Designed human TaqMan assays (Applied Biosystems, Foster City, CA) click here were used to quantify gene expression of osteocalcin (BGLAP), osteoprotegerin (TNFRSF11B), RANKL (TNFSF11), and Cbfa1 (RUNX2). Quantitative PCRs were carried out using ABI-Prism 7900 HT Fast Real-Time PCR System and a
TaqMan 5′-nuclease probe method (Applied Biosystems). Results were expressed as relative expression of each gene (versus β-actin gene expression), using arbitrary units according to the comparative CT (threshold cycle) method.20 All real-time PCR reactions for each sample were performed in triplicate. The primers used are listed in Table 1. Data are expressed as mean ± standard deviation (SD). All analyses were performed with the SPSS version 14.00 statistical package (SPSS Inc., selleck chemical Chicago, IL). Significant differences between any two groups were determined by Student t test or Mann-Whitney U test. When multiple groups were compared, analysis of variance was used, followed by a Tukey’s multiple contrast test, where applicable. A P value ≤0.05 was considered significant. Nonpassage human primary osteoblasts, after synchronization, displayed the characteristic pattern of gene expression and protein production of osteoblastic differentiation markers such as osteocalcin gene expression and alkaline phosphatase activity (data not shown). Increasing concentrations
of unconjugated bilirubin in the culture media resulted in a progressive decrease in cell viability, which was observed particularly at concentrations higher than 100 μM at 48 hours and higher than 50 μM at 72 hours (Table 2). The cell viability decrease was 36% and 56%, at 50 and 100 μM, respectively, compared with nontreated cells. Moreover, the presence of bilirubin (10 μM) resulted in significantly better cell viability Reverse transcriptase compared with no bilirubin in the experiments performed at 48 hours and in the plates without FBS. These effects on cell survival were partially prevented by the presence of 10% FBS, because the detrimental effect of bilirubin at 50 and 100 μM was completely abolished
in the experiments with FBS. Actually, in these latter experiments, the decreased cell viability was only observed with bilirubin at 1000 μM (Table 2). Serum samples from patients and healthy subjects were added at 2%, 10%, and 20% concentrations in culture medium. Cell viability significantly decreased in samples with increasing concentrations of sera from jaundiced patients at 72 hours (Table 3), with viability decreasing by 19%, 18%, and 33% at 2%, 10%, and 20% concentrations, respectively. No significant effect was observed at the other time points, although there was a trend in the experiments performed at 48 hours. Moreover, no effect on cell viability was observed in the experiments performed with samples from patients who had normal bilirubin levels.