免疫球蛋白结合蛋白在氟中毒大鼠骨组织中的表达研究

Immunoglobulin binding protein gene and protein expression in femur tissue of fluorosis rats

目的观察内质网应激标志性分子免疫球蛋白结合蛋白（BiP）在氟中毒大鼠股骨组织的表达，探讨内质网应激在氟骨症发病机制中的可能作用。方法60只Wistar大鼠，按体质量随机分成4组，每组15只。对照组饲以常规饲料（含钙量为0．79％），低钙组饲以自制低钙饲料（含钙量为0．063％），饮用自来水（氟化钠水平〈1mg／L）；高氟组和低钙高氟组分别饲以常规饲料、自制低钙饲料，饮用自来水中加氟（氟化钠水平为221mg／L）。实验期间动物自由进食、进水，每周测体质量1次。实验周期为12周。通过免疫组化和RT—PCR技术分析BiP在股骨组织中基因和蛋白水平的表达变化。结果高氟组、低钙组、低钙高氟组大鼠股骨矿物质水平[（0．131±0019）、（O．097±0．011）、（0．083±0．007）g／cm]均低于对照组[（0．159±0．029）g／cm，P均〈O．05]；低钙组、低钙高氟组的骨密度[（0．243士0．018）、（0．223±0．022）g／cm2]均低于对照组[（0．296±0．046）g／cm2，P均〈0．05]。免疫组化技术检测到低钙组和低钙高氟两组大鼠股骨内抗BiP阳性染色的成骨细胞较对照组显著增加，而且低钙高氟组明显比低钙组的阳性成骨细胞多。RT—PCR分析显示出低钙加氟组大鼠骨组织的骨桥蛋白（OPN）、骨钙蛋白（OCN）mRNA表达水平（1．3±0．20、1.31±0．11）高于对照组（0．82±0．16、0．85±0．15，P均〈0．05）；与加氟组（0．97±0．29）比较，低钙加氟组（1．36±0．20）大鼠骨组织的OPN表达明显提高（P〈0．05）；低钙组和低钙高氟组BiP基因表达（1．38±0．24、1．35±0．12）高于对照组（1．14±0．06，P均〈0．05）。结论投氟或联合低钙饮食刺激了大鼠股骨BiP基因和蛋白的表达，说明高氟或低钙高氟条件下大鼠骨组织出现不同程度的内质网应激。

Objective To observe the protein and gene expression of immunoglobulin binding protein （BiP） in the femur of fluoride-treated rats, and preliminarily study the possible role of endoplasmic reticulum stress in the pathogenesis of skeletal fluorosis. Methods Sixty Wistar rats were divided into 4 groups according to body weight, n = 15. The control and low-calcium groups were fed with normal diet（0.79% calcium） and low-calcium diet（0.063% calcium）, respectively, and both drank tap water（fluoride concentrations 〈 1 rag/L）. High-fluoride and coexposure to low-calcium groups were fed with conventional feed（0.79% calcium） and low-calcium diet （0.063% calcium）, respectively, and both drank tap water containing sodium fluoride（sodium fluoride concentration of 221 mg/L）. During experimental period, rats were measured body weight once a week with a stand diet and water available ad libitum. The experiment lasted for 12 weeks. The immunohistochemieal and reverse transcription - polymerase chain reaction （RT-PCR） techniques were used to detect the protein and gene expression of BiP in the femur of fluoride-treated rats and control subjects. Results The bone mineral contents of high fluoride, low- calcium and coexposure groups[ （0.131 ± 0019）, （0.097 ± 0.011 ）, （0.083 ± 0.007）g/cm] were lower than those of the control group [ （0.159 ±0.029）g/cm, all P 〈 0.05 ] ; the bone mineral density of low calcium and coexposure to fluoride group[ （0.243 ± 0.018）, （0.223 ± 0.022）g/em2j was lower than that of the control group[ （0.296± 0.046）g/cm2, all P 〈 0.05 ]. The immunohistochemical staining showed that the anti-BiP antibody positive osteoblasts were significantly increased in the low calcium diet and coexposure to fluoride groups than that in the control, and coexposure to fluoride elevated the positive cells than that in only low calcium diet group. The mRNA expression of osteopontin（OPN） and osteocalcin （OCN） in coexposure to fluoride with low-calcium group （1.36 ±0.20,1.31 ± 0.11 ） was higher than that of the control groups（0.82± 0.16, 0.85 ± 0.15, all P 〈 0.05） ; moreover, OPN expression significantly increased in this group than that of the only high fluoride group （0.97 ± 0.29, P 〈 0.05）. The mRNA expression of BiP in the low calcium and coposure to fluoride group （1.38 ±0.24,1.55 ±0.12） was significantly higher than that of the control group（ 1.14 ±0.06, all P 〈 0.05 ）. Conclusions Higher fluoride or coexposure to low calcium diet stimulates the gene and protein expression in rat femur BiP, indicating that varying degrees of endoplasmic reticulum stress is likely involved in the pathogenesis of rat skeletal fluorosis.