黄芪多糖诱导大鼠骨髓间充质干细胞分化的特性

Induction of Astragalus Polysaccharides on Differentiation of Rat Bone Marrow Mesenchymal Stem Cells

目的观察黄芪多糖对大鼠骨髓间充质干细胞(BMSCs)向神经细胞、脂肪细胞、成骨细胞和软骨细胞分化特性的影响,为开发有效而又低毒的分化诱导剂提供依据。方法采用全骨髓贴壁筛选法分离、纯化无特定病原体级Wistar大鼠BMSCs。用噻唑蓝(MTT)法筛选出黄芪多糖的合适浓度,取F3代细胞,采用随机数字表法分为对照组和诱导组(神经诱导、成脂诱导、成骨诱导、软骨诱导),采用甲苯胺蓝染色、油红O染色、茜素红染色检测神经细胞、脂肪细胞、软骨细胞表面特异性标记物。用Western Blot技术检测神经元特异性烯醇化酶(NSE)、脂蛋白酯酶(LPL)、胶原蛋白Ⅰ、胶原蛋白Ⅱ。分析黄芪多糖对F3代BMSCs向神经、脂肪、软骨和骨的诱导分化作用。结果 MTT显示,在1 g/L黄芪多糖诱导48 h下细胞增殖明显,甲苯胺蓝染色阳性,而油红O、茜素红染色阴性。Western Blot法检测显示NSE为阳性表达,LPL、胶原蛋白Ⅰ、胶原蛋白Ⅱ均为阴性表达。结论黄芪多糖可诱导大鼠BMSCs定向分化为神经细胞,而未向脂肪细胞、成骨细胞和软骨细胞分化。

Objective To investigate the effect of astragalus polysaccharides （APS） on the differentiation of rat bone marrow mesenchymal stem cells （BMSCs） to neurones, adipocytes, osteoblasts and chondrocytes, and provide basis for the development of effective and low toxic differentiation inducing agents. Methods BMSCs were isolated from SPF Wistar rats, purified, expanded and cultured to family 3. The appropriate concentration of APS was filtered out by MTT assay. The F3 cells were randomly divided into control group and induced group （neural induction, adipogenic induction, osteogenic induction, cartilage induction）. The effects of APS and classical chemical drugs on differentiation were measured by toluidine blue, oil red o and alizarin red staining. The protein expression of NSE, LPL, collagen Ⅰ and collagen Ⅱ were examined by Western Blot. Results MTT assay showed that 1 g/L APS promoted the proliferation better than other concentrations especially in 48 hours. The morphologic change of the cell from BMSCs was uniformly positive to toluidine blue staining, and was negative to oil red o and alizarin red staining. Western blot showed that the protein expression of the cell from BMSCs was positive for NSE but negative for LPL, collagen Ⅰ and collagen Ⅱ. Conclusion BMSCs induced by APS can differentiate to neurone and fail to differentiate to adipocytes, osteoblasts and chondrocytes in vitro.