Investigating The Influences Of Recipient Derived PBLAST2-hHGF Transfected Hepatic Oval Cells On Liver Grafts In Rats

Part One Establishment of Orthotopic Liver Transplantation Model in RatsObjective To investigate the establishing methods of orthotopic liver transplantation model in rats and provide a stable basic model for live tansplantation research.Methods Using Kamada two-cuff technique to establish rat orthotopic liver transplantation model.During pre-experiment,SD rats were executed with orthotopic liver transplantation,while in formal orthotopic liver transplantation experiment,DA and Lewis rats were used as donors and recipients respectively.After the operation, recipient survival rate and complications were observed.Results 310 rats orthotopic liver transplantation models were successfully established.Analyzing 60 model building operations,donor operation time was(20.5±4)min,donor truing time(8±3)min,recipient operation time(40±10.5)min,anhepatic time(13.4±3)min, operation success rate 97.3%,one week recipient survival rate 92.5%.Conclusion Successfully establishing rats orthotopic liver transplantation models needs the operators having some extent microsurgery techniques,rats abdominal anatomy information and sustained training for 4～6 months.Key factors for successful operation include right liver infusion methods,rigorous peritoneal hemostasis,skilled microvessel suture technique,shortened anhepatic time,improved fluid therapy, corrected acid and alkali balance during operation and rewarming after operation.Part Two Establishing Hepatic Oval Cells Proliferation Model in LewisRats and Isolating,Cultivating,Identifying,differentiating Hepatic Oval CellsObjective To activate hepatic oval cells(HOC) in Lewis rats by establishing 2-AAF/PH model,then isolating,purifying,cultivating and identifying HOC,inducing HOC differentiation into hepatocyte and biliary epithelial cell in vitro. Methods 20 male Lewis rats were fed with 2-AAF20mg/(kg.day) 5 days,then 2/3 volume hepatectomy and left lateral lobe hepatectomy was conducted respectively and continue fed with 2-AAF20mg/(kg.day) 7 days after the operations.Sacrificed rats,obtained liver tissue,and isolated,purified,cultivated and differentiated HOC.Flow cytometry was used to detect stem cell marker(c-kit,CD90 and CD34) on HOC surface,real-time fluorescence quantitative RT-PCR was used to detect ALB mRNA and CK-19mRNA in HOC,differentiated cell,liver tissue and extrahepatic biliary.Results Successfully established 2-AAF/PH HOC proliferation model in Lewis rats and isolated,purified,cultivated HOC.Then induced HOC differentiation into hepatocyte and biliary epithelial cell in vitro.C-kit,CD90 and CD34 positive rate were 99.8%,88.8%and 3.6%respectively in purified HOC.High level ALB mRNA and low level CK-19 mRNA were detected in HOC,while High level ALB mRNA and High level CK-19 mRNA were found in differentiated cell.Conclusion The establishment of 2-AAF/PH HOC proliferation model in Lewis rats was stable and feasible,and HOC isolated,purified from 2-AAF/PH HOC proliferation model could be detected to express stem cell marker.HOC obtained from male Lewis rats could be passage cultured stably,and differentiated into hepatocyte and biliary epithelial cell on this inducing condition,but mainly differentiated into biliary epithelial cell.Part Three pBLAST2-hHGF Plasmid Transfection in Hepatic Oval CellsObjective To establish pBLAST2-hHGF plasmid stablely transfected cell line in hepatic oval cells and observe the biological characteristics of transfection cells.Methods Liposome-mediated gene transfection was used to transfer pBLAST2-hHGF plasmid into the fourth generation HOC derived from male Lewis rats,then morphological changes,cell proliferation of transfection cells were observed under inverted phase contrast microscope.Proliferation and differentiation capability of transfection cells were measured and stable culture conditions were explored.Western blot was used to test hHGF expression on transfection cells.Results The fourth generation pBLAST2-hHGF/HOC could be passaged steadily to 14 generations without significant morphological changes under the culture conditions(SCF 20ng/ml,HGF 10ng/ml,EGF 10ng/ml and LIF 20ng/ml),while the growth and proliferation speed of transfection cells were faster than that of HOC.Removing growth factors mentioned above,pBLAST2-hHGF/HOC quickly differentiated into hepatocyte and biliary epithelial cell in vitro.hHGF protein could be tested in pBLAST2-hHGF/HOC by using western blot and it also could be tested with high level in waste culture fluid by using ELISA.Conclusion Liposome-mediated gene transfection in this experiment was reliable and the stable passage times of pBLAST2-hHGF/HOC was longer than that of HOC.pBLAST2-hHGF/HOC had the ability to secrete hHGF protein and this cell line could be used in following experiment. Part Four Investigating the Influences of Recipient Derived pBLAST2-hHGF/HOC on Liver Grafts in RatsObjective To study the located distribution of recipient derived pBLAST2-hHGF/HOC in liver grafts,then the influences of pBLAST2-hHGF/HOC on liver function,acute rejection,low immune response and long-term survival of recipient were researched.Methods Kamada two-cuff technique was used to establish rat orthotopic liver transplantation model and female DA rats,female Lewis rats were used as donors and recipients respectively.Three groups in this experiment were set up:control group(group A,only establishing rat orthotopic liver transplantation model);HOC transplant group(group B,1×10~6/ml HOC 1ml was injected into tansplanted liver via portal vein and hepatic artery respectively during the operation); pBLAST2-hHGF/HOC transplant group(group C,1×10~6/ml pBLAST2-hHGF/HOC 1ml was injected into tansplanted liver via portal vein and hepatic artery respectively during the operation).Therapeutic amount of tacrolimus(0.05mg/kg,twice per day,continuous for 8 days,1 day before operation and 7 days after operation,intragastric administrated) was administrated to recipients,then continuously half dose decreasing every day began from eighth day post-operation and stoped tacrolimus administration by thirteenth day post-operation.Liver tissues and blood samples were collected on 7th,14th,21th,28th,35th and 42th day after liver transplantation.SRY in situ hybridization and CD44,ICAM-1,Fas,CD40 immunohistochemical staining on liver tissue were tested and median survival time(MST) of recipients was calculated.Liver function(ALT,dBil,GGT,ALP,XCHE, ALB),blood cytokines(IL-2,IL-10,IFN-γ,,TNF-α,TGF-β,hHGF) and T cell subsets were tested.Observing liver pathologic changes and calculating acute rejection index. Results MST of recipient in group A was shorter than that in group B and group C,while it was shorter in group B than in group C.HOC and pBLAST2-hHGF/HOC mainly located distribution around portal area and central vein area,Sry positive rate in group C was continuously higher than that in group B since 7th day post-operation.ALT,DBil,GGT,ALP,IL-2,IFN-γ,,TNF-αand CD4+/ CD8+T cell ratio in group A were continuously higher than those in group B and C since 14th day post-operation respectively,and these indexes were continuously higher in group B than in group C since 14th～21th day post-operation respectively.ALB,XCHE,IL-10 and TGF-βin group A were continuously lower than those in group B and C since 14th day post-operation respectively,and these indexes were continuously lower in group B than in group C since 14th～21th day post-operation respectively.Acute rejection score in group A was continuously higher than that in group B and C since 14th day post-operation respectively,and it was continuously higher in group B than in group C since 21th day post-operation respectively.The intensity and range of CD44,ICAM-1,Fas,CD40 immunohistochemical staining on recipient liver tissue were continuously larger than those in group B and C since 14th day post-operation respectively,and these indexes were continuously larger in group B than in group C since 14th～21th day,post-operation respectively.Conclusion The proliferation and differentiation capability of recipient derived pBLAST2-hHGF/HOC in recipient liver were stronger than HOC,and they mainly located distribution around portal area and central vein area in recipient liver.Recipient derived pBLAST2-hHGF/HOC and their differentiated cells in recipient liver could secrete hHGF protein,then protected recipient liver and improved recipient liver function through HGF/C-Met pathway.Recipient derived pBLAST2-hHGF/HOC and their differentiated cells in recipient liver could effectively reduce acute rejection score,and this effect was stronger than HOC.Recipient derived pBLAST2-hHGF/HOC and their differentiated cells in recipient liver could effectively induce recipient's low immune response toward grafted liver,prolong recipient's survival time after liver tansplantation,these effects of pBLAST2-hHGF/HOC and their differentiated cells were stronger than those of HOC.