A Study Of The FLSPCs Transplantation Effectiveness On Different Acute Hepatic Failure Types Of F344Mice And A Model Of Acute Hepatic Failure With Catheterization Of Portal Venous Branch

The acute liver failure is one of the main death causes of patients with certain liverdiseases. The disease features symptoms including hepatic encephalopathy and fatal liverdamage. The liver transplantation is considered to be the most effective treatment. Thesurvival rate of transplantation is from50%to70%. Since the donor for livertransplantation is limited, more and more attentions are paid to the possibility of celltransplantation. Fetal liver stem/progenitor cells transplantation is one of the idealmethods. FLSPCs are obtained from embryonic-day (ED)-14rat fetuses then enriched andpurified by a three-step separation procedure previously reported by our group. To tracethe exact effect of FLSPCs on amending liver injury, by liposome-mediated transfection,the green fluorescent protein (GFP) gene was transferred into the FLSPCs. Animal modelsof drug-induced acute liver failure were made with carbon tetrachloride by peritoneal injection. The partial hepatectomies with85%sizes were performed on the mice toproduce models of acute liver failure of injury.After12hours the FLSPCs transfectedsuccessfully by GFP were transplanted into treated groups through caudal veins. Observethe liver-repair condition at different points. Differences are significant in hepaticfunctions (ALT, AST, TBIL and ALB) between treated groups and control groups12hours after transplantation. Compared to control group the indicators such as mortalityrates, hepatic pathology and coagulate function were gradually recovered in transplantgroups respectively72hours after transplantation..After study the feasibility andeffectiveness of FLSPCs treatment on different acute hepatic failure types of F344mice,we establish a Model of acute hepatic failure with catheterization of portal venous branchand make a study of the way of cell transplantation. we learn that stem cellstransplantations through ducts of portal venous branch improved the hepatic functions andpathology in acute hepatic injury model of F344rats as much as those through portal veindirectly. To conclude, the FLSCs derived by a three-step separation procedure are materialimproving the recovery of different acute hepatic failure types of F344mice. and themodel has advantages in repeated operation, fewer traumas and less complication and canbe treated whenever necessary.Objective1. To study the feasibility and effectiveness of FLSPCs treatment on different acutehepatic failure types of F344mice2. Establish a Model of acute hepatic failure with catheterization of portal venousbranch and make a study of the way of cell transplantation to evaluate the new model.Method1. The enrichment, purification and tracement of FLSPCsFLSPCs are obtained from embryonic-day (ED)-14rat fetuses then are enriched andpurified by a three-step separation procedure.To trace the exact effect of FLSPCs onamending liver injury, by liposome-mediated transfection, the green fluorescent protein(GFP) gene was transferred into the FLSPCs.2. Establishment of different acute hepatic failure types models, transplantation of FLSPCs and examination of liver repairEighty F344rats were randomly divided into4groups(n=20in each group). rats ofgroup A and B were induced acute liver injury with carhon tetrachloride at the dose of3ml/kg by peritoneal injection. The partial hepatectomies with85%sizes were performedon the mices of the C and D groups. After12hours, every mice of the group B and D weretransplanted into1×106cells through caudal veins. Control rats of A and C group recievedmedium of equal volumes. Mortality rates, ascites, hepatic functions such as ALT, AST,TBIL, ALB and coagulative function PT were measured at different points.Hepaticpathology was studied．3. Establish a rat model of acute hepatic failure with catheterization of portal venousbranch. make a study of the way of cell transplantation to evaluate the new model.Sixty F344rats were randomly divided into3groups(n=20in each group). Thepartial hepatectomy with85%sizes were performed on the rats of the E and F groups. Thesame operations and catheterizations of portal venous branch were carried out on the ratsof the G group. After the green fluorescent protein (GFP) gene was transferred into theFLSPCs, Every rats of the F group were transplanted into4×l05cells through the portalveins during the operations. Every rats of the G group were transplanted into the samecells through the ducts. Control rats in the E group received medium of equal volumes.Mortality rates and hepatic functions such as ALT AST were measured after72hours.Meanwhile, hepatic pathology was studied.Result1. FLSPCs were isolated successfully from ED-14rat liver and cultured as describedin our previous report. The FLSPCs multiply rapidly and the anchorage-dependent rate issignificant. The cell are small with monotonous sheet, and triangular in section with highnucleus cytoplasm ratio.2.12hours after transplantation differences are significant in hepatic functionsbetween treated groups(B and D) and control groups (A and C) respectively. The resultsshow that serous ALT, AST, TBIL and ALB of group A are (5404.17±376.94)U/L、(6823±348.99)U/L、(98.43±5.21)umol/L and (24.12±1.21)g/；while those of group B are(4807±244.62)U/L、(5290.67±289.68)U/L、(83.67±3.501)μmol/L、(26.33±1.21)g/L. Data of group C are (1946.3±106.87)U/L、(3834.5±230.05)U/L、(102.50±8.24)umol/L、(17.83±1.72)g/L；while those of group D are (1400.5±124.07)U/L、(2985±134.07)U/L、(83±7.61)umol/L、(21.33±1.86)g/L. With the time coursing the hepatic functions of treatgroups are improved rapidly. Coagulative function PT and ascites volume of treat groupsshow better performance than corresponding control groups. The degrees of pathologicalinjury were significantly different either. Pathological section of group A displays seriousstructural damaged, large death areas of hepatic cells and part of hepatic plates are injuredwhile pathological section of group B shows slighter injury degree than that of A group.Pathological section of group C displays few hepatic cells proliferation and inflammatorycells infiltration while D shows hepatic cells proliferate conspicuously and the recovery oflobular hepatis.3.72hours after different FLSPCs transplantation approaches by portal veins（group F）or ducts of new models（group G）data shows serousALT andAST of groupF were802.83±79.22)U/L、(1463.50±56.22)U/L and those of group G were(827.83±63.08)U/L、(1472.83±52.41)U/L, which were obviously higher than those of（1275.67±329.59)U/L、(2806.83±157.0)U/L (P<0.01) and indicate the ratio of hepaticfunctions improvement between group F and G is no different(P>0.05). Pathologyexamination of E group reveal that the remaining liver enlarge to compensate; dilatation ofhepatic sinusoidal and central vein, few hepatic cells proliferation and inflammatory cellsinfiltration can be observed. Pathological section of group F and G show morecompensatory enlargement and more proliferation of hepatic cells than that of group E;lobular hepatis structure is complete.It can indicate that hepatic functions improvementby transplantation through portal veins is as much as by transplantation through ducts ofnew models.Conclusion1. The FLSPCs derived by a three-step separation procedure are material improvingthe recovery of different acute hepatic failure types of F344mice.2. The model of acute hepatic failure with catheterization of portal venous branch hasadvantages in repeated operation, fewer traumas and less complication and can be treatedwhenever necessary.