Study on the Cell Cycle of Liver Cells during Rat Liver Regeneration
|Keywords||Rat liver regeneration cell proliferation cell cycle flow cytometry|
Objective: Methods to isolation and identification viable liver cells during liver regeneration were developed. The cell cycle of the regenerated liver cells were also studied.Methods: In this study, rat partial hepatectomy model was made according to the method of Higgins et al. Hepatocyte, biliary epithelial cells, kupffer cells, sinus endothelial cells, hepatic stellate cells and oval cells after PH 0h, 12h, 24h, 27h, 30h, 33h, 36h, 39h, 42h, 48h, 60h, 72h, 96h, and 120h were dispersed by two-step perfusion method, and then isolated by density gradient centrifugation with percoll. Immunocytochemistry method was applied to qualify the above isolated cells. Measurement of cellular DNA content and the analysis of the cell cycle were performed by flow cytometry and the commercial software available for these purposes. Regenerated hepatocyte in vitro cultivation has been measured by FCM.Results: Viability of hepatocyte, biliary epithelial cells, kupffer cells, sinus endothelial cells, hepatic stellate cells and oval cells isolated from 0h, 12h, 24h, 27h, 30h, 33h, 36h,39h, 42h, 48h, 60h, 72h, 96h, and 120h regenerated liver was above 95%, and the purity was above 90%. In regenerated liver, the cell cycle of hepatocyte was 30h approximately, and G1 6h, S 12h, G2/M 12h; biliary epithelial cell was 48h approximately, and G1 18h, S 24h, G2/M 9h; kupffer cell was 24h approximately, and G1 9h, S 9h, G2/M 6h; sinus endothelial cells was 33h approximately, and G1 9h, S 12h, G2/M 12h; hepatic stellate cell was 36h approximately, and G1 12h, S 12h, G2/M 12h; oval cells was 30h approximately, and G1 12h, S 12h, G2/M 6h.Regenerated hepatocyte cultured in vitro possessed the function of the hepatocyte in vivo. The cell cycle of it shorted slightly during culture, the cells entered the cell cycle at 24h after culture, and the hepatocyte proliferation emerges between 24 and 48h with the DNA synthesis peak at 72h.Conclusion: The characteristic of cell proliferation measured by FCM supply foundation for artificial liver in cell level. In regenerated liver, At first, hepatocyte prolifer, the cell cycle of hepatocyte was 30h approximately, kupffer cell was 24h, sinus endothelial cells was 33h, hepatic stellate cell was 36h, oval cells was 30h. At last, biliary epithelial cells prolifer, the cell cycle of biliary epithelial cell was 48h. The proliferation of cultured cells was slow down and the peak was put off, compared with the hepatocyte in vivo.