骨碎补水提液对大鼠MSCs骨向分化的影响及机制研究

Effects of Drynariae Rhizoma Water-extraction on the Ability of Osteogenic Differentiation and It's Mechanism

目的:通过观察骨碎补水提液对SD大鼠骨髓间充质干细胞(MSCs)骨向分化能力及转化生长因子β1(TGF-β1)、骨形态发生蛋白2(BMP-2)表达的影响,阐释其可能的诱导机制。方法:运用全骨髓贴壁法分离、纯化大鼠MSCs;运用MTT法检测不同浓度的骨碎补水提液对SD大鼠MSCs增殖活力的影响;以ALP活性及ALP染色阳性率确定骨碎补水提液最佳促MSCs骨向分化浓度,并以该浓度对MSCs骨向分化进行干预,按是否添加经典成骨诱导液将实验分为:空白对照组、经典成骨诱导液组、骨碎补水提液组、骨碎补水提液+经典成骨诱导液组,通过检测碱性磷酸酶(ALP)、Ⅰ型胶原(ColⅠ)、骨钙素(BGP)和钙化结节的表达,评价各组骨向分化能力;通过检测转化生长因子β1(TGF-β1)、骨形态发生蛋白2(BMP-2)的表达,探讨药物促MSCs骨向分化的作用机制。结果:骨碎补水提液最佳促MSCs增殖浓度为5μg/mL;最佳促MSCs骨向分化浓度为50μg/mL;经典成骨诱导液、骨碎补水提液及联合用药均能促进MSCs骨向分化及TGF-β1和BMP-2的表达。结论:补肾中药骨碎补能有效的促进MSCs增殖和骨向分化。在MSCs骨向分化过程中,上调TGF-β1、BMP-2的表达量可能是诱导各组促进MSCs骨向分化的作用机制之一。

Objective：To investigate the effects of Drynariae Rhizoma water-extraction on the ability of osteogenic differentiation and the expression of TGF-β1 and BMP-2 in inducing mesenchymal stem cells （MSCs） differentiation into osteoblast and its mechanism. Methods：MSCs were isolated and purified by differential time adherent method ;The most effective concentration of the Drynariae Rhizoma water-extraction on the proliferation and ostogenic differentiation of MSCs was confirmed by MTT method and the activity and positive staining of alkaline phosphatase （ALP）respectively;According to different induced condition, MSCs were divided into 4 groups： control group, classic group（ induced by the classic osteoblast-induced system）, Drynariae Rhizoma water-extraction group （induced by the most effective concentration of Drynariae Rhizoma water-extraction on differentiation into osteoblast） and Drynariae Rhizoma water- extraction ＋ classic group（ induced by the combination of classic osteoblast-induced system and the most effective concentration of Dry- nariae Rhizoma water-extraction on differentiation into osteoblast）. ALP, type I collagen （Col I ）, bone gla protein （BGP） and calcium nodes in each group were detected and compared to indicate the osteogenic differentiation of each group. TGF-β1, and BMP-2 were detected by ELISA. Results：The most effective concentration of the Drynariae Rhizoma water-extraction on the proliferation and osteogenic differentiation of MSCs was 5 μg/mL and 50 μg/mL, respectively. The classic group, Drynariae Rhizoma water-extraction group and Drynariae Rhizoma water-extraction ＋ classic group could promote the osteogenic differentiation of MSCs and increase the expression of TGF-β1 and BMP-2. Conclusion：The Drynariae Rhizoma water-extraction can promote the proliferation and osteogenic differentiation of MSCs. Epimedium water-extraction can improve the differentiation of MSC into osteoblast and its mechanism may be related to increasing the expression of TGF-β1 and BMP-2