Embryonic Stem Cells Secreted Cytokines Promoted Liver Fibrosis And Mechanisms

Part Ⅰ Embryonic stem cells secreted cytokines regulated liver fibrosis associated cellsObjective:Hepatic stellate cells (HSC) and hepatocytes play important roles in liver fibrosis, especially HSC, which can be activated in the early stage of liver fibrosis, becoming as myofibroblast-like cells. It can secrete proteins of extra cellular matrix (ECM), and inhibit the degradation of ECM. In order to explore the effect of embryonic stem cells to liver fibrosis, we firstly investigated its role to HSC.Methods:It will form teratoma if embryonic stem cell is directly transplanted into body. To avoid the tumorigenesis, we used the conditional culture medium (ESC-S) containg secretion from embryonic stem cells. After the treatment of ESC-S, we observed the changes of cell morphology, examined the difference of gene and protein expression. Results:ESC-S could activate HSC, promote the proliferation of HSC, the expression of α-SMA and desmin was highly up-regulated. Epithelial-Mesenchymal transition might play a role in liver fibrosis. The secretion of M1macrophage could activate HSC, but the secretion of M2c macrophage could inhibit HSC. When it came to hepatocytes, ESC-S could induce apoptosis of hepatocytes.Conclusion:ESC secreted cytokines could up-regulate HSC, while injured liver function. Part Ⅱ The influence of embryonic stem cells secreted cytokines to liver fibrosis in vivoObjective:We had realized that ESC-S could protome the activation and proliferation of HSC, to further investigate the effect, we did some study in vivo.Methods:We established the Carbon tetrachloride (CC14) induced liver fibrosis model. Firstly, we injected CC14and ESC-S together to find out the change of liver function. Then we injected ESC-S alone to explore the influence of ESC-S to liver fibrosis. We injected ESC-S three times a week, and lasted for6weeks.Results:In the ESC-S and CC14co-injected model, we found that HSC was more activated than that in control group, more collagen was produced too. While in the ESC-S injected model, the damage of liver function was more severe, and more macrophage was concentrated.Conclusion:ESC-S could not only initiate the process of liver fibrosis, enrich M1macrophage to liver and exacerbate liver function, but also could accelerate liver fibrosis once liver injury was caused by other factors. Part Ⅲ The possible mechanism of ESC-S induced liver fibrosisObjective:To explore the possbile mechanism of ESC-S induced liver fibrosis. Methods:We had found that ESC-S could down-regulate the gene expression of calcineurin and related signal proteins, so we assumed that lack of calcineurin played an importanat role in liver fibrosis. So we bought calcineurin knock out mice, CC14and common bile duct ligation were used to establish liver fibrosis model. Meanwhile,we exmined cytokines in ESC-S and possible singal pathways that might be involved.Results:CN-KO mice were more easy to get liver fibrosis compared to control group. More collagen was produced in CN-KO mice. We also found that vascular endothelial growth factor (VEGF) was mainly responsible for the profibrotic function of ESC-S. ESC-S could up-regulate TGF-β1in HSC, and TGF-β1could activate HSC via the transcription factor Smad2/3. It could also down-regulate calcineurin and increase STAT3in HSC, phosphorylated STAT3could activate HSC.Conclusion:Vascular endothelial growth factor played a critical role in ESC-S induced liver fibrosis. And, TGF-β1-Smad2/3and Calcineurin-STAT3signal pathways were mainly involved.