脂多糖调控Notch信号通路作用于人牙髓干细胞增殖、分化、凋亡的实验研究

Effects of lipopolysaccharide on proliferation,differentiation and apoptosis of human dental pulp stem cells by Notch signaling pathway

目的:探讨脂多糖调控Notch信号通路对人牙髓干细胞增殖、分化、凋亡的影响及机制。方法:从牙髓组织中分离出人牙髓干细胞(h DPSCs),CCK8实验检测0、0.1、1、10μg/ml的脂多糖处理h DPSCs 1、3、5、7 d后细胞的增殖情况;RTPCR检测1μg/ml的脂多糖处理h DPSCs 0、3、7、14、21 d后矿化相关基因ALP、DSPP、DMP1的mRNA表达情况;流式细胞仪检测1μg/ml的脂多糖处理h DPSCs 0、7、14、21 d后的细胞凋亡情况;Western blot检测Cleaved caspase3、Notch1、Hes1的蛋白表达。结果:不同浓度的脂多糖刺激h DPSCs 1、3、5 d后细胞的增殖均无显著差异,培养至第7天时,0.1、1、10μg/ml的脂多糖组细胞的增殖均显著低于0μg/ml的脂多糖组(P<0.01);脂多糖处理h DPSCs 3 d时ALP、DSPP、DMP1的mRNA表达与对照组比较差异均无统计学意义(P>0.05),7、14、21 d时ALP、DSPP、DMP1的mRNA表达均显著高于对照组(P<0.01);脂多糖处理h DPSCs 7、14、21 d时细胞的凋亡率及Cleaved caspase3、Notch1、Hes1蛋白表达均显著高于对照组(P<0.01),21 d时ALP、DSPP、DMP1的mRNA表达及细胞凋亡率和Cleaved caspase3、Notch1、Hes1蛋白表达均有下降趋势。结论:脂多糖可降低h DPSCs的增殖,促进其矿化和凋亡,其机制与激活Notch信号通路有关。

Objective： To investigate the effects and its mechanism of lipopolysaccharide on the proliferation,differentiation and apoptosis of human dental pulp stem cells by Notch signaling pathway. Methods： Human dental pulp stem cells（ h DPSCs） was isolated from dental pulp tissue; cell proliferation was detected after 0,0. 1,1,10 μg/ml treated cells for 1,3,5,7 days by CCK8 test; related mRNA expression ALP,DSPP,DMP1 gene was detected after 1 μg/ml lipopolysaccharide treated h DPSCs for 0,3,7,14,21 day by RTPCR; cell apoptosis was detected after 1 μg/ml lipopolysaccharide treated h DPSCs for 0,7,14,21 day by flow cytometry. Cleaved caspase3,Notch1,Hes1 protein expression was detected by Western blot. Results： Cell proliferation after different concentrations lipopolysaccharide stimulated h DPSCs for 1,3,5 days had no significant difference,significantly lower at 7 day than 0 μg/ml lipopolysaccharide group（ P〈0. 01）. ALP,DSPP,DMP1 mRNA expression lipopolysaccharide treated h DPSCs at 3 day compared with the control group had no statistical significance（ P〈0. 05）,significantly higher at 7,14,21 day than control group（ P〈0. 01）; cells apoptosis rate and Cleaved caspase3,Notch1,Hes1 protein expression lipopolysaccharide treatment h DPSCs at 7,14 and 21 day was significantly higher than the control group（ P〈0. 01）; ALP,DSPP,DMP1 mRNA expression and apoptosis rate and Cleaved caspase3,Notch1,Hes1 protein expression at 21 day was a downward trend. Conclusion： Lipopolysaccharide can decrease the proliferation of h DPSCs and promote its mineralization and apoptosis,which may be related to the activation of Notch signaling pathway.