| Summary: | 碩士 === 國立臺灣大學 === 動物科學技術學研究所 === 103 === According to official statistics, there are many people in Taiwan suffering from liver disease due to diet and lifestyle change in recent years. Most liver failure patients show the liver fibrosis symptoms in the beginning stage, because the liver is injured repeatedly and produce the number of hepatic extracellular matrix (ECM) through self-healing process. To treat the liver disease, the most effective way is to implement the liver transplantation. However, the orthotopic transplantation faces many difficulties, involving in the organ donor shortage, financial considerations, and also the requirement for lifelong immunosuppression. For above reasons, there is an urgent need to develop a new therapy for curing liver disease effectively.
Amniotic fluid is surrounding the embryo to protect the developing baby against with abdomen, moreover, is usually used to determine the fetus health by amniocentesis during pregnancy. In recent studies suggest amniotic fluid, where is full of shed fetal tissues and cells, is rich in stem cells (amniotic fluid stem cells, AFSCs). AFSCs perform not only high telomerase activity but also surprising proliferative ability than other stem cells from adult tissues. Most importantly, AFSCs, sharing the similar characteristics with ESC, can differentiate into three germ layer cells in vitro and express pluripotent stem cell marker, Oct4. Besides, in contrast to ESC, AFSCs are not teratogenic after transplantation and get rid of ethical issues. Therefore, it likely implies AFSCs are a new source to deal with the thorny problem of liver disease.
At of all, this study isolated the AFSCs from transgenic Ds-red pig in the tenth week pregnancy. The subculture purified Ds-red porcine AFSCs, determining surface antigen of by flow cytometry analysis, express CD44 and CD99 but not express CD31 and CD4a markers. Ds-red porcine AFSCs could be rise to osteoblasts, adipocytes and chondrocytes in differentiation potential test. In addition, porcine AFSCs could differentiate into hepatocyte-like cells in vitro by giving the appropriate growth factors (e.g. FGF and HGF) when cultured for thirteen days. The differentiated porcine AFSC cells transformed to a cubical shape and express liver-specific protein, albumin (ALB). These evidences indicate the potential to develop the therapy in liver disease by using porcine AFSCs.
The experiment in vivo utilized the male ICR mice treating by CCl4, a liver failure inducing drug, for 4 weeks and then the porcine AFSC cells are injected through portal vein. After treated CCl4 4weeks later, the serum detections perform a significantly high level of glutamic oxaloacetic transaminase (GOT/AST), glutamic pyruvic transaminase (GPT/ALT) and albumin (ALB) (P < 0.01). At the same time, the mice were randomly allocated to each trial group, including the single cells, sphere with diameter smaller than 40um (< 40um), between 40um and 70um (40-70um) and larger than 70um (> 70um), for cells injection surgery. After cells transplanted 4 weeks, liver enzyme level decrease significantly (P < 0.01) and ALB level show a tendency to a normal range (P <0.05) when compared with control group. Unfortunately, there is no difference between each group with cell treatment. The liver section were evaluated by Masson’s trichrome stain show the less liver scarring fibrosis in each cells transplantation group after cells transplanted 4 weeks. These results suggest the AFSCs could significantly improve the liver function of mice with fibrosis liver and prevent deterioration process. Furthermore, in order to figure out the migration of injected cells, the liver slide sections were observed at 24, 48, and 72 hours individually after cells transplanted. The transplanted Ds-red AFSCs distributed in interstitial space instead of located around liver vessels at 24 hours. Especially, by using immunostaining, the external AFSCs were found could form ALB expression cells in 72 hours group. Thus, above results implicate that transplanted cells may to affect the function of fibrosis liver.
In conclusion, the results indicate that transplanted AFSCs may to affect the fibrotic liver function. AFSCs derived from pig exhibit a positive potential in cell therapy of liver fibrosis, and it is considerable way to carry out cells injection via portal vein in patients.
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