Z Gastroenterol 2004; 42 - AB_1_31
DOI: 10.1055/s-2004-816056

Differential effects of primary and immortalized hepatocytes on liver mesenchymal cells in co-cultures

J Dudas 1, V Bordoni 2, R Novosyadlyy 1, JG Scharf 1, A Elmaouhoub 1, I Aprigliano 1, B Saile 1, M Tripodi 2, G Ramadori 1
  • 1Abt. Gastroenterologie und Endokrinologie, Uniklinikum Göttingen
  • 2National Institute of Infectious Diseases „L. Spallanzani“, IRCCS, Roma, Italy

Aims: Hepatocytes are in tight contact with the hepatic stellate cells (HSC) of the liver sinusoid and with the myofibroblasts (rMF) of the pericentral and perivenous area. Hepatocyte-derived signals may influence the behaviour of these cells.

Methods: HSC, rMF and hepatocytes (HC) were isolated from rat liver. HSC and rMF were co-cultured with primary HC or with met-murine hepatocytes (immortalized, untransformed cell line, MMH) using cell culture inserts (transwell culture), or were treated with MMH or HC conditioned media for two days. Co-cultured and medium treated HSC and rMF were analysed by cell counting, 3H-thymidine incorporation, flow cytometry, northern blot, real time PCR and TUNEL apoptosis detection. IGF-1 expression of MMH was proved by RT-PCR and sequencing. IGF binding proteins (IGFBP) in HC and MMH were detected by western-ligand blot. IGF-1 expression in MMH was suspended by transfection with antisense oligonucleotide.

Results: Primary hepatocytes induced significant increase of HSC-DNA synthesis, while cell number did not increase. In rMF DNA synthesis increase was lower and proliferation was not induced. On the other hand MMH induced a significant decrease of HSC DNA synthesis and cell number, which was accompanied by apoptosis (TUNEL positivity). Furthermore, MMH induced increase of bax and p53, and decrease of IGF-1 receptor and cyclin E1 mRNA expressions in HSC. Oppositely, MMH cells induced significant proliferation (increase in DNA synthesis and cell number) in rMFs. IGF-1 gene expression of MMH cells and HC was proved. Transfection of MMH cells with IGF-1 antisense oligonucleotide could partly suspend the effects on HSC. HC expressed IGFBPs: 1, 2 and 4, while MMH in addition expressed IGFBP-3, the latter being the most abundant.

Conclusions: HC in primary cultures exert different effect on HSC and rMF when compared to MMH cells. This difference could be due to a different IGFBP gene expression.

Schlüsselwörter

IGF-1 - co-culture - hepatic stellate cell - hepatocyte - myofibroblast