摘要
目的探讨慢性氟中毒大鼠骨骼损伤时细胞核转录因子-κB（NF-κB）相关基因表边改变与破骨细胞凋亡的关系，深入研究氟骨症的发病机制。方法健康SD大鼠36只，体质量100～120g，按体质量随机分为3组，每组12只。对照组饮用自来水（含氟量〈1mg／L），低氟组和高氟组分别饮用含5、50mg／L氟化钠的自来水。大鼠饲养8个月，建立慢性氟中毒模型，股动脉放血处死。取大鼠股骨干骺端，光镜观察骨组织形态学改变；采用酶联免疫吸附法检测血清抗酒石酸酸性磷酸酶5b（TRACP5b）水平：采用抗酒石酸酸性磷酸酶（TRAP）染色鉴定破骨细胞并计数；采用Real—timePCR和免疫组织化学方法检测骨组织中p50、IKBa和凋亡相关基因Bcl-2、Bax的mRNA和蛋白表达水平。结果染氟大鼠股骨干骺端呈骨质硬化表现。低氟组血清TRACP5b水平、破骨细胞数[（3．45±1．85）U／L、（6．75±1．29）个／切片]显著高于对照组．[（1．26±0．23）U／L、（3．92±1．38）个／切片，P均〈0．05]，高氟组[（2．74±1．85）U／L、（3．33±1．07）个／切片]较低氟组降低（P均〈0．05）。低氟组p50、IκBBα、Bcl-2、BaxmRNA表达水平（4．41±0．44、1．15±0．25、2．02±0．11、1．25±0．22）显著高于对照组（1．46±0．10、0．26士0．07、1．00±0．06、0．74±0．09，P均〈0．05），高氟组（0．69±0．09、0．14±0．03、0．95±0．08、0．62±O．08）较低氟组降低（P均〈0．05）。低氟组p50、IκBα蛋白表达水平（152．96±7．87、156．20±9．75）显著高于对照组（125．63±9．85、118．97±6．94，P均〈0．05），高氟组（120．56±9．57、114．50±7．61）较低氟组降低（P均〈0．05）；低氟组Bcl-2、Bax蛋白表达水平（170．61±6．60、160．77±7．66）和Bcl-2／Bax比值（1．07±0．08）较对照组（110．73±5．27、114．64±5．83、0．96±0．04）升高（P均〈0．05），高氟组（81．70±8．00、99．93±3．83、0．81±0．08）较对照组和低氟组降低（P均〈0．05）。p50、IκBα蛋白表达水平与Bcl-2／Bax比值呈正相关关系（r值分别为0．587、0．676，P均〈0.05）。结论慢性氟中毒可引起骨组织NF-κB相关基因表达改变以及破骨细胞凋亡，氟骨症的机制可能与NF—κB050和IKBa表达改变引起的破骨细胞凋亡失常有关。
Objective To investigate the relationship between change of relevant gene of nuclear factor kappa B （NF-κB） and osteoclast apoptosis in bone injury of rats with chronic fluorosis, and to reveal the mechanism Of skeletal fluorosis. Methods Thirty-six healthy SD rats, weighting 100 - 120 g, were randomly divided into three groups（twelve in each group）. Rats of control group were fed with tap water（NaF 〈 1 mg/L） and the experimental rats were exposed to NaF（low-dose group： 5 rag/L, high-close group： 50 mg/L） through drinking water to established chronic fluorosis model. All rats were killed at the eight month and metaphysic of femoral was collected. Bone tissues were stained with hematoxylin-eosin and observed under optical microscope. Serum content of tartrate-resistant acid phosphatase 5b（TRACP 5b） was detected by enzyme-linked immunosorbent assay（ELISA）. Osteoclast was identified and counted by tartrate-resistant acid phosphatase staining（TRAP）. The expression of pSO, IκBα, Bcl-2 and Bax＇s mRNA and protein of bone tissue was detected by Real-time PCR and immunohistochemistry.Results Bone sclerosis was observed under optical microscope. The content of TRACP 5b in serum and the number of osteoclast in the low fluoride group[ （3.45 ± 1.85）U/L, （6.75 ± 1.29）/slice] were significantly higher than that of the control [ （ 1.26 ± 0.23 ） U/L, （3.92 ± 1.38 ）/slice, all P 〈 0.05 ], but that of the high fluoride group [（2.74 ± 1.85）U/L, （3.33 ± 1.07）/slice] were lower than that of the low dose group（all P 〈 0.05）. The mRNA expressions of p50, IκBα, Bel-2 and Bax in low fluoride group（4.41 ± 0.44, 1.15 ± 0.25, 2.02 ± 0.11, 1.25 ± 0.22） were significantly higher than that of the control（1.46 ± 0.10, 0.26 ± 0.07, 1.00 ± 0.06, 0.74 ± 0.09, all P 〈 0.05）, but the high fluoride groups＇ （0.69 ± 0.09, 0.14 ± 0.03, 0.95 ± 0.08, 0.62 ± 0.08） were lower than that of the low dose group（all P 〈 0.05 ）. The protein expressions of p50 and IKBot in the low fluoride group （152.96 ± 7.87, 156.20 ± 9.75） were significantly higher than that of the control（125.63 ± 9.85, 118.97 ± 6.94, all P 〈 0.05）, but the high fluoride group（120.56 ± 9.57, 114.50 ± 7.61） were lower than the low dose group（all P 〈 0.05）. The protein expressions of Bel-2 and Bax（170.61 ± 6.60, 160.77 ± 7.66） and the ratio of Bcl-2/Bax （1.07 ± 0.08） were higher than the control（110.73 ± 5.27, 114.64 ± 5.83, 0.96 ± 0.04, all P〈 0.05）, but the high fluoride group（81.70 ± 8.00, 99.93 ± 3.83, 0.81 ± 0.08） were lower than that of the control and the low dose group （all P 〈 0.05）. There was a significant positive correlation between protein expression of p50, IκBα and Bcl-2/Bax （r = 0.587, 0.676, all P 〈 0.05）. Conclusions Chronic fluorosis can cause change of the relevant gene of NF-KB in rat bone tissues and osteoclast apoptosis. The mechanism of skeletal fluorosis might be related to the abnormal of osteelast apoptosis caused by changes of NF-κB p50 and IκBα.
出处
《中国地方病学杂志》
CAS
CSCD
北大核心
2012年第5期518-522,共5页
Chinese Jouranl of Endemiology
基金
科技部国际合作项目（2010DFB30530）
贵州省科技厅社会发展攻关项目（黔科合SY[2011]3006号）
