Therapeutic Effects Of Hepatocyte Transplantation By Using HGF Loaded PLA-O-CMC Nanoparticles On Treating Rats With Acute Liver Failure

Objective:To evaluate the therapeutic effect of intraperitoneal hepatocyte transplantation(HTx) using HGF loaded polylactic acid-O- carboxymethylated chitosan(PLA-O-CMC) nanoparticles on acute liver failure(ALF) rats and study the mechanism.Methods: ALF models of rats were prepared by D-gal intraperitoneal injection(1.2g/kg). 5ml rat hepatocytes routine embedded in collagen (5.0×10~7cells)(groupⅠ), 5ml rat hepatocytes co-cultured with PLA-O-CMC nanoparticles(5.0×107cells)(groupⅡ) and 5ml rat hepatocytes co-cultured with HGF loaded PLA-O-CMC nanoparticles(5.0×107cells)(groupⅢ) were transplanted into the abdominal cavity of SD rats at 48h after D-gal injection. As groupⅡ, added intravenous injection HGF 10μg/kg everyday for 7 days as comparison(groupⅣ) and 5ml composite cell culture fluid injection(groupⅤ) was used as the negative group. The 14th survival rate, hepatic function, liver pathological change, hepatocyte ultramicrostructure alteration as well as hepatocyte mitotic index of ALF rats and the pathological change of transplanted hepatocytes were observed. The recipient liver label index of Ki-67 antigen, proliferation cell nuclear antigen and bromodeoxyuridine incorporated rats were detected by immunohistochemical method. HGF concentration of ALF rats serum and liver tissue were detected by euzymelinked immunosorbent assay. Flow cytometry detected proliferation index of recipient hepatocytes and apoptosis rate, necrosis rate, survival rate of cultured and transplanted hepatocyte.Results: There was a significant difference in survival rate on 14th day among group I (50.00%), group II (68.75%), group III (81.25%), group IV (75.00%) and groupⅤ(18.75%) (P<0.05). Each transplanted group was higher than control group. After HTx 24h, the improving hepatic functional parameter of groupⅡ,Ⅲand groupⅣhave a significant difference with groupⅤ(P<0.05). The distinction occurred more obviously on 5th day when the ALB of groupⅢwas siginificantly higher than other groups(P<0.05). On 7th day, there was no significant defference between each group except ALT(P>0.05). The hepatic function and pathological damage restored most quickly and best in groupⅢ, follwed by groupⅣ, groupⅡ, groupⅠ, groupⅤ. The transplanted hepatocytes of groupⅢsurvived more long and less inflammatory infiltration. The hepatic lobules structure of groupⅢrecuperate more fast and hepatocyte caryocinesia obviously as so as rough endoplasmic reticulum and mitochondria occurred intensively. Each group showed liver regeneration when each transplanted group showed better result than control group and each nanometer-group better than un-nanometer- group. The hepatocyte mitotic index, labeling index of PCNA, Ki-67, BrdU and hepatocyte proliferation index of groupⅢwere higher than other groups as the difference in definite period had statistical significance (P<0.05). The HGF concentration of rats serum and liver tissue of each group heightened and become obviously on 3d after HTx. The HGF concentration of ALF rats liver tissue in groupⅢwas higher than others as well as the HGF concentration of ALF rats serum in groupⅣwas higher than others(P<0.05). As time prolonged, the motility rate descended as so as the necrosis rate and apoptosis rate rised. The motility rate of groupⅢwas the highest and the apoptosis rate was the lowest among all groups. The difference on 3th day occured more obviously as the motility rate in groupⅢandⅣwere higher than groupⅠ, furthermore in groupⅢwas higher than in groupⅡ. At the same time the apoptosis rate in groupⅢandⅣwere significantly lower than groupⅠ, in groupⅢwas lower than in groupⅡtoo(P<0.05). Conclusion: Intraperitoneal transplantation of HGF loaded PLA-O-CMC nanoparticle-attached rats hepatocytes can increase the survival rate of ALF rats induced by D-gal and can improve the recipient hepatic function significantly. It can promote recipient liver regeneration and inhibit transplanted hepatocytes apoptosis which showed satisfactory therapeutical effect.