Role of PI3K/Akt Pathway in MMC Induced Apoptosis of Liver Stem Cells / 华中科技大学学报(医学版)

Objective To investigate the role of PI3K/Akt pathway in mitomycin(MMC) induced apoptosis of liver stem cells.Methods Rat liver stem ceils were stimulated with MMC,and the effect of MMC on the apoptosis rate of WB-F334 cells at different time points(6,12,24 h),as well as the effects of different concentrations of (0.2,0.4,0.6,0.8,1.0 mg/mL) MMC on the cytotoxicity of WB-F334 cells were evaluated;moreover,cells were treated with p38 MAPK inhibitor and PI3K/Akt inhibitor,and the roles of MAPK and PI3K/Akt signaling pathway in MMC mediated apoptosis of WB-F334 cells were explored.Results The apoptosis rate of the MMC-treated WB-F334 increased with time(P＜0.05).Compared with the un-treated control group,different concentrations of MMC had obvious cytotoxicity on liver stem cells,and the cytotoxicity increased with concentration.Western blotting results showed that Akt and MAPK in WB-F334 cells were significantly phosphorylated 15 min after MMC stimulation;the degree of phosphorylation decreased with time,and phosphorylation disappeared after 60 min,suggesting that the p38 MAPK,PI3K/Akt pathway can be activated by MMC;furthermore,when p38 MAPK inhibitor was added to MMC treated cells,the apoptosis rate of p38 MAPK inhibitor treated cells showed no significant difference compared to the un-treated cells(P＞0.05),indicating that the MAPK pathway had no significant effect on MMC induced WP,-F334 cell death;however,when the PI3K/Akt inhibitor(API-2)was added,the apoptosis rate of API-2 treated cells was significantlv decreased compared to the un-treated cells(P＜0.05),indicating that the PI3K/Akt pathway had a significant effect on MMC induced apoptosis of WB-F334 cells(P＜0.05).Conclusion Stimulation of MMC can induce apoptosis of hepatic stem cells WB-F334 through the activation of the PI3K/Akt signaling pathway.

Mechanism of the transforming growth factor-â induction of fibronectin expression in hepatic stem-like cells

Transforming growth factor-â1 (TGF-â1) plays an important role in the fibrogenic process in the liver. The aim of the present study was to explore the action of TGF-â1 on fibronectin expression in rat hepatic stem-like cells and the underlying mechanisms. The level of fibronectin expression was determined in hepatic stem-like cells (WB cells) before and after TGF-â1 stimulation by RT-PCR and Western blot methods. Using immunogold transmission electron microscopy and the Western blot method, we observed the result of the expression and the distribution of cAMP, phosphorylated Smad3 and Smad7 before and after TGF-â1 treatment. The levels of fibronectin expression in both mRNA and protein increased 4- to 5-fold after TGF-â1 stimulation, reaching an optimum level after 8 h and then gradually falling back. Similarly, TGF-â1 stimulation resulted in an increase of cAMP in WB cells, peaking at 8 h. After treatment with TGF-â1 for 24 h, the expression of cAMP gradually decreased. In addition, we found that TGF-â1 treatment also contributed to the increased expression and to changes in cellular distribution of phosphorylated Smad3 (translocation from the cytoplasm to the nucleus) and Smad7 (translocation from the nucleus to the cytoplasm) in WB cells. The present study demonstrates that TGF-â is involved in the fibrogenic process in hepatic stem cells through up-regulation of fibronectin expression, and the mechanisms underlying this process may be associated with the activation of cAMP and Smad pathways.

Effects of in vitro suspension culture in soft agar medium on differentiation of embryonic hepatic stem cells / 中华肝胆外科杂志

Objective To develop an ideal cultural method to amplify embryonic hepatic stem cells and inhibit their differentiation in vitro. Methods Suspension of ED 14 Fischer (F) 344 rat em-bryonic hepatic stem cells was prepared by collagenase digestion and mechanical disaggregation. Then cells were divided into two groups randomly. The cells in group 1 were seeded into type I collagen-coated plates by adherent culture while those in group 2 were seeded into soft agar medium by suspen-sion culture. After culture for 2 weeks, the morphology and ultrastructure of cells in both groups were observed and compared by inverted microscope and transmission electron microscope, respectivley.The expression of CD90. 1 and CD49F, the two specific stem cell surface markers, was tested by flow cytometry to manifest the establishment of embryonic hepatic stem cells. Alkaline phosphatase stai-ning was used to detect stem cell differentiation. Result Embryonic hepatic stem cells in group 2 were characterized by higher nucleus-cytoplasm ratio and less cell organelles, higher expression of CD90. 1 and CD49F, and stronger positive reaction for alkaline phosphatase staining compared with those in group 1. Moreover, the cells in group 1 showed significant differentiation features. Conclusion Em-bryonic hepatic stem cells cultured suspendedly in soft agar medium experience less differentiation than those adherently cultured in serum-added culture medium, and can proliferate and form clone ball with a specific stem cell feature.

Growth pattern and phenotype identification of hepatic stem cells from mice / 中华肝胆外科杂志

Objective To study the proliferation and differentiation patterns of hepatic stem cells from mice cultured in vitro isolated identify their characteristics.Methods Mice were divided into 5 groups according to the pregnant (embryo days,ED13,ED15 and ED18) or born age (day0 and 3).Hepatic stem cells were isolated and cultured in vitro.The amount of the stem cells as well as their growing situation and differentiation pattern were observed and compared among different groups and markers of stem cells (CD117,CD90.1,CD49f,c-Met),hepatic cells(AFP) were used to identify the cultures.Resulls The cells with best situation of growing as colonies were obtained from ED15 group.Their expression of specific markers suggested that they were hepatic stem cells.The stem cells isolated from ED15 mice in subculturing proliferated in line pattern and differentiated in reverse line pattern.The expression of AFP varied as normal distribution as cell differentiation development.Conclusion Most cells have characteristics of hepatic stem cells isolated from fetal liver and the number of these cells decreases gradually as embryo duration prolongs.The hepatic stem cells proliferate in line pattern and differentiate into hepatic cells after in vitro culture.

Hepatic Stem Cell Transplantation in Liver Failure

Whole or partial liver transplantation has become one of the main treatment strategies for hepatic failure. The availability of a donor liver is the single most limiting factor in liver transplantation. In cell therapy, liver cells have been used clinically to bridge patients to whole organ transplantation and/or as an alternative to whole organ transplants. The crucial property that defines a stem cell is its ability to give rise to a large family of descendants, and at least some hepatocytes do exhibit this pleuripotency. Thus hepatocyte-like cells derived from the bone marrow, embryonal stem cells, or placental sources can also be used. Stem cells of many types have been prodded and cajoled by combinations of growth factors, matrix and culture modifications to identify conditions that favor differentiation or transdifferentiation of stem/progenitor cells into hepatocytes. Additional studies are needed to determine whether hepatocyte-like cells derived from the bone marrow or other sources express the full complement of hepatic functions. If a sufficient number of differentiated hepatocytes can be produced from these stem cells, they could be useful in clinical transplantation programs.

Research Progress and Clinical Value of Hepatic Stem Cells and Its Relations with Liver Cancer / 中国普外基础与临床杂志

Objective To investigate the clinical outlook of hepatic stem cells and its relations with liver cancer. Methods The literatures of recent years on the studies of hepatic stem cells were reviewed. Results Liver cancer may consist of cells of various differentiation grades and it may result from the perodifferentiated hepatic stem cells or abnormal differentiated cells. Conclusion The hypothesis of hepatic stem cells has been identified extensively. Further study maybe helpful for revealing the origin, carcinogenesis of hepatic cancer, and may also be useful for the understanding of the mechanism of metastasis.

Origin of hepatic stem cells in human hepatocellular carcinoma / 第三军医大学学报

Objective To investigate the activation, distribution, origin, and expression of hepatic stem cells(HSC)in different histological types of primary liver carcinomas. Methods The histological and immunohistochemical features of 94 cases of hepatocellular carcinoma (HCC), 12 cases of intrahepatic cholangiocarcinoma (ICC) and 10 cases of mixed hepatocarcinoma were examined by HE staining and immunohistochemistry SP method, with 5 cases of sclerotic liver and 4 cases of normal liver tissues as control. Results HSC expression was observed and the transfor mation from HSC to carcinoma cell was also noted in the liver. CK7, CK19, c-kit, Thy-1, and AFP were found expressed in different types of hepatic carcinomas and the greatest intensive expression was found in the mixed hepatocarcinoma (P