氟在内质网应激引起原代培养成釉细胞自噬与凋亡中的作用

Roles of fluoride in autophagy and apoptosis of primary rat ameloblasts induced by endoplasmic reticulum stress

目的探讨氟在内质网应激引起原代培养成釉细胞自噬与凋亡中的作用及相关机制。方法分离SD大鼠上下颌磨牙牙胚中的成釉细胞进行原代培养。待细胞生长稳定后,采用0.8 mmol/L、1.6 mmol/L NaF分别干预体外培养成釉细胞24 h和48 h,倒置显微镜及透射电镜观察细胞形态及其超微结构。1.6 mmol/L NaF作用于成釉细胞24 h和48 h,利用realtime PCR和Western blot法分别检测不同时间点下成釉细胞XBP1s mRNA、GRP78和CHOP蛋白的表达水平。结果倒置显微镜下观察,0.8 mmol/L NaF干预成釉细胞24 h和48 h,与对照组相比均未见明显差异。1.6 mmol/L NaF作用于成釉细胞48 h凋亡细胞数明显多于同浓度氟作用于细胞24 h时。透射电镜结果显示,NaF干预成釉细胞24 h,0.8 mmol/L NaF组细胞粗面内质网池样扩张,线粒体肿胀,体积增大,电子密度降低,溶酶体丰富,自噬样结构可见。1.6 mmol/L NaF组细胞内自噬样结构明显增加。1.6 mmol/L NaF作用下,成釉细胞内质网应激相关蛋白XBP1s mRNA、GRP-78及CHOP蛋白表达水平48 h时间点较24 h时间点均明显增加(P<0.01)。结论氟可导致成釉细胞内质网应激,启动自噬,随着氟处理时间延长,激活凋亡信号通路,诱导细胞凋亡。

Objective To investigate the effects and mechanisms of fluoride on endoplasmic reticulum（ ER） stress-induced autophagy and apoptosis of primary rat ameloblasts in vitro. Methods Ameloblasts were isolated from tooth germ of maxillomandibular molar and cultured in vitro. Ameloblasts were treated with 0. 8 mmol / L and 1. 6 mmol / L NaF for 24 h and 48 h,respectively. Cell morphology and ultrastructure were observed under phase contrast microscopy and TEM after the cell growth reached to a stable stage. After ameloblasts were treated with 1. 6 mmol / L NaF for 24 h and 48 h,XBP1 s mRNA levels,expression of GRP78 and CHOP proteins were measured by real-time PCR and Western blot at each time point. Results Under the phase contrast microscope,the cell morphology showed no obvious difference after treated with 0. 8 mmol / L NaF between 24 h and 48 h. After treated with 1. 6 mmol / L NaF,the number of apoptotic cells statistically higher at 48 h than that at 24 h. The TEM results showed the extension of rough endoplasmic reticulum cistern,swelling of mitochondria,enlarged cell body,decreased electron density,increased lysosome,and appearance of autophagic structure after treated with 0. 8 mmol / L NaF for 24 h. Compared with 0. 8 mmol / L NaF group,autophagic structures increased in 1. 6 mmol / L NaF group. After treated with 1. 6 mmol / L NaF,ER stress-related XBP1 s mRNA,GRP-78,and CHOP proteins were significantly higher at 48 h than that at 24 h（ P〈0. 01）. Conclusion The results suggest that fluoride could cause ER stress of ameloblast and induce the autophagy,thus lead to apoptosis as the treatment continues.