Shark Liver Parenchymal Cell Culture And Membrane Separation And Purification Studies

Plasma membrane play important roles in numerous biological phenomina including cell development, differention, implantation, morohogenesis, tumor metastasis, cell-cell and cell substratum recognition, microbe infection, etc. To study the actions of the surface glycans in tumor metastasis, two experiments about the sugar chains in the plasma membrane of Lip Shark (Chiloscyllium plagiosurri) were carried out, including the isolation of Lip Shark hepatocytes and the isolation and purification of hepatocytes membrane of Lip Shark.Three isolation methods of Lip Shark hepatocytes were compared, including mechanical disruption, enzymatic digestion,and organ perfusion. In mechanical distruption, different culture conditions, including medium, incubation temperature, osmobility, serum were compared. And good yields could be obtained when cultured in L-15 medium supplemented with 20% fetal bovine serum and 0. 0351g/ml NaCl at 27℃ However, much higher yields were got with organ perfusion.The hepatocytes were broken through swelling. The plasma membrane preparation was purified by the method of sucrose density gradient. The purity of plasma vesicles were assessed by electron microscopy and marker enzyme assay. By the procedure, the specific activity of final purified plasma membrane could be increased to 7.51U/g Proten and was 7 times more than the crude extract. The purified plasma membrane was checked by polyacrylamide gel electropheresis in the presence of sodium dodecyl sulfate (SDS-PAGE), and showed five bands. Their molecular was 95.8KD, 85.6KD, 77.2KD, 59.2KD, and 8KD.