氟对体外培养LS8细胞增殖、氧化应激与凋亡的作用研究

Effect of Fluoride on Cell Proliferation, Oxidative Stress and Apoptosis in LS8 Cells

目的:观察氟化物对小鼠成釉细胞样细胞(LS8细胞)增殖、氧化应激以及凋亡的影响。方法:取对数生长期的LS8细胞,在培养液中分别加入终浓度为0、0.5、1、2、4和8mM的氟化钠(NaF)溶液,培养24h和48h后,CCK-8检测细胞增殖;倒置显微镜观察细胞形态;生化方法检测细胞丙二醛(MDA)含量及超氧化物歧化酶(SOD)活性;qRT-PCR检测过氧化氢酶(Cat)、谷胱甘肽过氧化物酶1(Gpx-1)和谷胱甘肽S转移酶(Gst)mRNA水平;流式细胞术检测细胞凋亡;Western blotting检测caspase-9和caspase-3蛋白表达改变。结果:过量氟抑制LS8细胞增殖,且随着NaF浓度增加,细胞增殖呈下降趋势;过量氟显著增加LS8细胞内MDA含量,降低SOD活性及Gpx-1、GstmRNA表达水平;过量氟引起LS8细胞形态改变和细胞凋亡,且伴随着caspase-9,caspase-3表达的显著增加。结论:过量氟抑制LS8细胞增殖,扰乱细胞的氧化与抗氧化平衡并诱发氧化应激,持续的氧化应激最终导致细胞凋亡。

Objective To investigate the effects of fluoride on cell proliferation, oxidative stress and apoptosis in ameloblast-like cells(LS8 cells). Methods LS8 cells were treated with NaF at 0, 0.5, 1, 2, 4, 8 mM for 24 and 48 h, respectively. The effect of fluoride on cell viability, apoptosis and cell changes were assayed by CCK-8, flow cytometry and microscope, separately. Some well-known regulators of oxidative stress and mitochondrial pathway of apoptosis were assessed by biochemical assay, qRTPCR and western blotting. Results The results showed significantly decreased of cell viability and increased cell apoptosis in NaF-treated LS8 cells. Besides, NaF treatment in LS8 cells markedly decreased MDA content, while increased SOD activity, the mRNA level of Gpx-1 and Gst, and the protein levels of cleaved caspase-9 and cleaved caspase-3. Conclusion Fluoride exposure could suppress cell proliferation, disorder the balance between oxidative system and anti-oxidant system and cause oxidative stress, and finally result in apoptosis of LS8 cells.