Cell-cell communication between multiple-cell populations is believed to be important for the activation of many cellular functions. Previously, we showed that, in the rat hepatoma cell line Fao, grown in a layered co-culture system with a human umbilical vein endothelial cell (HUVEC) sheet, the expression of albumin and apolipoprotein A-I increased time-dependently for 10 days and was maintained at a significantly higher level than Fao without the HUVEC sheet. Because the gene-expression profile of hepatocytes and HUVECs under double-layered co-culture has not previously been elucidated, in the present study, we examined the difference in messenger ribonucleic acid expression between Fao or HUVEC monolayer cells and double-layered co-cultured Fao cells/HUVECs using suppression subtractive hybridization. More than 200 transcripts were differentially screened to ensure unique expression. The expression levels of genes in the co-cultured and monolayer cells were determined using SYBR Green I real-time reverse transcription polymerase chain reaction with species-specific primers. We found 23 genes that showed at least a 2-fold difference in expression level. Five hepatocyte-specific genes-α1-acidglycoprotein, α2-microglobulin, hepcidin, transferrin, and trausthyretin-were identified from the Fao cells. Two cell-surface protein genes-bone morphogenetic protein receptor type II and CD82-which may be related to cell-cell communication, showed greater expression in the HUVECs co-cultured with Fao cells. These results indicate that many hepatocyte and endothelial cell functions increase in intensity upon layered co-culture.
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