染氟小鼠成骨细胞系MC3T3-E1细胞中还原型谷胱甘肽和氧化型谷胱甘肽观察

Level of reduced glutathione and oxidized glutathione in a mouse bone cell line MC3T3-E1 cells exposed to fluoride

目的观察染氟小鼠成骨细胞系MC3T3-E1细胞中还原型谷胱甘肽（GSH）和氧化型谷胱甘肽（GSSG）的水平。方法采用噻唑蓝（MTT）法检测不同染氟条件下[F-剂量分别为0（对照）、0．5、1．0、2．0、4．0、8．O、12．O、20．0mg／L]MC3T3-E1细胞1、2、4、10d的细胞活性。另外，按染氟剂量，将MC3T3一E1细胞分为O（对照）、2、8、20mg／L组，分别染氟2、4、10d，采用高效液相色谱-串联四极杆质谱技术检测细胞内GSH、GSSG和谷氨酰胺（Gin）水平。结果染氟1d时2．0mg／L组细胞活性（O．57±0．05）明显高于对照组（0．49±0．03．P〈0．01）；染氟4d时8．0、12．0mg／L组细胞活性（0．49±0．07、0．47±0．09）明显低于对照组（0．63±0．06，P〈0．05或〈0．01）；染氟10d时8．0mg／L组细胞活性（1．52±0．29）明显高于对照组（0．86±0．23），而20．0mg／L组细胞活性（0．54±0．07）明显低于对照组（P均〈0．01）。染氟2、10d时20mg／L组细胞中GSH水平[（13．92±4．63）、（0．53±0．30）μmol／L]明显低于相应的对照组[（26．42±3．67）、（24．85±5．68）μmol／L，P均〈0．01]。染氟2d时2mg／L组、染氟4d时8mg／L组、染氟10d时8mg／L组细胞内GSSG水平[（1．12±0．62）、（2．13±0．62）、（2．97±1．30）μmol／L]明显高于相应的对照组[（0．55±0．22）、（1．46±0．46）、（1．35±0．50）μmol／L，P〈0．05或〈0．01]。染氟4d时2mg／L组和染氟10d时8、20ms／L组细胞内Gin水平[（62．80±17．41）、（122．26±19．51）、（19．38±8．11）μmol／L]明显低于相应的对照组[（83．28±14．32）、（147．154-16．95）μmol／L，P均〈0．05或〈0．01]。结论染氟能明显改变成骨细胞内的GSH、GSSG和Gln水平，从而影响细胞内氧化还原平衡态。

Objective To observe the level of reduced glutathione（GSH） and oxidized glutathione（GSSG） in a mouse bone cell line MC3T3-E1 cells exposed to fluoride. Methods MTT method was used to detect cell viability of MC3T3-E1 cells exposed to varying concentrations and periods of fluoride [ F- concentration： 0 （control）, 0.5, 1.0, 2.0, 4.0, 8.0, 12.0, 20.0 mg/L; F- periods： 1, 2, 4 and 10 days]. The Xevo TQ MS was employed to test the levels of GSH, GSSG and glutamine （Gln）. Results The MC3T3-E1 cell viability was significantly higher in the 2 mg/L group（0.57 ± 0.05） 1 day after the exposure compared to the respective control（0.49 ± 0.03, P 〈 0.01）; conversely, cell viability was markedly lower in the 8 mg/L（0.49 ± 0.07） and 12 mg/L（0.47 ± 0.09） groups 4 days after the exposure in comparison to the control（0.63 ± 0.06, P 〈 0.05 or P 〈 0.01）. The cell viability in the 8 mg/L group（1.52 ± 0.29） 10 days after the exposure was significantly higher than that in the control group （0.86 ± 0.23, P 〈 0.01）, however, the value in the 20.0 mg/L group（0.54 ± 0.07） was significandy lower（P 〈 0.01 ）. The level of cell GSH decreased significantly in the 20 mg/L groups 2 days [ （13.92 ± 4.63）μmol,/L ] and 10 days[（0.53 ± 0.30）μmol/L] after exposure compared to the respective controls[（26.42 ± 3.67）, （ 24.85 ± 5.68.） μmol/L, all P 〈 0.01 ]. The level of cell GSSG markedly increased in the 2 mg/L group 2 days [ （1.12 ± 0.62）μmol/L ] and the 8 mg/L group 4 days [ （2.13 ± 0.62） μmol/L] after exposure compared to the controls [ （0.55 ± 0.22）, （1.46 ± 0.46）μmol/L, all P 〈 0.05]. The similar change was observed in the 8 mg/L group[ （2.97 ± 1.30） μmol/L] 10 days after exposure compared to the control [（1.35 ± 0.50）μmol/L, P 〈 0.05]. The level of Gln decreased significantly in the 2 mg/L group[ （62.80 ± 17.41 ）μmol/L] 4 days and in the 8 and 20 mg/L groups 10 days[ （122.26 ± 19.51）, （1938 ± 8.11）μmol/L] after exposure compared to the controls[ （83.28 ± 14.32）, （147.15 ± 16.95） μmol./L , all P 〈 0.05 or P 〈 0.01 ]. Conclusions Fluoride exposure can significantly promote the changes of GSH, GSSG and Gin levels in the osteoblast, thus affecting the intracellular redox equilibrium.