PBC liver tissues and other liver tissues and observed its staining intensity by immunohistochemistry. The results showed that 1F9 expression had relation with PBC progression, but not other liver diseases. It had close relationship with PBC in two aspects: First, it located in endsome capsule in normal liver tissues; second, in PBC liver tissues, it not only located in endsome capsule at canalicular membrane, but also distributed in cytolysosome.4. The results of immunoprecipitation and immunoblotting revealed that 1F9 antibody recognized 120 kDa proteins.5. We obtained 41 proteins through LC–MS/MS spectrum analysis and 7 gene fragments through cDNA library screening. Bioinformatics analysis of these genes and proteins showed that Calsyntenin might be the target antigen of 1F9.【Conclusions】1. Four monoclonal antibodies on hepatocyte canaciluar membrane were obtained by hybridoma technique.2. 1F9 expression strengthened gradually with PBC development and 1F9 expressed disorderly in parts of PBC liver tissues. All these results revealed 1F9 had a close relationship with PBC.3. Calsyntenin might be the candidate molecule.