自噬抑制抑制根尖乳头干细胞的成骨分化
[Inhibition of autophagy suppresses osteogenic differentiation of stem cells from apical papilla].
作者信息
Huang Ying, Xiong Huacui, Chen Ke, Zhu Xiaobin, Yin Xiaoping, Liang Yun, Luo Wei, Lei Qiyin
机构信息
Stomatology Center, Shunde Hospital, Southern Medical University (The First People's Hospital of Shunde), Foshan 528308, China.
Stomatological Hospital, Southern Medical University, Guangzhou 510000, China.
出版信息
Nan Fang Yi Ke Da Xue Xue Bao. 2019 Jan 30;39(1):106-112. doi: 10.12122/j.issn.1673-4254.2019.01.17.
OBJECTIVE
To investigate the effects of autophagy on osteogenic differentiation of stem cells from the apical papilla (SCAPs) in the presence of tumor necrosis factor- (TNF-) stimulation .
METHODS
SCAPs treated with TNF- (0, 5, and 10 ng/mL) with or without 5 mmol/L 3-MA were examined for the expression of autophagy marker LC3-Ⅱ using Western blotting. The cells were transfected with GFP-LC3 plasmid and fluorescence microscopy was used for quantitative analysis of intracellular GFP-LC3; AO staining was used to detect the acidic vesicles in the cells. The cell viability was assessed with CCK-8 assays and the cell apoptosis rate was analyzed using flow cytometry. The cells treated with TNF- or with TNF- and 3-MA were cultured in osteogenic differentiation medium for 3 to 14 days, and real- time PCR was used to detect the mRNA expressions of osteogenesis-related genes (ALP, BSP, and OCN) for evaluating the cell differentiation.
RESULTS
TNF- induced activation of autophagy in cultured SCAPs. Pharmacological inhibition of TNF--induced autophagy by 3-MA significantly decreased the cell viability and increased the apoptosis rate of SCAPs ( < 0.05). Compared with the cells treated with TNF- alone, the cells treated with both TNF- and 3-MA exhibited decreased expressions of the ALP and BSP mRNA on days 3, 7 and 14 during osteogenic induction ( < 0.05) and decreased expression of OCN mRNA on days 3 and 7 during the induction ( < 0.05).
CONCLUSIONS
Autophagy may play an important role during the osteogenic differentiation of SCAPs in the presence of TNF- stimulation.
目的
探讨在肿瘤坏死因子-α（TNF-α）刺激下自噬对根尖乳头干细胞（SCAPs）成骨分化的影响。
方法
用或不用5 mmol/L 3-甲基腺嘌呤（3-MA）处理SCAPs，并用肿瘤坏死因子-α（0、5和10 ng/mL）刺激，通过蛋白质免疫印迹法检测自噬标志物LC3-Ⅱ的表达。用绿色荧光蛋白-LC3质粒转染细胞，并用荧光显微镜对细胞内绿色荧光蛋白-LC3进行定量分析；用吖啶橙染色检测细胞内酸性囊泡。用CCK-8法评估细胞活力，并用流式细胞术分析细胞凋亡率。将用肿瘤坏死因子-α或肿瘤坏死因子-α与3-MA处理的细胞在成骨分化培养基中培养3至14天，并用实时聚合酶链反应检测成骨相关基因（碱性磷酸酶、骨桥蛋白和骨钙素）的信使核糖核酸表达，以评估细胞分化。
结果
肿瘤坏死因子-α诱导培养的SCAPs发生自噬激活。3-MA对肿瘤坏死因子-α诱导的自噬的药理学抑制显著降低了SCAPs的细胞活力并增加了其凋亡率（P<0.05）。与单独用肿瘤坏死因子-α处理的细胞相比，在成骨诱导的第3、7和14天，用肿瘤坏死因子-α和3-MA处理的细胞碱性磷酸酶和骨桥蛋白信使核糖核酸的表达降低（P<0.05），在诱导的第3和7天骨钙素信使核糖核酸的表达降低（P<0.05）。
结论
在肿瘤坏死因子-α刺激下，自噬可能在SCAPs的成骨分化过程中发挥重要作用。
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