不同浓度氟对山羊成骨细胞周期和凋亡的影响

The Influence of Fluoride on Cell Cycle and Apoptosis of Cultured Caprine Osteoblasts

为了探讨氟化物对反刍动物成骨细胞的细胞周期和凋亡的影响,本研究以初生山羊股骨骨膜为材料分离成骨细胞,接种于含15%FBS的DMEM培养液,于37℃、5%CO2的培养箱中培养。通过形态观察（Hoechst33342/PI双重染色）、DNA ladder电泳、透射电镜、流式细胞仪等方法检测氟对成骨细胞的调控作用。结果表明,高氟（1.0×10^-3及2.5×10^-3mol/L）使成骨细胞G0/G1期减少,S期细胞增多,抑制成骨细胞周期由合成DNA的S期向G2M期转化,使细胞停滞在S期。1.0×10^-5-2.5×10^-3mol/L氟可使DNA断裂,诱导成骨细胞凋亡,其中1.0×10^-4mol/L氟所致早期和晚期凋亡率最高,分别为3.33%和2.92%。本试验证实氟可诱导成骨细胞凋亡。

In order to determine whether fluoride would induce apoptotic cell death, our research group monitored DNA fragmentation and the cellular morphology with Hoechst33342/PI double staining,DNA ladder and transmission electron microscope （TEM）. The level of apoptosis was analyzed with a fluorescence-activated cell sorter （FACS） by the Annexin-V-FITC and the propidium iodine （PI） double staining method. Cell-cycle analysis was also examined with FACS using PI staining. The experimental results were as follows： Ultrastructural features of osteoblasts in control group exhibited clear nuclear envelope, large nucleolus, even karyoplasms, abundant endoplasmic reticulum and mitochondria. When fluoride was more than 1.0 × 10^-4 mol/L, cellular membrane disintegration, cytoplasm condensation, chromatin compaction or fragmentation, endoplasmic reticulum （ER） expansion, nucleus shrinkage or periphery were observed. Fluoride at 1.0 × 10^-5 - 2.5 × 10^-3 mol/L induced apoptosis with DNA fragmentation. FACS cell-cycle analysis demonstrated that fluoride caused potent G0/G1 arrest. Few cells could be found either in the S phases or the G2/M, thus inhibiting the transformation from S phase into G2/M phase under high levels of fluoride. The highest apoptosis rate of earlier and later stages were induced by 1.0× 10^-4 mol/L fluoride（3.33% and 2. 92% respectively）. In conclusion, the results revealed that fluoride can induce apoptosis in osteoblast.