Objectives
Effects of insulin-like growth factor 1 (IGF1), fibroblast growth
factor 2 (FGF2) and bone morphogenetic protein 2 (BMP2) on the expression
of genes involved in the proliferation and differentiation of osteoblasts
in culture were analysed. The best sequence of growth factor addition
that induces expansion of cells before their differentiation was
sought.
Methods
Primary human osteoblasts in in vitro culture
were treated with IGF1, BMP2 or FGF2 (10 ng/ml) for 24 hours (IGF1)
or 48 hours (BMP2 and FGF2). Experiments were performed during the
exponential growth phase with approximately 1e7 cells per 75 cm2 flask.
mRNA was reverse transcribed directly and analysed using RT-PCR
Taqman assays. Expression levels of key genes involved in cell growth
and differentiation (CDH11, TNFRSF11B, RUNX2, POSTN, ALP, WNT5A,
LEF1, HSPA5, FOS, p21) were monitored using RT-PCR with gene-specific
Taqman probes.
