Effect Of Small Hepatocytes Of Rats Transplantation On Acute Liver Injury

It has been proven that hepatic stem cells, which are activated when the liver is injured, play important role in development and repair of tissue and organ. Hepatic stem cells can differentiate into mature hepatic cells or biliary epithelia cells. Research on hepatic stem cells will benefit cell-transplantation for liver diseases, hepatic tissue engineering and gene therapy. But in normal condition the number of hepatic stem cells is small and remains steady. Small hepatocytes, which is known as comparatively more mature, take 1.5-2.0 percent of all the hepatic cells. When the liver is seriously injured and proliferation of mature hepatic cells are inhibited, repair of the liver depends on hepatic stem cells which reside in the portal vein area. In recent years more attention has been paid to therapeutic application of stem cells for hepatic diseases of terminal stage. In our experiment,model of liver cirrhosis and proliferation of SHCs was established by diethylinitrosamine. Then SHCs were isolated, identified and cultured. Finally, the effect of transplantation of SHCs on acute liver injury was observed.Aims :1. To investigate an more effective method of isolation, cultivation of SHCs, and examine the phenotype of them. 2. To study the effect of SHCs transplantation on liver injury induced by CCl4 and 2/3PH.Methods:1.20 SD rats were selected to be fed with bait vessel containing DEN to establish the model of hepatic cirrhosis. Improved collagenase dabbling digestive method was used to isolate small hepatocytes and then the cells were primarily cultivated. Observe cell morph under light microscope and ultramicrostructure of cell under electron microscope. Growth curve and cell cycle were determined. AFP,CK8,CK19,CK7,CK18 monoclonal antibody were used for the immunocytochemistry staining.2.SHCs were stained with PKH26 membrane fluorchrome. 30 SD rats of the same line were randomly divided into three groups, which were blank control group (B), CCl4+2/3PH injured group(M) and SHCs transplantation group (T). Rats of group B were intraperitoneally injected with normal saline, while group M and group T were intraperitoneally injected with CCl4. 24 hours later, all rats were anaesthetized by intraperitoneally injection of butaylone, then disinfection and operation were done. 2/3PH were done in group M and T. Then group B and group M were injected with normal saline via portal vein, and group T were injected with SHCs suspension. 4 weeks later, in all three groups the level of serous ALT and AST were tested, and pathological sections of liver were observed. Frozen sections of group T were observed under fluorescence microscope.Results:1. Primary cultured SHCs adhered to plastic surface within 24h. And SHCs which had clear smaller chromatospherite and less cytoplasm compared to normal hepatocytes were observed under light microscope. Under electron microscope manipulus short microvilli on the SHCs'surface and high karyoplasmic ratio were observed as the presentation of dedifferentiation. Immunocytochemistry staining of AFP,CK8,CK19,CK7 and CK18 presented the color of buffy as positive.2. Under fluorescence microscope hepatic cells which differentiated from the SHCs and could construct hepatic plates should be observed. In group T under fluorescence microscope hepatic plates which constructed by hepatic cells were observed.3. SHCs transplantation decreased the level of serous ALT and AST. Serous ALT and AST of group B were (13.67±9.74)U/L and (13.49±9.39)U/L, while those of group M were (270.90±11.57)U/L and (222.75±17.49)U/L, which were obviously higher than those of group B (P<0.01). Serous ALT and AST of group T were (149.60±12.35)U/L and (160.12±17.56)U/L, which were obviously higher than those of group B (P<0.01) and lower than those of group M (P<0.01).4 . The degrees of pathological injury were significantly different. Pathological section of group M displayed a lot of inflammatory cells, large death areas of hepatic cells and part of hepatic plates are injured. But pathological section of group T displayed some inflammatory cells and edema hepatic cells, fatty degeneration of hepatic cells but the hepatic plates were completed.Conclusion:1.A simple but effective method of isolation and cultivation of SHCs has been established.2. Transplantation of SHCs has therapeutic effects on acute liver injury induced by CCl4+2/3PH, and SHCs take part in the regeneration and repair of liver.