氟对人成骨样细胞株凋亡的影响

Effect of NaF on apoptosis in human osteoblast-like cell line

目的研究氟化钠(NaF)对人成骨样细胞株凋亡的影响及其机制。方法四甲基偶氮噻唑蓝(MTT)法测定细胞的增殖能力,萤光染剂(Hoechst 33342)染色,DNA梯形(DNA Ladder)琼脂糖电泳,流式细胞仪分析检测细胞凋亡,RT-PCR半定量分析抗凋亡蛋白Bcl-2和促凋亡蛋白Bax mRNA表达,显色法检测凋亡相关激酶半胱氨酸蛋白酶3(Caspase-3)的活性。结果NaF(4,6,8×10-3mol/L)可以剂量依赖性地抑制细胞增殖(P<0.01),Hoechst33342染色、琼脂糖电泳和流式细胞仪均可检测到氟对成骨细胞凋亡的诱导作用。RT-PCR结果显示:NaF可以剂量依赖性地抑制Bcl-2 mRNA表达(P<0.01),提高Bax mRNA表达(P<0.01),并显著增强Caspase-3的活性(P<0.01)。结论NaF可以显著的抑制人成骨样细胞株增殖和诱导其凋亡,并可能是通过抑制Bcl-2表达,促进Bax表达和提高Caspase-3活性来诱导人成骨样细胞株凋亡。

Objective To study the apoptotic effect of sodium fluoride （NaF） and its mechanisms in human osteoblast cell line OS - 732. Methods The effects of NaF on osteoblast cell proliferation and apoptosis were detected by methyl thiazolyl tetrazolium （MTT）, Hoecbst33342 fluorescence staining, agarose gel electrophoresis, flow cytometry （FCM）, semiquantitative RT - PCR and caspase - 3 activity assays. Results After cells were treated with NaF （4, 6, 8×10^-3 mol/L）for 24 h, the proliferation of OS- 732 cells measured by MTT methods was inhibited in a dose dependent manner（ P 〈 0.01 ）. The morphological changes and the apoptotic peaks were observed by fluorescence staining and FCM respectively. In addition, the expression of Bcl- 2 mRNA decreased whereas the expression of Bax mRNA increased measured by semi - quantitative RT - PCR. The activity of Caspase - 3 also increased after cells were treated with NaF （6 ×10^- 3 tool]L）for 24h（ P 〈 0.01 ）. Finally, DNA fragmentation was observed when cells were treated with NaF （4, 6, 8×10^-3 mol/L）for 48 h. Conclusion NaF inhibited the proliferation of osteoblast cell line OS- 732 and induced the cells to undergo apoptosis by increasing the activity of caspase - 3, and the expression of Bax while decreasing the expression of Bcl - 2.