刺五加苷诱导大鼠间充质干细胞成骨分化的作用

Osteogenic differentiation of rat bone marrow stem cells induced by acanthopanax senticosus glycoside

目的探讨刺五加苷诱导大鼠间充质干细胞(BMSCs)成骨分化的作用。方法提取SD大鼠的BMSCs,培养3代后采用流式细胞术鉴定表面抗原CD45、CD29、CD90的表达;在经典成骨培养液中加入不同浓度的刺五加苷而分为9组:A组(1×10^(–4)mol/L),B组(1×10^(–5)mol/L),C组(1×10^(–6)mol/L),D组(1×10–7mol/L),E组(1×10^(–8)mol/L),F组(1×10^(–9)mol/L),G组(1×10^(–10)mol/L),H组(经典成骨组),I组(阴性对照组)。采用细胞计数试剂盒(CCK-8)检测细胞增殖能力,反转录定量PCR(RT-qPCR)检测骨形成蛋白(BMP)表达情况,筛选出刺五加苷的最佳诱导浓度。以最佳诱导浓度培养12d后,RT-qPCR检测成骨分化相关基因Runt相关转录因子(RUNX)、成骨细胞特异性转录因子(OSX)、骨涎蛋白(BSP)、骨钙素(OCN)的mRNA表达水平,Western blotting检测Notch跨膜受体蛋白1(Notch1)、毛发增强分裂蛋白(Hes1)的蛋白表达水平;第21天时行矿化钙结节茜素红染色。结果第3代BMSCs表面抗原符合干细胞鉴定标准。CCK-8检测结果显示,A、B两组在培养120h后明显抑制了BMSCs的增殖,后期培养中将舍弃A、B两组;而RTqPCR结果显示,添加有刺五加苷的C～G组中,E组BMP表达量最高(4.91±0.46),因此选择1×10^(–8)mol/L作为刺五加苷的最佳诱导浓度进行后续实验。RT-qPCR检测结果显示,E组OSX表达量(30.72±1.96)明显高于I组(1.02±0.27)和H组(9.99±0.59,P<0.05),BSP表达量(8.15±0.47)高于I组(1.09±0.31)和H组(6.03±0.8,P<0.05),OCN表达量(5.91±0.68)高于I组(1.18±2.91)和H组(3.05±0.53,P<0.05),RUNX表达量(1.99±0.09)虽高于I组(1.02±0.19),却低于H组(2.51±0.06,P<0.05)。Westernblotting检测结果显示,E组成骨诱导后Notch1表达量(4608±103)较I组(2638±308)高,却低于H组(5218±182,P<0.05),Hes1表达量(8885±17)较I组(6241±461)增高,却低于H组(12 289±629,P<0.05)。茜素红染色结果显示刺五加苷浓度为10–8mol/L时矿化钙结节多于经典成骨组,提示该浓度刺五加苷成骨诱导效果较经典成骨组更佳。结论刺五加苷能协同地塞米松促进BMSCs的成骨分化,其作用可能与Notch通路有关。

Objective To investigate the effect of acanthopanax on inducing osteogenesis of rat bone marrow stem cells (BMSCs). Methods BMSCs were extracted from SD rats, and the surface antigens CD45, CD29 and CD90 were identified by flow cytometry at the third generation. Different concentrations of acanthopanax were added to the classical osteogenic medium to make it being 9 groups: A(1×10^–4mol/L), B(1×10^–5mol/L), C(1×10^–6mol/L), D(1×10^–7mol/L), E(1×10^–8mol/L), F(1×10^–9mol/L), G(1×10^–10mol/L), H(classical osteogenic group), and I(negative control group). The cell counting kit CCK-8 was used to detect cell proliferation, RT-qPCR was performed to detect the mRNA expression of bone morphogenetic protein (BMP), and then the optimal concentration of acanthopanax was selected and used to the later experiments. On the 12th day of culturing with optimal concentration, RT-qPCR was performed to detect osteogenic differentiation-related gene expression: RUNX, OSX, BSP and OCN. Western blotting was used to detect the levels of transmembrane receptor protein 1 (Notch1) and hairy enhancer of split 1 (Hes1). On the 21th day of culturing, the mineralized calcium nodules were stained with alizarin red. Results The surface antigens of the third generation BMSCs were consistent with the stem cell identification criteria. CCK-8 results indicated that the proliferation of BMSCs was inhibited in group A and group B 120h after cultivation, so the two groups were discarded in the later culture. RT-qPCR results showed that among groups C-G with acanthopanax, the expression of BMP in group E (1×10^–8mol/L) was the highest (4.91±0.46), so 1×10^–8mol/L was selected as the optimal concentration of acanthopanax to finish the later experiments. The results of RT-qPCR showed that the expression of OSX was significantly higher in group E (30.72±1.96) than in group I (1.02±0.27) and group H (9.99±0.59, P<0.05);the expression of BSP (8.15±0.47) was higher than in group I (1.09±0.31) and group H (6.03±0.8, P<0.05);and the expression of OCN (5.91±0.68) was higher than in group I (1.18±2.91) and group H (3.05±0.53, P<0.05). However, the expression of RUNX was higher in group E (1.99±0.09) than in group I (1.02±0.19, P<0.05), but was lower than in group H (2.51±0.06, P<0.05). Western blotting suggested that the Notch1 in group E (4608±103) was higher than in group I (2638±308), but lower than in group H (5218±182, P<0.05);Hes1 expression in group E (8885±17) was higher than in group I ( 6241±461), but lower than in H group (12289±629, P<0.05). The alizarin red staining indicated that the number of mineralized calcium nodules was higher in group E than in group H, suggesting that the osteogenic effect in group E (with acanthopanax concentration of 10^–8mol/L) is better than in group H. Conclusion Acanthopanax may cooperate with dexamethasone to promote the osteogenesis of BMSCs, which may be related to the Notch signaling pathway.