Annexin V/PI Detection And Fas Expression Of Apoptosis In Adult Primary Hepatocyte During Its Culture After Isolation And Cryopreservation

Objective: To study apoptosis of in vitro cultured adult primary hepatocyte after its isolation and cryopreservation, in order to explore the apoptosis feature of in vitro cultured hepatocytes.Methods: We isolated hepatocytes from benign liver tissue of adult patients removed by surgery using modified collagenase perfusion, cryopreservated hepatocytes with freeze-stored liquid;adult primary hepatocytes freshly isolated and recovered from cryopreservation are counted by blood count plate,trypan blue staining technology and fluorescence activated cell sorting(FACS) were used to detect the viability of these two groups of hepatocyte; During the in vitro cultivation of the two groups, we detected the apoptosis state of hepatocyte by Annexin V/PI with fluorescence microscopy and Annexin V/PI with flow cytometry at 1d, 2d, 3d, 5d, 7d, 9d, and observed the Fas expression state of the in vitro cultured freshly isolated hepatocyte by immunohistochemical method at 1d, 2d, 3d, 5d, 7d, 9d.Results: 1.There were no obvious morphological changes between freshly isolated hepatocyte and recovered hepaptocyte from cryopreservation; 2.The quantity of fresh adult primary hepatocytes isolated by modified collagenase perfusion reached more than 109; 3.Viability detection of fresh adult primary hepatocytes with trypan blue staining and FACS was respectively 92.7% and 93.5%, and that of recovered hepaptocyte from cryopreservation was 80.6% and 81.4%; 4. The number of apoptosis in cultured freshly isolated hepatocytes observed by Annexin V/PI with fluorescence microscopy increased, and which increased significantly after the 3rd day; 5. The apoptotic rate detection by Annexin V/PI with flow cytometry during the cultivation of adult primary hepatocyte shows: the apoptotic rate of all hepatocytes increased gradually, and which increased significantly after the 3rd day in freshly isolated hepatocytes and after the 2nd day in recovered hepatocytes from cryopreservation(P<0.05); 6. Fas expression increased gradually with time and increased significantly after the 3rd day in freshly isolated hepatocytes(P<0.05).Conclusion:1. Modified collagenase perfusion technique for adult primary hepatocytes isolatation has a simple, convenient and time-saving advantage, the hepatocyte isolated has a high quantity, purity and Viability.2. Hepatocyte with freeze-stored liquid has a high viability after recovered from cryopreservation and a nice activity in long-term storage.3. The apoptotic rate of hepatocytes from isolation and cryopreservation increased gradually.4. The apoptotic rate and the number of Fas expression increased5. significantly at the 3rd day in freshly isolated hepatocyte. The apoptotic rate increased significantly at the 2nd day in recovered hepatocyte from cryopreservation, which prompts that time limits may be the best time for the clinical application of hepatocyte transplantation.