淫羊藿次苷对犬颌骨间充质干细胞增殖与成骨分化的影响

目的 研究淫羊藿次苷Ⅱ（Icarisid Ⅱ,ICSII）对体外培养的比格犬颌骨间充质干细胞（alveolar bone-derived stem cells, AMSCs）增殖与成骨分化的影响。方法 取比格犬颌骨骨片,体外分离培养出dAMSCs,传代至第4代,做多向分化鉴定。以10^-5、10^-6、10^-7、10^-8、10^-9 mol/L浓度的ICSII刺激dAMSCs后,取1、3、5、7 d的细胞,分别用CCK-8及碱性磷酸酶（ALP）试剂盒检测dAMSCs的增殖及ALP活性;用10^-6 mol/L的ICSII处理dAMSCs后14 d,作ALP染色,21 d时作茜素红染色,以判断ICSII对细胞分化及矿化能力的影响。在成骨诱导及10^-6 mol/L的ICSII药物诱导第4、8天后,用蛋白免疫印迹试验分析成骨相关蛋白OSX、Runx-2及OCN的表达量;在诱导第6天时,利用RT-PCR分析成骨相关基因OSX、bFGF、Runx-2及OCN的表达情况。采用SPSS 22.0软件包进行统计分析。结果 原代培养的dAMSCs贴壁生长,呈梭形,具备多向分化能力;ICSII在不同浓度下均可促进dAMSCs的增殖,其ALP活性亦有提高,且在实验浓度时可观察到钙结节形成。各成骨相关基因在ICSII诱导后表达量有不同程度增加,均高于完全培养基组。随着时间延长,加药组Runx-2蛋白表达量有所下降, OCN蛋白表达量逐渐增多,与对照组差距加大;OSX的蛋白表达量虽高于对照组,但无统计学差异。结论 ICSII可促进dAMSCs的增殖及成骨分化。

Effects of Icarisid Ⅱ on proliferation and osteogenic differentiation of alveolar bone-derived stem cells in Beagle dogs

PURPOSE: To investigate the effect of Icarisid Ⅱon proliferation and osteogenic differentiation of alveolar bone-derived stem cells(AMSCs) in Beagle dogs. Methods: Alveolar bone-derived stem cells were isolated and cultured from the jaw bone of Beagle dogs in vitro. After dAMSCs were stimulated by ICSII at 10^-5, 10^-6, 10^-7, 10^-8 and 10^-9 mol/L, the proliferation and alkaline phosphatase (ACP) activity of dAMSCs were detected by CCK-8 and ALP kit at 1, 3, 5 and 7 days. ALP staining, Alizarin red staining, Western blot and RT-PCR were performed at different time points to evaluate the osteogenic capacity of cells. SPSS22.0 software package was used for statistical analysis. Results: Primary dAMSCs adhered to the wall, showing spindle shape and multidirectional differentiation ability. ICSII can promote the proliferation of dAMSCs at different concentrations. The results showed that ALP activity was also increased, and the formation of calcium nodules was observed at the experimental concentration. The expression levels of each osteogenic gene increased, which were significantly higher than that of the complete medium group. With the extension of time, runx-2 protein expression decreased in the experimental group, OCN protein expression gradually increased, and the gap between the experimental group and the control group widened. OSX protein expression was higher than that of the control group, but there was no significant difference. Conclusions: ICSII can promote the proliferation and osteogenic differentiation of dAMSCs.