Primary rat hepatocyte isolation and culture regulates the SREBP/SREBP target gene profile

The major limitation of using primary hepatocytes is the rapid dedifferentiation of hepatocytes during isolation and culture, whereby most of the liver-specific functions are lost. Despite the many efforts that have been made to optimize the culture condition, current primary cultured hepatocyte preparations still only retain partial hepatocyte phenotype observed in liver in vivo. To further optimize primary hepatocytes it is essential to understand the key factors that underlie the dedifferentiation process during primary hepatocyte culture. Up or down-regulation of key transcription factors has been observed during primary hepatocyte culture, which could lead to dramatic change of downstream target genes. In my study, the influence of primary hepatocyte culture on the expression of a more ubiquitously expressed transcription factor family called sterol regulatory element binding protein (SREBP) was examined. SREBP transcriptionally regulates genes involved in glucose metabolism, lipognesis and cholesterolgenesis, which are the three important functions of liver. I compared the expression of SREBP and putative SREBP target genes in two culture media- one of which was a basic support for primary hepatocyte culture (MM) and the second of which was . designed to sustain hepatocytes in a more differentiated state for a prolonged period (Hepatozyme™). I showed that mRNA encoding SREBP and putative SREBP target genes decreased during culture in MM, but that Hepatozyme™ selectively improved mRNA expression for SREBP-2 and NF-Y and all the putative SREBP target genes which contain SRE/NF-Y composite elements in their promoters (FAS, 514, HMGCR and squalene synthase). Genes that lacked the SRE/NF-Y structure within their promoters (SREBP-1a, GK, L-PK and PEPCK) showed a decreased mRNA profile during culture in both medium conditions. Transcription factors involved in the transcriptional activation of GK, PEPCK and SREBP-1a (including LXR-a, HNF-4a and Sp1) also exhibited a decreased profile during culture in both medium conditions. My data indicates that improved expression of specific transcription factors during primary hepatocyte culture could underlie the recovery of a subset of downstream target genes. However, the number of improved transcription factors was small, which suggests that further improvement of the expression of transcription factors would be an important aspect for the maintenance of primary hepatocyte gene expression. My studies have provided an approach of understanding the relationship between the status of key transcription factors with that of downstream target genes during primary hepatocyte culture, and this could be a good starting point of understanding a broader aspect of key transcription factors that is involved in the maintenance of global gene expression during primary hepatocyte culture.EThOS - Electronic Theses Online ServiceGBUnited Kingdo