Identification and characterization of brain isoform glycogen phosphorylase as a novel marker of rat hepatic progenitor cells

• Main Authors: Yu-Wen Huang, 黃育文
• Other Authors: Hsuan-Shu Lee
• Format: Others
• Language: en_US
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/53691600545932315880

博士 === 國立臺灣大學 === 生物科技研究所 === 103 === The liver is constantly renewing itself under normal conditions. With adult stem cells having been discovered in many of the body''s organs, one might assume that the tremendous regenerative capacity of the liver is contributed by liver stem/progenitor cells. Until now, at least four types of liver cells are considered as candidate of liver stem cells, including oval cells, hepatic stellate cells (also known as Ito cells), liver epithelial cells (liver epithelial cell), mesenchymal stem cells. However, it hasn’t been so easy to isolate in mammals due to limited liver progenitor cell markers. An adult rat liver progenitor cell line Lig-8 has been established, which is characterized like liver epithelial cell. In the induction of sodium butyrate, these cells were shown able to differentiate into both hepatocytes and bile duct cells expressing albumin and cytokeratin-19, the markers of respective cell types. We previously generated a monoclonal antibody using the whole Lig-8 cells as immunogen. The yielded monoclonal antibody, named as Ligab, belongs to IgG subclass G1 and κ-light chain. It specifically reacted to Lig-8 cells but it did not react to a rat hepatoma cell line H4IIE. Furthermore, the expression of Ligab antigen in Lig-8 cells declined under sodium butyrate (SB)-induced differentiation was found. We took advantage of Ligab antibody to immunoprecipitate the target antigen, and identify glycogen phosphorylase (GP) in Ligab immunoprecipitates by using liquid chromatography combined with tandem mass spectrometry. Three GP isoforms exist in mammals: the brain isoform (GPBB), liver isoform (GPLL), and muscle isoform (GPMM). Three isoform-specific polyclonal antibodies and antiserum were used to examine the existence of these GP isoforms in the liver progenitor cells. Immunoblotting results showed that GPBB was expressed in two liver progenitor cell lines Lig-8 and WB-F344. The level of GPBB expression decreased in the WB-F344 cells under sodium butyrate (SB)-induced cell differentiation, which was consistent with roles of GPBB as a liver progenitor cell marker. The short hairpin RNA (shRNA)-mediated GPBB knockdown followed by glucose deprivation test shows that GPBB aids in liver progenitor cell survival under the low glucose conditions. Furthermore, shRNA-mediated GPBB knockdown followed by SB-induced cell differentiation shows that reducing GPBB expression delayed liver progenitor cell differentiation. In conclusion, GPBB is a novel liver progenitor cell marker, which facilitates liver progenitor cell survival under the low glucose conditions and cell differentiation.