尼古丁抑制人牙髓干细胞增殖和分化

Nicotine inhibits proliferation and differentiation of human dental pulp stem cells

目的探讨尼古丁的刺激对人牙髓干细胞增殖、分化的影响。方法培养人牙髓干细胞,流式细胞计量术鉴定细胞表面抗原;用不同浓度的尼古丁(10-4、10-3和10-2 mol/L)刺激牙髓干细胞,培养0、1、2、3和4 d后,CCK8法检测细胞增殖能力;茜素红染色法检测细胞分化过程中矿化结节形成,RT-qPCR和免疫印迹(Western blot)检测牙本质涎磷蛋白(DSPP)、碱性磷酸酶(ALP)、骨桥素(OPN)及细胞外信号调节激酶(ERK)、c-Jun氨基末端激酶(JNK)、p38(p-ERK、p-JNK、p-p38)等MAPK通路相关蛋白表达。结果培养第3、4天时,与对照组相比,尼古丁刺激时A值显著降低(P<0.05);与对照组相比,尼古丁刺激时矿化结节形成数、DSPP、ALP、OPN mRNA和蛋白、p-ERK、p-JNK、p-p38蛋白表达显著降低(P<0.05)。结论尼古丁抑制人牙髓干细胞增殖、成骨分化能力。

Objective To investigate the effects of nicotine stimulation on proliferation and differentiation of human dental pulp stem cells.Methods Human dental pulp stem cells were culture and cell surface antigen was identified by flow cytometry.The cultured dental pulp stem cells were stimulated by nicotine(10-4,10-3 and 10-2 mol/L)at different concentrations,after 0,1,2,3 and 4 d of culture,the proliferation of cells were detected by CCK8 assay;alizarin red staining to detect the formation of mineralized nodules during cell differentiation,RT-qPCR and Western blot were used to detect the expression of dentin sialophosphoprotein(DSPP),alkaline phosphatase(ALP),osteopontin(OPN)and the expression of MAPK pathway-related proteins such as extracellular signal-regulated kinase(ERK),c-Jun N-terminal kinase(JNK),p38(p-ERK,p-JNK,p-p38).Results On the 3rd and 4th day of culture,the A value of nicotine stimulation was significantly lower than that of control group(P<0.05);compared with the control group,the number of mineralized nodules,the expression of DSPP,ALP,OPN mRNA and protein,the expression of p-ERK,p-JNK and p-p38 protein were significantly decreased when nicotine was stimulated(P<0.05).Conclusions Nicotine inhibits the proliferation and osteogenic differentiation of human dental pulp stem cells.