Therapeutic effects of hepatocyte transplantation via two different intrasplenic approaches in acute hepatic failure rats / 中国组织工程研究

ABSTRACT

BACKGROUND: Hepatocyte transplantation has achieved some success in animal experiments for the treatment of metabolic diseases and acute liver failure. However, the clinical efficacy of hepatocyte transplantation is unsatisfactory. The difference between the experimental results and the clinical efficacy may be related to the hepatocyte transplantation approach. OBJECTIVE: To evaluate the therapeutic effects of hepatocyte transplantation by the intubation via the intubation of the splenic artery and intrasplenic injection in rats with acute hepatic failure, providing more optimal transplantation approaches and methods. METHODS: Hepatocytes were isolated and cultured by the modified Seglen's method (two-step). Acute hepatic failure was induced by D-gal in Sprague-Dawley rats (provided by the Experimental Animal Center of Chongqing Medical University in China). After 24 hours, an intubation tube was inserted into the splenic artery in 65 rats with acute hepatic failure, which was successful in 60 rats. Then these 60 rat models were randomly divided into three groups (n=20 per group). Intrasplenic injection group received about 2×107 hepatocytes through intrasplenic injection and 0.4 mL of Hank's solution through the splenic artery. Splenic artery group received 0.4 mL of Hank's solution through intrasplenic injection and 2×107 hepatocytes through the splenic artery. Model group received 0.4 mL of Hank's solution through intrasplenic injection and 0.4 mL of Hank's solution through the splenic artery. Survival rate and liver function of the rats was observed within 14 days after transplantation. The distribution of CFDA-SE-labeled hepatocytes transplanted via the splenic artery was observed under fluorescence microscope at 7 days after transplantation, and meanwhile, the distribution and proliferation of transplanted hepatocytes in the spleen were observed using hematoxylin-eosin staining. Synthesis of albumin in the spleen was observed by immunofluorescence staining. RESULTS AND CONCLUSION: (1) 80％-90％ hepatocytes survived after isolation. (2) At 14 days after transplantation, the survival rates of rats in the three groups were significantly different intrasplenic injection group> splenic artery group> model group. (3) The liver function of rats was significantly improved in the intrasplenic injection group and the splenic artery group, especially in the former group. (4) CFDA-SE-labeled hepatocytes (green fluorescence) were scattered in the rat spleen and liver at 7 days after transplantation via the splenic artery. (5) At 14 days after transplantation, immunofluorescent staining of albumin demonstrated some positive cells in the rat spleen in the intrasplenic injection group and splenic artery group. (6) At 7 days after transplantation, transplanted hepatocytes were concentrated and colonized in the red pulp of the spleen. In conclusion, hepatocyte transplantation through catheterization of the splenic artery via carotid route can improve the survival of rats with acute hepatic failure and ameliorate the hepatic function, but intrasplenic injection is significantly superior to the injection via the splenic artery.