Effect Of Atorvastatin Calcium On Proliferation And OPG/RANKL System Of Osteoblast

Background:Statins is used to lower blood cholesterol at present clinically,but a retrospectie study showed that statins can not only make older women blood cholesterol decline,but also can make their hip density increase significantly. It hints that statin can improve the bone mineral density. The OPG/RANKL system is a newly discovered ,which plays an important role in the pathogenesis and treatment of osteoporosis.Objective:To research the effect of different concentrations atorvastatin calcium on cell proliferation and OPG(osteoprotegerin), RANKL(receptor activator of nuclear factor-κΒligand), M-CSF(macrophage-colony stimulating factor) gene expression of osteoblast in vitro,to explore the molecular mechanism of atorvastatin calcium anti osteoporosis,and to provide the experiment basis for clinical drug use. Methods:(1) The crinial bones were digesting by mixed enzyme which contains enzyme andⅠtype collagenase.The osteoblast were purified by selective plating technique. The osteoblasts were identified through morphlogy observation, alkaline phosphatase dyeing, collagenⅠimmunohistochemical staining. (2) The second generation of osteoblasts is employed. With different concentrations of atorvastatincalcium(10^-9 ,10^-8 ,10^-7,10^-6,10^-5mol/L)intervention osteoblasts, the cell proliferation is assayed using MTT after 24h,48h,72h. (3)With different concentrations of atorvastatin calcium(10^-9 ,10^-8 ,10^-7,10^-6,10^-5mol/L) intervention osteoblasts,the gene expression of OPGmRNA,RANKLmRNA, M-CSFmRNA was measured by Real-time fluorescence quantitative PCR. (4) Statistical methods: Experimental data are used mean±standard deviation to show ,SPSS17.0 statistical software is applied for statistical analysis.Single factor analysis of variance (ANOVA) is used for comparison between groups,when P﹤0.05, there were significant differences. Results:(1)The cultured cells have the typical features of osteoblast.The cells are triangle, polygonal, long spindle shape, etc.and mostly are mononuclear cells, have large pseudopodia.Alkaline phosphatase dyeing collagenⅠimmunohistochemical staining are both positive.(2) The result of MTT method shows that it hasn't statistically significant differences (P > 0.05) with different concentrations of atorvastatin calcium (10^-9 ,10^-8 ,10^-7,10^-6,10^-5mol/L) processing rat osteoblasts 24 hours. The medication groups'OD value are greater than the control group with different concentrations of atorvastatin calcium (10^-9 ,10^-8 ,10^-7.10^-6,10^-5mol/L) processing rat osteoblasts 48,72hours, it has statistically significant differences while the meication concentration are10-9 ,10-8 ,10-7 ,10-6mol/L (P﹤0.05), but it has not statistically significant differences among medication groups. (3) The result of real-time fluorescence quantitative polymerase chain reaction (PCR) shows that atorvastatin calcium (10-9 ,10-8 ,10--7,10-6,10-5mol/L) can promote OPG and M-CSF gene mRNA expression, restrain RANKL gene mRNA expression, and this effect may be concerned with medication'concentration and time.Conclusion:(1)A large quantity and activity of the good cells were obtained by mixed enzyme digestion method. High purity osteoblasts were obtained by selective plating technique that can remove impurities such as fibroblast cells.(2)Atorvastatin calcium can promote the osteoblast proliferation.Atorvastatin calcium can promote OPG and M-CSF gene's expression of osteoblast, inhibit RANKL gene's expression of osteoblast. These may be the important mechanism that atorvastatin calcium resistance of osteoporosis.