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-α (TNF-α) stimulation in vitro.
Methods: SCAPs treated with TNF-α (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-α or with TNF-α 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-α induced activation of autophagy in cultured SCAPs. Pharmacological inhibition of TNF-α-induced autophagy by 3-MA significantly decreased the cell viability and increased the apoptosis rate of SCAPs (P < 0.05). Compared with the cells treated with TNF-α alone, the cells treated with both TNF-α and 3-MA exhibited decreased expressions of the ALP and BSP mRNA on days 3, 7 and 14 during osteogenic induction (P < 0.05) and decreased expression of OCN mRNA on days 3 and 7 during the induction (P < 0.05).
Conclusions: Autophagy may play an important role during the osteogenic differentiation of SCAPs in the presence of TNF-α stimulation.
目的: 探讨抑制自噬对根尖乳头干细胞成骨分化水平的影响。
方法: 5、10 ng/mL肿瘤坏死因子-α（TNF-α）分别处理根尖乳头干细胞（SCAPs），对照组不做处理，检测自噬相关蛋白LC3-Ⅱ表达水平，GFP-LC3质粒转染并检测细胞内GFP-LC3数目，吖啶橙染色检测酸性囊泡情况。TNF-α，TNF-α+3-MA分别处理SCAPs，检测LC3-Ⅱ的表达水平，GFP-LC3质粒转染并检测GFP-LC3数目，CCK-8法检测细胞活力，流式细胞仪检测细胞凋亡。TNF-α、TNF-α+3-MA分别处理SCAPs，对照组不做处理，诱导成骨向分化，qRT-PCR检测分化第3、7、14天时成骨相关基因碱性磷酸酶（ALP）、骨涎蛋白（BSP）、骨钙素（OCN）的表达。
结果: TNF-α可诱导SCAPs自噬活化：TNF-α组LC3-Ⅱ/β-actin水平较对照组升高且具有浓度依赖性（P < 0.05），TNF-α组GFP-LC3数目较对照组升高且具有浓度依赖性（P < 0.05），TNF-α组酸性囊泡较对照组增多。3-MA可抑制TNF-α诱导的SCAPs自噬活化：TNF-α+3-MA组LC3-Ⅱ/β-actin水平及细胞内GFP-LC3数目较TNF-α组显著降低（P < 0.05），并下调细胞活力（P < 0.05），上调细胞凋亡水平（P < 0.05）。抑制自噬导致SCAPs成骨分化的抑制：TNF-α+3-MA组ALP、BSP表达量在分化第3、7、14天均较TNF-α组降低（P < 0.05），OCN的表达量在第3、7天较TNF-α组降低（P < 0.05）。
结论: TNF-α可诱导SCAPs自噬水平的活化；自噬可能对TNF-α作用下的SCAPs起细胞保护作用，对抗细胞凋亡；自噬的抑制将下调TNF-α作用下SCAPs的成骨向分化水平，提示自噬在TNF-α作用下SCAPs的成骨分化过程中具有重要作用。
Keywords: autophagy; differentiation; stem cells from apical papilla; tumor necrosis factor-α.