香豆雌酚对新生大鼠成骨细胞增殖分化的影响

Effect of coumestrol on the proliferation and differentiation of neonatal rat osteoblasts

目的:观察香豆雌酚对大鼠成骨细胞(OBs)增殖分化的影响,初步探讨香豆雌酚对OBs的作用机制。方法:用不同浓度香豆雌酚干预OBs,根据不同时间分别用酶消化法测碱性磷酸酶活性和羟脯氨酸含量、放免法测骨钙素含量及半定量RT-PCR法检测OBs内OPG/RANKL基因的表达。结果:香豆雌酚呈浓度和时间依赖性促进大鼠OBs增殖及碱性磷酸酶表达;干预12 h,各浓度组羟脯氨酸含量呈浓度依赖性增高,其中10-8mo.lL-1作用最大(P=0.022);培养12 h,OBs表达骨钙素呈浓度依赖性,24 h达高峰,其中10-8mol.L-1有显著性差异(P=0.012),48 h有所下降;不同浓度、时间组香豆雌酚均能促使OBs增加表达OPG mRNA,同时有抑制RANKL mRNA表达的作用,其在24 h 10-8mol.L-1浓度作用强度最大(P=0.038),10-5mol.L-1则呈现抑制作用。结论:香豆雌酚能促进OBs的增殖分化,其作用呈浓度和时间依赖性,且至少部分有OPG/RANKL途径的参与。

Objective To observe the effect of coumestrol on the proliferation and differentiation of neonatal rat osteoblasts and study the mechanism of coumestrol to the osteoblasts （OBs）. Methods The effects of coumestrol to the OBs were observed according to the different time and concentration. Alkaline phosphatase activities and Hydroxyproline concentration were measured by enzyme digestion. Osteocalcin （OC） was measured by radioimmunity. Total cellular RNA was isolated with TRIzol. Semiquantitative RT-PCR method was used to determine the expression of osteoprotegerin （OPG） mRNA and RANKL mRNA. Results Coumestrol resulted in a dose- and time-dependent increase in cell proliferation as measured by MTr, and stimulated expression of alkaline phosphatase activity. Hydroxyproline contents increased by coumestrol dose-dependently. Coumestrol （ 10^-8 mol·L^- l ） increased OC expression at 24 h （ P = 0.012 ）. Coumestrol （10^-8 mol·L^- l） increased OPG mRNA expression （P =0.015 ） and decreased RANKL mRNA expression （P = 0. 038 ） after co-treat for 24 h. Conclusion Coumestrol has an enhancing effect on the proliferation and differentiation of neonatal rat osteoblasts by dose- and time-dependently, at least in part, by stimulating OPG/RANKL expression in osteoblasts.