低氧浓度促进人牙髓干细胞增殖及神经分化

Hypoxia promotes proliferation and neural differentiation of human dental pulp stem cells

目的探讨不同氧浓度对人牙髓干细胞(hDPSCs)增殖及神经分化的影响,为hDPSCs的临床应用提供依据。方法无菌收集正畸减数牙及完整拔除的智齿,组织块酶消化法分离培养人牙髓干细胞,随机分为4组,设置氧浓度分别为1%、5%、10%及21%。5-乙炔基-2’脱氧尿嘧啶核苷(EdU)标记法检测各组人牙髓干细胞的增殖,CCK-8法检测细胞活力;观察各组人牙髓干细胞神经诱导后的形态学变化,细胞免疫荧光法检测各组人牙髓干细胞神经诱导后细胞巢蛋白(Nestin)的表达。结果人牙髓干细胞鉴定结果示CD90^(+)、CD146^(+)、CD34^(-);体外培养24 h时,5%O_(2)组人牙髓干细胞增殖率及细胞活力显著高于1%O_(2)、10%O_(2)和21%O_(2)组;神经诱导5 d,各组细胞形态均诱导为神经元样,5%O_(2)组细胞突起数及Nestin的表达显著高于1%O_(2)、10%O_(2)和21%O_(2)组。结论5%O_(2)及10%O_(2)均可促进人牙髓干细胞增殖,并可促进诱导的人牙髓干细胞向神经细胞分化,5%O_(2)促进人牙髓干细胞增殖、神经分化尤为显著。

Objective To investigate the effects of different oxygen concentrations on the proliferation and neural differentiation of human dental pulp stem cells(hDPSCs),and to provide a basis for the clinical application of dental pulp stem cells.Methods The orthodontic reduced teeth were collected aseptically,and the pulp stem cells were isolated and cultured by enzyme digestion of tissue masses.They were randomly divided into 4 groups and treated with different oxygen concentrations:1%O_(2),5%O_(2),10%O_(2) and 21%O_(2).5-ethynyl-2’deoxyuridine(EdU)labeling method to detect the proliferation of dental pulp stem cells in each group,CCK-8 method to detect cell viability;observation of the morphological changes of dental pulp stem cells in each group after neural induction,immunofluorescence cytochemistry the method was used to detect the expression of Nestin in each group of dental pulp stem cells after neural induction.Results The results of dental pulp stem cell identification showed CD90^(+),CD146^(+),CD34^(-);When cultured for 24h in vitro,the proliferation rate and cell viability of human dental pulp stem cells in the 5%O_(2) group were significantly higher than those in the 1%O_(2),10%O_(2) and 21%O_(2) groups;after nerve induction for 5 days,the cell morphology in each group was induced to be neuron-like and the number of cell protrusions and the expression of Nestin protein in the 5%O_(2) group were significantly higher than those in the 1%O_(2),10%O_(2) and 21%O_(2) groups.Conclusion Both 5%O_(2) and 10%O_(2) can promote the proliferation of dental pulp stem cells and the differentiation of induced dental pulp stem cells into nerve cells,5%O_(2) is more liable to promote the proliferation and neural differentiation of dental pulp stem cells.