氟对软骨细胞印度刺猬蛋白、甲状旁腺激素相关肽、跨膜蛋白表达及细胞增殖与凋亡的影响  

Effects of fluoride on the expression of Indian hedgehog, parathyroid hormone-related peptide, smoothenedprotein and mRNA and on proliferation and apoptosis of chondrocytes

作　　者：陶欣[1] 朱志坚[1] 邓超男[1] 

机构地区：[1]贵州医科大学病理学教研室,贵阳550004

出　　处：《中华地方病学杂志》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）

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�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：

关 键 词：氟 软骨细胞 Ihh信号通路 细胞增殖 细胞凋亡 

分 类 号：R599[医药卫生—内科学]