雷帕霉素对小鼠腭突细胞生物学特性的影响

摘要/Abstract

摘要： 目的：研究雷帕霉素对小鼠腭突细胞（MEPCs）生物学特性的影响。方法：取胎龄13.5天的小鼠，分离培养MEPCs，加入不同浓度（0、5、10、50、100 nmol/L）的雷帕霉素，观察MEPCs活性、凋亡、迁移、成骨情况，选择最佳作用浓度。Western? blot法观察在此浓度下不同的作用时间（0、3、6、12、24、48 h）自噬标志物LC3Ⅱ和p62/SQSTM1表达水平的变化。结果：6 h内，雷帕霉素促进MEPCs增殖，10 nmol/L雷帕霉素作用效果最明显；而6 h后，雷帕霉素抑制MEPCs增殖，其作用呈时间依赖性，作用时间越长，抑制增殖作用越强。6 h内，雷帕霉素抑制MEPCs凋亡，其中10 nmol/L抑制效果最明显；而6 h后，雷帕霉素促进细胞凋亡。细胞划痕结果表示，雷帕霉素促进MEPCs迁移，作用时间越长、浓度越高，促进迁移作用越强。雷帕霉素处理组抑制MEPCs成骨分化，碱性磷酸酶、茜素红染色及钙离子浓度结果显示，随着成骨诱导时间的延长，抑制作用增强，RT-PCR结果显示成骨相关基因的表达低于对照组。雷帕霉素作用下，随着作用时间的延长，LC3Ⅱ的表达明显增加，p62/SQSTM1的表达与对照组相比明显降低，呈时间依赖性，雷帕霉素促进了自噬。结论：雷帕霉素可能是通过促进自噬影响腭突细胞的生物学特性。

关键词: 雷帕霉素, 腭突细胞, 自噬, 小鼠

Abstract: Objective： To study the effects of rapamycin on the cytological properties of mouse embryonic palatal cells (MEPCs). Methods： MEPCs were isolated from E13.5 mice embryos and treated with different concentrations of rapamycin (0, 5, 10, 50, 100 nmol/L) for 0, 3, 6, 12, 24, 48 h to observe cell viability, apoptosis, migration, and osteogenic differentiation capacity, while the most effective concentration was selected. Western blot method is used to detect LC3Ⅱ and p62/SQSTM1 expression level in the concentration. Results： Compared with the control group, rapamycin promoted cell proliferation, and the effect of 10 nmol/L rapamycin was the most obvious in 6 h. After 6 h, the inhibition of rapamycin was in a time-dependent manner for MEPCs proliferation. The longer the time was, the stronger the inhibition. Compared with the control group, rapamycin inhibited cell apoptosis and 10 nmol/L rapamycin had the most obvious effect in 6 h, and after 6 h, rapamycin promoted cell apoptosis. Rapamycin significantly promoted MEPCs migration in a dose- and time-dependent manner. Rapamycin inhibited the osteogenesis of MEPCs which was shown by ALP activity, Alizarin red staining and calcium ion in a time-dependent manner; RT-PCR assays showed that the expression of bone sialoprotein and osteocalcin was lower than the control group. Compared with control group, LC3 Ⅱ expression was increased, the expression of p62/SQSTM1 was significantly decreased, rapamycin induced autophagy in a time-dependent manner. Conclusions： Rapamycin can affect the cytological properties of palatal cells, which may be related to the induction of autophagy.

Key words: rapamycin, embryonic palatal cells, autophagy, mouse

姚亚霞杜娟. 雷帕霉素对小鼠腭突细胞生物学特性的影响 [J]. , 2019, 10(2): 62-67.

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