ABSTRACT
Aim To observe the effect of epidermal growth factor (EGF) on cell growth under different culture conditions using the real-time cell analyzer and EGF as a tool medicine to promote cell growth,and to provide reference for establishing pharmacokinetic model of IEC-6 cell growth (proliferation).Methods IEC-6 cell was inoculated on E-Plate 16 plate at a density of 1 × 104 cell/well and cultured in DMEM with 10％ serum for 24 h,then replaced by serum-free DMEM culture (serum starvation)for 20 h,then the effects of different culture conditions on cell growth as well as EGF efficacy were observed.Results ① When the serum concentration was 10％,the cell growth index of EGF group(1,10,100 μg · L-1) after drug administration 24 h,48 h and 72 h was P ＞ 0.05 compared with the blank group,suggesting that 10％ serum culture could not reflect the efficacy of EGF.② When the serum concentration was 0％,EGF (1,10 μg · L-1) improved cell growth inhibition caused by serum-free cultivation,but could not recover it to normal level (the EGF group after drug administration 24 h,48 h and 72 h was P ＜0.01 compared with 5％ serum),which suggested that serum-free culture could not reflect the EGF efficacy.③ 0％,0.5％,1％ serum had different effects on cell growth,of which 0.5％ serum could neither have obvious inhibition on cell growth,nor reflect the EGF effect due to promoting cell growth for a long time.④When the serum concentration was 0.5 ％,the cell growth index of EGF groups after drug administration 24h,48h and 72h was P ＜0.01 compared with the blank group,suggesting that 0.5％ serum culture could better reflect EGF efficacy.⑤The efficacy of EGF (10 μg· L-1) in promoting cell growth was confirmed by repeated validation of 0.5 ％ serum.Condusions A reference scheme of the IEC-6 cell growth (proliferative) pharmacological experimental model is established in the real-time cell analyzer:cells are cultured in DMEM with 10％ serum for 24h,then in serum-free DMEM (serum starvation) for 20h,then after the adding of reagent,cells are cultured in DMEM with 0.5％ serum for 48-72 h to observe its effect on cell growth (proliferation).
ABSTRACT
Objective To observe the effects of recombinant human parathyroid hormone 1 to 34(referred to as hPTH) on the expression level of alkaline phosphatase(ALP) and bone gla protein(BCP) in human osteosarcoma cell line SaOS-2(referred to as SaOS-2 cells). Methods SaOS-2 cells were subcultured and treated with 1, 10 and 100 nmol/L hPTH for 12, 24 and 48 h. Total cellular RNA was extracted, cDNA was synthesized by reverse doses of hPTH, different duration of action, and their interaction on the expression level of ALP mRNA of SaOS-2 cells was significantly different(F = 29.32, 2.92, 7.64, all P < 0.05). The expression level of ALP mRNA(0.78 ± 0.43, 0.71 ± 0.05, 0.75 ± 0.19, 0.76 ± 0.14) of SaOS-2 cells after treatment with 0, 1, 10 and 100 nmol/L hPTH for 48 h was lower than those of treated for 12 h(1.01 ± 0.16, 1.37 ± 0.38, 1.49 ± 0.16, 2.52 ± 0.70, all P< 0.05) and 24 h (1.80 ± 0.47, 1.30 ± 0.36, 1.27 ± 0.17, 1.17 ± 0.11, all P< 0.05). The expression level of ALP mRNA of SaOS-2 cells after treatment with 100 nmol/L hPTH for 12 hours was higher than that of the control(P < 0.05); the expression level of ALP mRNA of SaOS-2 cells after treatment with 1, 10 and 100 nmol/L hPTH for 24 h interaction on the expression level of BGP mRNA of SaOS-2 were significantly different (F = 8.26, 10.33, 5.51, all P< 0.05). The expression level of BGP mRNA(1.17 ± 0.28, 0.98 ± 0.08, 0.92 ± 0.17 and 0.84 ± 0.59) of SaOS2 cells after treatment with 0, 1, 10 and 100 nmol/L hPTH for 48 h was lower than those of treated for 12 h( 1.01 ± 0.14, 1.21 ± 0.18, 1.34 ± 0.30, 1.68 ± 0.62, all P< 0.05), and 24 h(1.71 ± 0.35, 1.41 ± 0.47, 1.28 ± 0.31 and 1.01 ± 0.18, all P < 0.05). The expression level of BGP mRNA of SaOS-2 cells after treatment with 100 nmol/L hPTH for 12 h was higher than that of those groups treated with 0 and 1 nmol/L hPTH(all P< 0.05). The expression level of BGP mRNA of SaOS-2 cells after treatment with 10 and 100 nmol/L hPTH for 24 h and 48 h was lower than those of the control(all P < 0.05). The expression level of BGP mRNA of SaOS-2 cells after treatment with 100 nmol/L hPTH for 24 hours was lower than that the group treated with 1 nmol/L hPTH(P < 0.05). Conclusions In vitro, hPTH significantly enhances osteogenic activities of human osteoblast in a short time, however, with prolonged stimulation time, osteogenic activity can show a downward trend.
ABSTRACT
Objective To investigate the effect of recombinant human bone morphogenetic protein 2 (rhBMP-2) on the osteogenic activities of human osteosarcoma cell line SaOS-2. Methods SaOS-2 cells were exposed to rhBMP-2 for 12,24,48 h at 0(control) ,2,20,200 μg/L, respectively. The mRNA expression of alkaline phosphatase(ALP) and bone gla(BCP) were detected by real time polymerase chain reaction. Results The mRNA expression of ALP and BGP of SaOS-2 cells increased gradually with rhBMP-2. The mRNA expression of ALP of the 20 μg/L group exposed for 48 h(1.60 ± 0.64), and the 200 μg/L group exposed for 12,48 h(1.70 ± 0.41, 1.80±0.19) were significantly higher than those of control (12 h: 0.80±0.25, 48 h: 0.74±0.21, allP<0.05). The mRNA expression of BGP of the 2 μg/L group exposed for 24 h(1.67 ± 0.33), the 20 μg/L group exposed for 12,24 h(2.42 ± 0.13,1.82 ± 0.14) and the 200 μg/L group exposed for 12,24 h(1.46 ± 0.11,1.24 ± 0.07) were significantly higher than those of control( 12 h: 1.01 ± 0.14, 24 h: 0.84 ± 0.12, all P< 0.05). Conclusions rhBMP-2 can promote the mRNA expression of ALP and BGP of SaOS-2 cells. They have a dose-response relationship, but represent a different dose-response effect.