不同浓度黄芩苷对人根尖乳头干细胞成骨分化的影响

目的:探讨不同浓度黄芩苷对人根尖乳头干细胞(stem cells from apical papilla,SCAPs)成骨分化的影响。方法:选取具备稳定传代能力的第3代SCAPs,采用Live/Dead细胞荧光染色,检测细胞在0、10、20、50、100μmol/L不同黄芩苷浓度干预培养24 h后的存活情况及细胞活力;然后将SCAPs分为普通培养基组(DMEM)和成骨诱导培养基组(ODM),根据黄芩苷的不同浓度,将实验组分为5个实验亚组,依次为0、10、20、50、100μmol/L,利用碱性磷酸酶(ALP)染色法和ALP定量检测法,研究不同浓度黄芩苷对SCAPs培养7、14 d早期成骨分化的影响。应用茜素红染色法,定量检测不同浓度黄芩苷对SCAPs培养21 d后成骨矿化的影响。利用MTT法,检测不同浓度黄芩苷对SCAPs生长的影响。采用SPSS 20.0软件包对数据进行统计学分析。结果:活/死细胞荧光染色结果表明,经不同浓度药物干预处理后,细胞活力旺盛,无明显异常;但黄芩苷药物浓度高于50μmol/L时,细胞死亡数目有上升趋势。ALP定量检测结果显示,ODM组诱导分化第14天时,20μmol/L浓度组的ALP活性显著高于对照组(P<0.05),100μmol/L浓度组的ALP活性显著低于对照组(P<0.05)。DMEM组培养14 d后,20μmol/L浓度组的ALP活性显著高于对照组(P<0.05)。茜素红定量显示,ODM组中100μmol/L实验组的A值显著低于对照组(P<0.05);20μmol/L的黄芩苷浓度组是促进SCAP生长的最适浓度。结论:高于50μmol/L浓度的黄芩苷对SCAPs的成骨分化表现出明显抑制作用,而低浓度黄芩苷,尤其是20μmol/L的黄芩苷对SCAPs成骨分化及细胞生长有促进作用。

Effect of baicalin on osteogenic differentiation of stem cells from human apical papilla

PURPOSE: To investigate the effect of baicalin on osteogenic differentiation of stem cells from human apical papilla(SCAPs). METHODS: The third-passage SCAPs with stable passage ability were selected. After culturing for 24 hours in a medium containing 0, 10, 20, 50, 100 μmol/L of baicalin, cell viability in each group was measured by live/dead fluorescence staining. Then the SCAPs were divided into normal medium group(DMEM) and osteogenic induction medium group(ODM), and the experimental groups were further divided into 5 subgroups according to different concentrations of baicalin, i.e. 0, 10, 20, 50 and 100 μmol/L. Alkaline phosphatase(ALP) staining and ALP quantitative assay were used to detect the effects of different concentrations of baicalin on early osteogenic differentiation of SCAPs for 7 and 14 days; the effect of different concentrations of baicalin on osteogenic mineralization of SCAPs was determined by alizarin red staining after 21 days of culturing. MTT assay was used to detect the effects of baicalin at different concentrations on the growth of SCAPs. SPSS 20.0 software package was used for statistical analysis of the data. RESULTS: The results of live/dead cell fluorescence staining showed that SCAPs were vigorous after treatment in each concentration group, but when the concentration of baicalin was higher than 50 μmol/L, the number of dead cell increased. ALP quantitative results showed that ALP activity in the 20 μmol/L concentration group was significantly higher than that in the control group on the 14th day after induced differentiation in ODM group (P<0.05), and ALP activity in 100 μmol/L concentration group was significantly lower than that in the control group(P<0.05). After 14 days of DMEM culture, ALP activity of 20 μmol/L group was also significantly higher than that of the control group(P<0.05). Quantitative analysis of alizarin red showed that the A value of the 100 μmol/L group was significantly lower than the control group in ODM. 20 μmol/L baicalin was the optimum concentration to promote growth of SCAPs. CONCLUSIONS: Baicalin with a concentration higher than 50 μmol/L can significantly inhibit osteogenic differentiation of SCAPs, while low concentration of baicalin, especially 20 μmol/L of baicalin, has a promoting effect on SCAPs osteogenesis differentiation and proliferation.