氟对原代培养大鼠成釉细胞增殖、凋亡的影响

The effects of fluoride on proliferation and apoptosis of primary cultured rat ameloblast

目的探讨不同浓度氟对体外培养的原代大鼠成釉细胞增殖、凋亡的影响。方法分离出生4d的SD大鼠上下颌磨牙牙胚成釉细胞进行原代培养。采用0．0（对照组）、0．4、0．8、1．6、3．2、6．4mmol／L氟化钠（NaF）作用24、48、72h，倒置显微镜下观察细胞形态，免疫组织化学法鉴定成釉细胞，3-（4，5．二甲基噻唑．2）．2，5-二苯基四氮唑溴盐（MTT）法检测各时间点细胞增殖情况。细胞经1．6mmol／LNaF处理24、48h或经50ixmoL／Lcaspase泛抑制剂Z．VAD．FMK预处理1h后给予1．6mmol／LNaF处理48h。流式细胞术检测细胞凋亡。蛋白免疫印迹法（Westernblot）检测caspase．3及多聚（ADP核糖）聚合酶（PARP）表达。采用统计软件SigmaStatV3．5对数据进行统计学分析。结果①细胞形态：成釉细胞密度较低时呈多角形，密度较大时呈铺路石样，细胞核明显，具有上皮来源细胞的典型形态特征。②免疫组织化学染色：培养的细胞细胞角蛋白14（CKl4）和成釉蛋白（AMBN）表达呈阳性，符合成釉细胞细胞免疫学特征。@MTr检测：NaF对成釉细胞增殖效应呈剂量和时间依赖性。低浓度NaF（0．4、0．8mmol／L）组经24h处理，细胞增殖指数（1．38±0．11、1．29±0．13）均显著高于对照组（1．00±O．00，P均〈0．05）；经48h处理，各处理组间细胞增殖比较差异无统计学意义（对照组、0．4、0．8mmol／L组增殖指数分别为l｜00±0．00、1．16±0．14、0．94±0．07，P均〉0．05）；经72h处理，0．4、0．8mmol／L组细胞增殖指数（0．87±0．03、0．80±0．04）均显著低于对照组（1.00±0．00，P均〈0．05）。中等浓度（1．6mmol／L）组经24h处理，细胞增殖指数（0．90±0．08）与对照组比较，差异无统计学意义（P〉0．05）；经48、72h处理，细胞增殖指数（0．38±0．03、0．26±0．04）均低于对照组（P均〈0．01）。高浓度（3．2、6．4mmol／L）组经24、48、72h处理，细胞增殖指数（3．2mmol／L：0．574-0．14、0．084-0．03、0．004-0．00；6．4mmol／L：0．1l±0．04、0．00±0．oo、0．00±0．00）均显著低于对照组（P均〈0．01）。④流式细胞术检测细胞凋亡：1．6mmol／LNaF处理细胞24、48h，细胞凋亡率[（5．80±2．03）％、（17．45±4．97）％]均高于对照组[（2．59±0．95）％，P均〈0．05]。caspase泛抑制剂Z．VAD．FMK预处理组细胞凋亡率[（9．43±3．79）％]低于1．6mmol／LNaF组[（18．26±3．39）％，P〈0．05]。@Westernblot检测：1．6mmol/LLNaF处理细胞48h可引起easpase．3及PARP活化。结论过量氟可抑制成釉细胞体外增殖，通过激活caspase级联反应引起细胞凋亡。

Objective To investigate the effects of fluoride at different concentrations on proliferation and apoptosis of primary rat ameloblast in vitro. Methods Ameloblasts were isolated from tooth germ of 4 days SD rat maxillomandibular molar and cultured in vitro. Cells were treated with NaF at 0.0 （control group）, 0.4, 0.8, 1.6, 3.2 and 6.4 mmol/L for 24, 48 and 72 h, respectively. Inverted microscope was used to observe cell morphology; immunochemistry method was used to identify ameloblasts; 3-（4, 5-dimethylthiazole-2）-2, 5-diphenyl tetrazolium bromide （MrIT） assay was applied to measure cell viability at each time point. The cells were treated with 1.6 mmol/L NaF for 24 and 48 h, or after 50 mol/L caspase pan-inhibitor Z-VAD-FMK pretreatment 1 h, 1.6 mmol/L NaF treatment for 48 h. Cell apoptosis was then tested by flow cytometry. In addition, activation of caspase-3 and poly （ADP-ribose） polymerase （PARP） were assessed by Western blotting to explore potential involvement of caspase activation in NaF-induced apoptosis. All data analysis was performed using SigmaStat V 3.5 software. Results （!）Primary rat ameloblasts were in polygonal shape at low density and appeared like paving stone at high density with obvious nucleus, showing typical morphological characteristics of cells with epithelial origin. ②The results of immunochemistry assay indicated that the cultured cells were positive in cytokeratin 14 （CK14） and ameloblastin （AMBN） staining, in accordance with the immunocytochemical characteristics of ameloblasts. ③The effects of NaF on ameloblast proliferation were in a dose- and time-dependent manner. For low dose NaF （0.4 and 0.8 mmol/L） groups, ceils treated for 24 h had significantly higher cell proliferation rates than that of the control group （0.0 mmol/L, P 〈 0.05）, the proliferation indexes for 0.0, 0.4 and 0.8 mmol/L groups were 1.00 ± 0.00, 1.38 ± 0.11 and 1.29 ± 0.13, respectively; the same doses of NaF had no obvious influence on cell proliferation at 48 b （1.00 ± 0.00, 1.16 ± 0.14 and 0.94 ± 0.07, P 〉 0.05）; cell proliferation indexes at 72 h were significantly lower than that of the control group （0.87 ± 0.03 and 0.80 ± 0.04, P 〈 0.05）. Medium dose of NaF （1.6 mmol/L） did not cause obvious alterations in cell proliferation at 24 h （0.90 ± 0.08,＇P 〉 0.05）; while cell proliferation indexes at 48 and 72 h were obviously reduced than that of the control group （0.38 ± 0.03 and 0.26 ± 0.04, P 〈 0.01）. For high NaF concentration （3.2 and 6.4 mmol/L） groups, cell proliferation indexes were significantly decreased at all time points compared with control cells, the rates for 3.2 mmol/L groups were 0.57 ± 0.14, 0.08 ± 0.03 and 0.00±0.00, respectively, and the rates for 6.4 mmol/L groups were 0.11 ± 0.04, 0.00±0.00 and 0.00 ± 0.00, respectively （P 〈 0.01）. ④Flow cytometry was used to detect apoptosis. The results showed that treatment with 1.6 mmol/L NaF resulted in significantly increased apoptosis in ameloblasts at both 24 h [（5.80±2.03）%] and 48 h [（17.45±4.97）%] compared to the control group [（2.59±0.95）%, P 〈 0.05]. In cells pre-treated with pan-caspase inhibitor Z-VAD-FMK, NaF-induced apoptosis was significantly lower than that of cells treated with only 1.6 mmol/L NaF [（9.43±3.79）% vs （18.26±3.39）%, P 〈 0.05]. ⑤Cleavage of caspase-3 and PARP was detected in ameloblasts treated with 1.6 mmol/L NaF for 48 h. Conclusion Overdose fluoride could inhibit proliferation and induce apoptosis via activation of caspase cascade in primary cultured rat ameloblasts.