当归多糖对高糖状态下大鼠骨髓间充质干细胞成骨向分化的影响

Effect of Angelica sinensis polysaccharide on the osteogenic differentiation of bone marrow mesenchymal stem cells of rats with high glucose levels

目的探讨当归多糖(ASP)对高糖状态下大鼠骨髓间充质干细胞(BMSCs)成骨向分化的影响。方法培养并收集第3代BMSCs进行成骨成脂分化诱导鉴定。将BMSCs分为3组进行培养:正常对照组(葡萄糖浓度5.5 mmol·L-1)、高糖组(葡萄糖浓度25.5 mmol·L-1)、ASP+高糖组(葡萄糖浓度25.5 mmol·L-1+40 mg·L-1 ASP)。CCK8检测各组BMSCs的增殖活性,茜素红染色和碱性磷酸酶活性检测成骨活性。实时荧光定量聚合酶链反应检测成骨标记基因Runt相关转录因子2(Runx-2)、锌指结构转录因子(Osx)、骨钙蛋白(OCN)、Ⅰ型胶原酶(COL-Ⅰ)mRNA及Wnt/β-catenin信号通路关键因子CyclinD1及β-catenin的mRNA表达。建立2型糖尿病大鼠模型,将大鼠分为3组:正常对照组(正常大鼠)、糖尿病组(糖尿病大鼠)、糖尿病+ASP组(糖尿病大鼠,ASP喂养),制备大鼠胫骨骨缺损,进行组织学检测,观察骨缺损修复情况。结果高糖组、ASP+高糖组的BMSCs增殖高于正常对照组(P<0.05),高糖组和ASP+高糖组二者之间无统计学差异(P>0.05)。高糖组中BMSCs钙结节数量、碱性磷酸酶活性及Runx-2、OCN、Osx、COL-Ⅰ、CyclinD1、β-catenin的mRNA表达均低于正常对照组和ASP+高糖组(P<0.05),正常对照组和ASP+高糖组之间无统计学差异(P<0.05)。组织学检测结果显示,糖尿病组骨小梁数量少于正常对照组和糖尿病+ASP组(P<0.05),而正常对照组和糖尿病+ASP组二者之间无统计学差异(P>0.05)。结论 ASP可促进高糖状态下大鼠BMSCs的成骨分化及2型糖尿病大鼠的骨缺损修复,这种促进作用可能与Wnt/β-catenin信号通路的激活有关。

Objective This study aims to evaluate the effect of Angelica sinensis polysaccharide (ASP) on the osteogenic differentiation of the bone marrow mesenchymal stem cells (BMSCs) of rats with high glucose levels. Methods Rat BMSCs were isolated and identified by osteogenic and adipo-genic differentiation. Then, the BMSCs were divided into three groups as follows: normal control group (5.5 mmol·L ^-1 glucose), high glucose group (25.5 mmol·L ^-1 glucose), and ASP+high glucose group (25.5 mmol·L^-1 glucose +40 mg·L -1 ASP). The proliferation activities of the BMSCs were detected by CCK8. Alizarin red staining, and alkaline phosphatase activity were used in the examination of osteogenic activity. Quantitative real time-polymerase chain reaction was used to detect the expression levels of the osteogenic genes (Runx2, Osx, OCN, Col-Ⅰ) and the key factors of Wnt/β-catenin signal pathway (CyclinD1,β-catenin). In vivo, a type 2 diabetes rat model was established. The rats were divided into three groups, namely, the normal control group (normal rats), diabetes group (diabetic rats), diabetes+ASP group (diabetic rats, ASP feeding). Then, the tibia bone defect was established. The repair of bone defects in each group was observed through histological examination. Results The proliferation of BMSCs was higher in the high glucose group and ASP+high glucose group than in the normal control group (P<0.05). No significant difference was observed between the high glucose group and ASP+high glucose group (P>0.05). The number of calcium nodules of BMSCs;alkaline phosphatase activity;and the mRNA expression of Runx2, OCN, Osx, Col-Ⅰ, CyclinD1,β-catenin in the high glucose group were lower than those in the normal control and ASP+high glucose groups (P<0.05). No significant difference was observed between the normal control and ASP+high glucose groups (P>0.05). The bone mass was significantly lower in the bone defect of the diabetes group than in the bone defect of the normal control or diabetes+ASP group (P<0.05). No statistical difference was found between the normal control and diabetes+ASP groups (P>0.05). Conclusion ASP can promote the osteogenic differentiation of rat BMSCs under high glucose culture and induce bone regeneration in rats with type 2 diabetes. These features may be related to the activation of the Wnt/β-catenin signaling pathway.