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重庆医科大学附属口腔医学院、口腔疾病与生物医学重庆市重点实验室、重庆市高校市级口腔生物医学工程重点实验室,重庆,401147
修回日期:2020-03-05,
录用日期:2020-03-18,
纸质出版日期:2023
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谭玺. “Sirt1-自噬”在低氧诱导小鼠成牙骨质细胞凋亡中的作用[J]. 重庆医科大学学报, 2023,48(1):24-29.
Tan Xi. Role of "Sirt1-autophagy" on hypoxia-induced apoptosis in mice cementoblasts[J]. Journal of chongqing medical university, 2023, 48(1): 24-29.
目的: 初步研究“Sirt1-自噬”在低氧诱导小鼠成牙骨质细胞OCCM-30凋亡中的作用。方法: 体外培养小鼠成牙骨质细胞,随机分为对照组、低氧组、低氧+Sirt1激活剂白藜芦醇组、低氧+自噬抑制剂3-MA组、低氧+白藜芦醇+Sirt1抑制剂ex527组、低氧+白藜芦醇+3-MA组,MDC染色观察自噬溶酶体变化;LC3 Ⅱ免疫荧光检测自噬体变化;透射电镜观察自噬体;Western blot检测Sirt1、LC3Ⅱ、p62等蛋白表达;流式细胞仪检测细胞凋亡情况;不同组间结果数据采用单因素方差分析,组间采用LSD-t分析。结果: 低氧组较对照组自噬溶酶体含量(P=0.000)及自噬体数量(P=0.000)明显增加,LC3Ⅱ蛋白表达明显增加(P=0.001),p62蛋白表达明显减少(P=0.000),细胞凋亡率增加(P=0.000);低氧+白藜芦醇组较低氧组自噬溶酶体(P=0.019)及自噬体数量(P=0.000)、LC3 Ⅱ蛋白表达(P=0.049)、Sirt1蛋白表达(P=0.000)明显增加,p62蛋白表达明显减少(P=0.000),细胞凋亡率减少(P=0.021);低氧+白藜芦醇+ex527组较低氧+白藜芦醇组自噬溶酶体(P=0.000)及自噬体数量(P=0.000)、LC3 Ⅱ蛋白表达(P=0.000)、Sirt1蛋白表达(P=0.000)明显减少,p62蛋白表达明显增加(P=0.000),细胞凋亡率增加(P=0.000);低氧+白藜芦醇+3-MA组较低氧+白藜芦醇组自噬溶酶体(P=0.000)及自噬体数量(P=0.000)、LC3 Ⅱ蛋白表达明显减少(P=0.000),p62蛋白表达明显增加(P=0.000),细胞凋亡率增加(P=0.000),Sirt1蛋白表达减少(P=0.146),差异无统计学意义。结论: 自噬相关分子可能是低氧环境下Sirt1的下游作用分子,自噬在低氧诱导小鼠成牙骨质细胞(OCCM-30)凋亡中起保护作用。
Objective: To investigate the role of "Sirt1-autophagy" on the hypoxia-induced apoptosis in OCCM-30 cementoblasts of mouse. Methods: OCCM-30 cells were cultured in vitro and were randomly divided into the control group
the hypoxia group
the hy-poxia + resveratrol group
the hypoxia + 3-MA group
the hypoxia + resveratrol + ex527 group and the hypoxia + resveratrol + 3-MA group. Changes of autophagy lysosomes in each group were observed by MDC staining and changes of autophagosome light chain Ⅱ (LC3 Ⅱ) were detected by immunofluorescence assay. The expression of Sirt1
LC3 Ⅱ and p62 proteins was detected by Western blot. Cell apoptosis was detected by flow cytometry. Results of different groups were analyzed by one-way ANOVA and different groups were analyzed by LSD-t. Results: Compared with the control group
the content of autophagy lysosomes (P=0.000) and the number of autophagosomes (P=0.000) in the hypoxia group were significantly increased
the expression of LC3 Ⅱ (P=0.001) protein was signifi-cantly increased
the expression of p62 protein was significantly decreased (P=0.000)
and the apoptosis rate was increased (P=0.000). Compared with the hypoxia group
the number of autophagy lysosomes (P=0.019) and autophagosomes (P=0.000)
and the expression of LC3 Ⅱ protein (P=0.000) and Sirt1 protein (P=0.049
P=0.000) in the hypoxia + resveratrol group were significantly increased
the expression of p62 protein (P=0.000) was signifi-cantly decreased
and the apoptosis rate was decreased (P=0.021). Compared with the hypoxia + resveratrol group
the number of autophagy lysosomes (P=0.000) and autophagosomes (P=0.000)
and the expression of LC3 Ⅱ (P=0.000) and Sirt1 (P=0.000) in the hypoxia + resveratrol + ex527 group were sig-nificantly decreased
the expression of p62 protein (P=0.000) was significantly increased
and the apoptosis rate was increased (P=0.000). Compared with the hypoxia + resveratrol group
the num-ber of autophagy lysosomes (P=0.000) and autophagosomes (P=0.000)
and the expression of LC3 Ⅱ (P=0.000) protein expression in the hypoxia + resveratrol + 3-MA group were significantly decreased
the expression of p62 protein (P=0.000) was increased
the apoptosis rate was increased (P=0.000)
and the expression of Sirt1 protein (P=0.146) was decreased
with no statistically significant difference. Conclusion: Autophagy-related molecules may be downstream acting molecules of Sirt1 in the hypoxic environment and autophagy plays a protective role in hypoxia-induced apoptosis of OCCM-30.
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