锶盐联合淫羊藿苷对大鼠骨髓基质干细胞成骨及成脂分化影响的研究

The effects of strontium ranelate and icariin on the differentiation of mesenchymal stem cells in vitro

[目的]探索锶盐联合淫羊藿苷对超生理剂量糖皮质激素作用下大鼠骨髓基质干细胞(mesenchymal stem cells,MSCs)成骨、成脂分化的影响,并探讨Wnt/β-catenin信号通路在其中的调控作用。[方法]采用全骨髓培养法获取1周龄SD大鼠乳鼠MSCs,传至第3代时分为对照组、激素(终浓度:10-6mol/L)组、激素+淫羊藿苷(终浓度:10μg/L)组、激素+雷奈酸锶(终浓度:3 mmol/L)组及激素+淫羊藿苷+雷奈酸锶组。培养满21 d后进行HE、油红O、茜素红及ALP染色检测,RT-PCR检测Wnt信号通路相关因子(DKK-1、Wnt7b、GSK-3β、β-catenin、Runx-2和PPAR-γ2)基因的mRNA相对表达量。所有实验重复3次。[结果]所有组的细胞培养至21 d后,油红O染色结果显示激素组低倍镜下出现片状红染区,高倍镜下可见红染圆形脂滴,明显多于其他组;茜素红染色结果显示激素组未见明显紫红色阳性区域,而其他组可见片状阳性区域,由集落为中心放射性向四周生长,明显多于激素组,且联合组的阳性结果更明显;ALP染色结果显示激素组未见明显紫色阳性区域,而其他组可见片状阳性区域,明显多于激素组;RT-PCR结果显示激素组的Wnt相关因子(Wnt7b、GSK-3β和β-catenin)及成骨相关因子Runx-2显著低于其他组(P<0.05),而激素组的Wnt信号通路特异性抑制因子DKK-1及成脂相关因子PPAR-γ2显著高于其他组(P<0.05)。[结论]超生理剂量地塞米松可以通过抑制Wnt信号通路,诱导MSCs成脂分化,抑制其成骨分化,这可能与激素诱发股骨头坏死的发生、发展密切相关。而淫羊藿苷及锶盐可以在一定程度上减少超生理剂量糖皮质激素对Wnt信号通路的抑制作用,进而促进MSCs成骨分化、抑制其成脂分化,且二者联合应用的效果更好,这为研究激素性股骨头坏死早期防治提供了新的思路及方法。

[ Objective] This study aimed to explore the effects and mechanism of strontium ranelate and icariin on the dif- ferentiation of MSCs induced by super physiological dose of glucocorticoid in vitro. [ Methods ] The whole marrow method was used to culture the MSCs from one -week old SD rat, and the cells were subeuhured to the third generation in the cell culture bottles or 6 wells culture clusters and then were randomly devided into 5 groups： ①the control group, cultured in DMEM （L） medium with 10% FBS; ②the dexamethasone group, cultured in DMEM （L） medium with 10 ～ 6 mol/L dexamethasone; ③ the icariin group, culture in DMEM （L） medium with dexamethasone and 10μg/L icariin; ④the strontium ranelate group, cultured in DMEM （L） medium with dexamethasone and 3mmol/L strontium ranelate. [ Results ] The red oil O staining showed that there were red stained areas in the dexamethasone group, lipid droplets in the cytoplasm were stained into red, while no red stain areas were observed in other groups. The alizarin red staining showed that there were few positive stained areas in the dexamethasone group, while more positive stained areas were observed in other groups, especially in strontium ranelate and icariin groups. The alkaline phosphatase staining showed that there were few positive stained areas in the dexamethasone the strontium ranelate and icariin can partially suppress the adipogenic differentiation of MSCs and make the MSCs differentiating into osetoblasts. Moreover, combined application of strontium ranelate and icariin has a better effect.