摘要
目的 观察氟对软骨细胞印度刺猬蛋白（Ihh）、甲状旁腺激素相关肽（PTHrp）、跨膜蛋白（Smo）表达和细胞增殖与凋亡的影响。方法原代培养乳鼠关节软骨细胞，传第3代后按染氟（氟化钠，NaF）剂量分为0（对照），5、10、20、40mg／L染氟组。采用噻唑蓝（MTr）法测定24、48、72h细胞增殖率；流式细胞术法检测24、48、72h对照组和40mg／L染氟组细胞凋亡情况；蛋白质免疫印迹法（Westernblot）和反转录PCR（RT—pCR）法检测48hIhh、PTHrp、Smo蛋白及mRNA表达。结果与对照组比较，24、48、72h5mg／L染氟组细胞增殖率明显升高，差异有统计学意义[（1．10±0．08）％比（1．17±0．07）％、（1．13±0．08）％比（1．20±0．06）％、（1．15±0．08）％比（1．16±0．08）％]；40mg／L染氟组细胞增殖率在48、72h显著降低[（0．72±0．11）％、（0．68±0．04）％，P均〈0．05]。与对照组比较，40mg／L染氟组软骨细胞凋亡率在24、48、72h逐渐增高[（1．47±0．05）％、（19．874-3．03）％、（25．30±1．28）％、（45．73士4．63）％，F=123．328，P〈0．01]。与对照组比较，5、lO、20、40mg／L染氟组在48hIhh、PTHrp、Smo蛋白及mRNA表达均明显增高（Ihh蛋白：0．77±0．08比0．98±0．07、1．23±0．06、1．37±0．07、1．34±0．07；PTHrp蛋白：0．68±0．04比0．89±0．05、0．83±0．05、1．29±0．05、1．16±0．08；Smo蛋白：0．37±0．01比0．64±0．06、0．67±0．03、0．96±0．06、0．69±0．06，IhhmRNA：0．77．4-0．05比0．98±0．05、1．09±0．05、1．27±0．03、1．46±0．06；PTHrpmRNA：0．67±0．07比0．97±0．05、1．07±0．08、1．37±0．05、1．45±O．05：SmomRNA：0．45±0．03比O．63±0．04、0．71±0．05、0．81±0．01、1．00±0．02，P均〈0．05）。结论低氟剂量对软骨细胞起增殖作用，而高氟剂量则起抑制作用。随时间的延长，氟可促进软骨细胞凋亡；随染氟剂量增高，软骨细胞中Ihh信号通路Ihh、PTHrp、Smo的蛋白及其mRNA表达逐渐升高。氟上调软骨细胞中Ihh信号通路以及促进软骨细胞的增殖与凋亡，并共同参与软骨细胞的损伤过程。
Objective To study the proliferation and apoptosis and investigate the expression of Indian hedgehog （Ihh）, parathyroid hormone-related peptide （PTHrp）, smoothened （Smo） protein and mRNA in the cultured rat primary chondrocytes exposed to different doses of NaF. Methods The third generation articular chondrocytes of neonate rat were cultured in vitro and treated with 0 （control）, 5, 10, 20 and 40 mg/L of fluoride. The proliferation activities of cells at different times （24, 48 and 72 h） were tested by Thiazolyl Blue Tetrazolium Bromide （MTT）. The apoptosis rate was determined by flow cytometry. The expressions of protein and mRNA of Ihh, Smo and PTHrp at 48 h were determined by Western blotting and semi-quantitative RT-PCR, respectively. Results After exposed to 5 mg/L of iluoride for 24, 48 and 72 h, the proliferation rates were significantly increased [（1.17±0.07）%, （1.20± 0.06）%, （1.16 ± 0.08）%] compared with those of control group [（1.10±0.08）%, （1.13 ± 0.08）%, （1.15 ± 0.08）%], but the proliferation activity at 48 and 72 h in 40 mg/L group [（0.72 ± 0.11）%, （0.68 ±0.04）%] was significantly lower than those in control group （all P 〈 0.05）. Compared with the control group, apoptosis rate of cartilage cell in fluoride treatment group increased gradually [（1.47± 0.05）%, （19.87± 3.03）%, （25.30 ± 1.28）%, （45.73 ± 4.63）%, F = 123.328, P 〈 0.01]. Western blot analysis and RT-PCR results showed that the Ihh, PTHrp, Smo mRNA and protein expression increased in the fluoride groups at 48 h （Ihh protein： 0.77 ±0.08 vs. 0.98 ± 0.07, 1.23± 0.06, 1.37 ± 0.07, 1.34 ± 0.07; PTHrp protein： 0.68 ± 0.04 vs. 0.89 ± 0.05, 0.83 ± 0.05, 1.29 ± 0.05, 1.16 ± 0.08; Smo protein： 0.37 ± 0.01 vs. 0.64 ± 0.06, 0.67 ±0.03, 0.96 ± 0.06, 0.69 ± 0.06; Ihh mRNA： 0.77 ± 0.05 vs. 0.98 ± 0.05, 1.09 ± 0.05, 1.27 ± 0.03, 1.46 ± 0.06; PTHrp mRNA： 0.67 ±0.07 vs. 0.97 ± 0.05, 1.07 ± 0.08, 1.37 ± 0.05, 1.45 ± 0.05; Smo mRNA： 0.45 ± 0.03 vs. 0.63 ± 0.04, 0.71 ± 0.05, 0.81 ± 0.01, 1.00 ± 0.02, all P 〈 0.05）. Conclusions Low doses of fluoride can promote the proliferation of chondrocytes cultured in vitro, and high doses of fluoride can promote the apoptosis of chondrocytes cultured in vitro. The expression of Ihh signaling pathway RNAs and proteins of the cartilage cells are increased following increased levels of fluoride. The results suggest that fluorine .has activated the Ihh signaling pathway in chondrocytes and promoted the proliferation and apoptosis processes which might be involved in chondrocytes injury.
出处
《中华地方病学杂志》
CAS
CSCD
北大核心
2016年第2期83-88,共6页
Chinese Journal of Endemiology
基金
国家自然科学基金（81260419）
教育部博士点基金（博导类）（20125215110001）
关键词
氟
软骨细胞
Ihh信号通路
细胞增殖
细胞凋亡
Fluorine
Chondrocytes
Ihh signaling pathway
Cell proliferation
Apoptosis
