Effects Of Arsenic Trioxide On The Proliferation, Apoptosis And The Expression Of BMP-2 Gene In Osteoblasts Cultured In Vitro

Objective:The aim of this study was to observe the impacts of different doses of arsenic trioxide for morphological characteristics on osteoblasts cultured in vitro, Detect the impacts of arsenic trioxide for proliferation and apoptosis of osteoblasts and for BMP-2 gene expression of osteoblasts; We investigate the mechanism of arsenic for growth and metabolism of osteoblasts to provide the experimental evidence of the toxicological effects and application of pharmacology of arsenic trioxide.Methods:Different concentrations of As2O3(100μmol/L, 10μmol/L, 1μmol/L,0.1μmol/L) acted osteoblasts cultured in vitro, we made the following test:Observe the morphology of osteoblasts; MTT Assay tested the proliferation of osteoblasts; observed apoptotic morpho-logy of osteoblasts by fluorescence microscope; AnnexinV-FITC/PI double staining and TUNEL Assay tested apoptosis of osteoblasts; RT-PCR tested the BMP-2 gene expression of osteoblasts.Results:1) Morphological observation showed that:osteoblAstic nuclear of 100μmol/L hAs caryolysis and cytoplAsmic vacuolization phenomenon,and it can not distinguish between the cytoplAsm and nucleus, some osteoblasts of 10μmol/L become smaller and shrink, nuclear condensation, fragmentation, cell membrane folds, and depression; the large number of osteoblasts of 1μmol/L was split phase; osteoblasts.of 0.1μmol/L are close connection.2)MTT Assay showed that OD Value of 100μmol/L and 10μmol/L groups was Significantly lower than the control group, OD Value in 72hours was lower than OD Value in 48hours,which had Statistical difference (P<0.01) OD Value of 1μmol/L group Significantly higher than the control group. OD Value in 72hours was higher than OD Value in 48hours, which had Statistical difference (P<0.01). Comparing OD Value of 0.1μmol/L group in 72hours with 48hours had no Statistical difference(p>0.05), the same As control group;3)AnnexinⅤ-FITC/PI and TUNEL assays showed that compared with the control group(8.13%,12.12%), apoptotic rate (10μmol/L)increased which had Statistical difference (P<0.01), apoptotic rate (1μmol/L) reduced which had Statistical difference (p<0.05).apoptosis rate (0.1μmol/L)had no Statistical difference(P>0.05);4)RT-PCR assay showed that BMP-2 gene expression of 10μmol/L group in 72hours was significantly lower than in 48 hours and BMP-2 gene expression of 10μmol/L group was Significantly lower than the control group which had Statistical difference (P<0.01);BMP-2 gene expression of 1μmol/L group hAs no significantly difference between in 48 hours and in 72h and the same as control group (P>0.05). BMP-2 gene expression of 0.1μmol/L group in 72hours was significantly higher than in 48 hours and BMP-2 gene expression of 10μmol/L group was Significantly higher than the control group which had Statistical difference (P<0.01)Conclusions:There is a bidirectional effect of As2O3on the proliferation and apoptosis of rat osteoblasts cultured in virtro:the osteoblasts treated with 1μmol/L AS2O3 it's proliferation is promoted,the vitality is increased and the cell apoptosis is decreased; while treated with 10μmol/L As2O3,the cell growth is inhibited,the vitality is decreased and the cell apoptosis is increased; A certain concentration of As2O3 (0.1μmol/L) can enhance the expression of osteoblast BMP-2 gene and promote the function of osteoblastic bone formation, while with the higher concentration (10μmol/L) it inhibits the expression of BMP-2 gene, decreases the bone formation capacity.