雷奈酸锶对卵巢切除大鼠BMSCs成骨分化及BMP-2表达的影响

Effects of strontium ranelate on the osteogenic differentiation and expression level of BMP-2 in BMSCs from ovariectomized rat

目的探讨雷奈酸锶(SR)对卵巢切除大鼠骨髓基质干细胞成骨分化及骨形态发生蛋白-2(BMP-2)表达的影响。方法3月龄SD大鼠24只,分别接受假手术(Sham组,n=8)和双侧卵巢切除(OVX,n=16)手术,术后8周OVX大鼠分为2组,分别接受安慰剂(OVX+V组)或SR(0.9g·kg-1·d-1,OVX+SR组)治疗3个月。实验结束后处死所有大鼠,检测第4腰椎骨密度、最大载荷、弹性模量等生物力学性能,取股骨和胫骨骨髓细胞进行体外培养并向成骨细胞诱导分化,第25天共聚焦显微镜观察BMP-2的表达及分布,第28天提取细胞RNA及蛋白,RT-PCR及Western blot法检测BMP-2的表达,细胞培养第32天von kossa染色观察细胞外基质矿化能力。结果 (1)骨密度:OVX+V组显著低于Sham组和OVX+SR组(P<0.05),OVX+SR组显著低于Sham组。(2)最大载荷:OVX+V组显著低于OVX+SR组(P<0.05),OVX+SR组显著低于Sham组(P<0.05);弹性模量:OVX+V组显著低于Sham组和OVX+SR组(P<0.05)。(3)共聚焦显微镜观察各组细胞BMP-2的表达及胞内分布无明显差异。(4)RT-PCR及Western blot法检测BMP-2在各组的表达差异无统计学意义。(5)von kossa染色观察各组矿化面积百分比差异无统计学意义(P>0.05)。结论 SR可部分改善卵巢切除大鼠骨量丢失和力学性能,对其骨髓基质干细胞体外成骨分化能力及BMP-2的表达无显著影响。

Objective To reveal the effects of strontium ranelate on the osteogenie differentiation and expression level of BMP-2 in BMSCs from ovariectomized rat. Methods A total of 24 SD rats were randomly divided into 3 groups of 8 animals in each..Sham group （n=8）,the other 16 rats were subjected to ovariectomized（OVX） groups, 8 weeks after ovariectomy operation, OVX rats were administered with strontium ranelate （0.9 g · kg-1 · d-1 , OVX＋ SR） or vehicle as control（OVX＋ V） ,all rats were sacrificed 3 months later. The bone mineral density and biomechanical properties were analyzed in 4th lumbar vertebrae. Bone mar- row cells were harvested and cultured in vitro in osteogenic-differentiation induced medium,the expression and cellular distribution of bone morphometric protein-2 were analyzed by confocal microscope on 25th day, realtime PCR and Western blot were used to as- sess the expression levels of BMP-2 on 28th day,yon kossa staining was performed on 32th day to observe the extracellular minerli- zation. Results （1）BMD：OVX rats showed markedly decreased BMD comparing to Sham and OVX＋SR group（P〈0.05）. （2）Bio- mechanical test： The maximal load in OVX＋ V group was significantly lower than OVX＋ SR group（P〈0.05）, and the maximal load in OVX＋SR group was significantly lower than Sham group（P〈0.05）. Elastic modulus in OVX＋V group was significantly lower than Sham and OVX＋ SR groups. （3）No significant difference between any two groups were found of the distribution of BMP-2 by confocal microscope（P〈0.05）, similar results were found in real-time PCR and Western blot analysis for the expressioia levels of BMP-2,as well as the detection of extracellular matrix mineralization by von kossa staining. Conclusion Strontium rane- late could partially prevent the bone.loss and the deterioration of the biomechanical properties in OVX rat,but could not affect the osteogenic differentiation and expression level of BMP-2 in BMSCs.