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Effects of Pathologic Microenvironmental Cytokines Derived from Cholestatic Cirrhosis on Liver Stem Cell Differentiation
Wang Jian1,2, Kang Quan1,2*, Luo Qing2, Yang Bo1,2, Xiao Cheng1,2, Li Zhipeng1,2, Gong Mengjia2, Bi Yang2
1Department of Hepatology, Children’s Hospital of Chongqing Medical University, Chongqing 400014, China; 2Laboratory of Stem Cell Biology and Therapy, Children’s Hospital of Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders, China International Science and Technology Cooperation Base of Child Development and Critical Disorders, Chongqing Key Laboratory of Pediatrics, Chongqing 400014, China
Abstract: The aim of this study is to investigate the changes of cytokines derived from pathologic microenvironment of cholestatic cirrhosis in different time points of common bile duct ligation mices， and to find the optimal combination of cytokines to induce liver stem cells HP14-19 efficiently to differentiate into mature hepatocytes in vitro. The Balb/c mices underwent choledochal ligation (BDL) to simulate the microenvironment of cholestatic cirrhosis， the levels of EGF， HGF and TGF-α in liver tissue of mice with common bile duct ligation detected by immunohistochemistry. The mouse embryonic liver stem cells HP14- 19 cells were employed in this study. ALB-Gluc assay was performed to evaluate ALB synthesis ability at different concentrations and different time points. qRT-PCR and Western-blot were used to detect the expression of differentiated cell markers AFP， CK18， ALB. ICG uptake and PAS staining were carried out to detect the metabolism and synthesis function of induced HP14-19 cells. Mouse choledochal ligation can successfully simulate cholestatic cirrhosis， and increase the degree of cirrhosis with the ligation time. Compared with the control， the activity of ALB-Gluc in HP14-19 cells was enhanced after treatment of EGF (10 ng/mL)， HGF (20 ng/mL) and TGF-α (20 ng/mL) alone， and markedly enhanced in EGF (10 ng/mL)， HGF (20 ng/mL)， TGF-α (20 ng/mL) in combined treatment. The results of qRT-PCR and Western blot showed that ALB and CK18 expression increased as the growth of the time and AFP level was opposite， and ICG uptake and PAS staining were significantly increased. The results suggested that cholestasis of liver cirrhosis pathological microenvironment-derived cytokines could effectively promote the differentiation of liver stem cells into mature hepatocytes， and it might have a certain guiding role in the treatment of cholestatic cirrhosis through cytokines combined with liver stem cell transplantation.
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