香豆雌酚对大鼠骨髓基质干细胞增殖和成骨分化的调控研究

Effect of the phytoestrogen coumestrol on the proliferation and osteogenic differentiation of rat bone marrow stromal cells

目的观察香豆雌酚（coumestrol）对大鼠骨髓基质干细胞（bone marrow stromal cells BMSCs）增殖和成骨分化的影响，探讨香豆雌酚对骨髓基质干细胞的调节及其对骨骼系统的影响。方法用不同浓度（0，10^-10～10^-5mol·L^-1）香豆雌酚干预6—8周雌性SD大鼠的骨髓基质干细胞，并设17β-E2（17β雌二醇，10^-8mol·L^-1）为阳性对照，在干预不同时间分别用MTT法测细胞增殖率、Von kossa染色鉴定钙化结节形成、全自动生化仪测碱性磷酸酶（Alkaline phosphatase，ALP）活性、酶消化法测羟脯氨酸（Hydroxyproline，Hyp）含量、放免法测骨钙素（Osteocalcin，OC）含量。并用Western-Blot法测干预7天时骨保护素（Osteoprotegerin，OPG）蛋白的表达，用Reahime-PCR法测干预7天、14天时OPG、RANKL（receptor activator of nuclear factor-κB ligand，核因子κB受体活化因子配基）基因的表达。结果①细胞增殖：干预24、48、72h，10^-8、10^-7mol·L^-1香豆雌酚均能显著促进大鼠骨髓基质干细胞的增殖。而10^-5mol·L^-1香豆雌酚在干预24、72h抑制细胞增殖。②成骨分化：10^-8mol·L^-1香豆雌酚干预25天后开始出现钙化结节；10^-8、10^-7mol·L^-1香豆雌酚干预组ALP在14、21天时显著增加、Hyp在28天时显著增加，10^-8mol·L^-1香豆雌酚干预组OC表达在28天时显著增加，高浓度（10^-5mol·L^-1）则呈现抑制作用。③OPG／RANKL表达：干预7、14天，10^-7mol·L^-1香豆雌酚能增加骨髓基质干细胞OPGmRNA及其蛋白的表达，提高OPG／RANKL基因的相对表达。结论香豆雌酚能促进大鼠骨髓基质干细胞的增殖和成骨分化，并能通过影响OPG／RANKL的表达来发挥骨骼系统的保护作用。

Objective To study the effect of coumestrol on proliferation and osteogenic differentiation of bone marrow stromal cells（BMSCs） of Sprague-Dawley rat, and to discuss the regulative effect of coumestrol on bone marrow stromal cells and skeletal system. Methods BMSCs obtained from 6 - S-week-old female SD rat were incubated in presence of various concentrations of coumestrol （0, 10^-10 - 10^-5 mol·L^-1）, or 10^-8 mol·L^-1 17β- E2 for positive control. MTT assay was used to determine the proliferation state of cells at the 24th, 48th, 72th hour under the treatment. Calcium nodes were seen by Von Kossa staining. Alkaline phosphatase （ALP） activity was detected by automatic biochemistryAnalyzer. Hydroxyproline （Hyp） was measured by enzyme digestion. Osteocalcin （OC） was measured by radioimmunity （RI）. Western-blot assay was used to determine the Osteoprotegerin （OPG） protein at the 7th day. RealTime-PCR assay was used to determine the OPG and RANKL mRNA expression level of the cells at the 7th and lgth day. Results （1） Cell proliferation： after treated for 24, 48,72 h, 10^-8, 10^-7 mol·L^-1 treatment groups have significantly higher level and for 24,72h, 10^-5 mol·L^-1 treatment group has significantly lower level than the negative control group （ P 〈 0.0001 ）. （2） osteogenic differentiation： BMSCs induced to osteogenesis formed mineralized nodular structures after 25 days in 10^-8 mol·L^-1 coumestrol group; Cell ALP activity was significantly increased at the lgth and 21th day treated with 10^-8, 10^-7 mol·L^-1 coumestrol; Hyp content was increased by 10^-8 ,10^-7 mol·L^-1 coumestrol at the 28th day, but was lower than the 17β-E2 group, and 10^-5 mol·L^-1 coumestrol significantly decreased the Hyp content; 10^-8 mol·L^-1 coumestrol increased OC expression at the 28th day, and 10^-5 mol·L^-1 coumestrol decreased it（ P 〈 0.0001）. （3）OPG/RANKL expression： 10^-7 mol·L^-1 coumestrol significantly increased OPG protein expression and the ratio of OPG/RANKL gene expression at the 7th and 14th day. Conclusions coumestrol has an enhancing effect on the proliferation and osteogenic differentiation of bone marrow stromal cells, and it also can protect the whole skeletal system by regulating OPG/RANKL expression.