Ying Huang1, Huacui Xiong2,3, Ke Chen2,3, Xiaobin Zhu2, Xiaoping Yin4, Yun Liang2,3, Wei Luo3, Qiyin Lei3. 1. Stomatology Center, Shunde Hospital, Southern Medical University (The First People's Hospital of Shunde), Foshan 528308, China. 2. Stomatological Hospital, Southern Medical University, Guangzhou 510000, China. 3. Guangzhou Women and Children's Medical Center, Guangzhou 510623, China. 4. Affiliated Hospital of Guilin Medical University, Guilin 541000, China.
Abstract
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.
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 &lt; 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 &lt; 0.05) and decreased expression of OCN mRNA on days 3 and 7 during the induction (P &lt; 0.05). CONCLUSIONS: Autophagy may play an important role during the osteogenic differentiation of SCAPs in the presence of TNF-α stimulation.
Authors: Austin Nuschke; Melanie Rodrigues; Donna B Stolz; Charleen T Chu; Linda Griffith; Alan Wells Journal: Stem Cell Res Ther Date: 2014-12-17 Impact factor: 6.832
Authors: Takehiro Torisu; Kumiko Torisu; In Hye Lee; Jie Liu; Daniela Malide; Christian A Combs; Xufeng S Wu; Ilsa I Rovira; Maria M Fergusson; Roberto Weigert; Patricia S Connelly; Mathew P Daniels; Masaaki Komatsu; Liu Cao; Toren Finkel Journal: Nat Med Date: 2013-09-22 Impact factor: 53.440