Objective To observe the expression of fibronectin in bone of fluorosis rats and in vitro cultured osteoblast,and to study the role of fibronectin in pathogenesis of chronic fluorosis.Methods Male and female Wistar rats 144 were randomly divided into four groups,which were designated as the control group(normal diets,n =36),fluoride group(normal diets + 100 mg/L fluoride,n =36),lower calcium monophagia group (synthetic diets,n =36) and lower calcium monOphagia with fluoride group(synthetic diets + 100 mg/L fluoride,n =36).Rats were sacrificed 4 and 8 months after beginning of the experiment,respectively,and femur tissue was fixated and paraffin-embedded.The osteoblast isolated from calvaria of neonatal rats was treated with different dose of fluoride(0,1,2,4 mg/L fluoride,respectively) for 48 and 72 h,cell culture supernatant and cells were collected,respectively.The cranial osteoblasts were cultured in vitro and divided into four groups according to different concentration of fluoride added,which were 0(control group),0.01,1.00,and 10.00 mg/L groups.These cells were treated with mineralized induced medium at day 2 and cultured for 3 more weeks whereafter,and then the slides were fixed in alcohol.The expression of fibronectin in rat femur tissue was detected by immunohistochemistry (IHC),and fibronectin mRNA expression was determined by in situ hybridization; the fibronectin levels in supernatant of cultured osteoblast was examined by enzyme-linked immunosorbent assay(ELISA),and the expression of fibronectin mRNA in osteoblasts was detected with RT-PCR; skull mineralized nodule formation of osteoblasts was observed under a light microscopy after stained with 0.1％ red alizarin liquid.Results Little expression of fibronectin (brown granules under light microscope) could be seen in femur tissue of fluorosis rats of control group and lower calcium monophagia group; but abundantly expressed in fluoride group and lower calcium monophagia with fluoride group; the fibronectin was also expressed in osteoblasts,bone cells and bone marrow cells with less red particles in the control group and lower calcium monophagia group,but more in the fluoride group and lower calcium monophagia with fluoride group.The expression of fibronectin protein in supernatant of cultured osteoblasts was significantly increased in the group of 4 mg/L fluoride at 48 h(0.108 ± 0.042,t =0.764,P＜ 0.05) compared with control group(0.081 ± 0.010); the value was also significantly increased in 1,2,4 mg/L groups at 72 h(0.089 ± 0.010,0.087 ± 0.012,0.098 ± 0.023; t =0.765,0.704,0.996; all P ＜ 0.05) compared with control group (0.070 ± 0.014) ; the expression of fibronectin mRNA was much higher in 1,2,4 mg/L groups at 48 h (0.61 ±0.06,0.77 ± 0.07,0.77 ± 0.07) and 72 h(1.61 ± 0.14,2.54 ± 0.20,2.75 ± 0.22) compared with control group [0.48 ± 0.04(t =0.111,0.182,0.182,all P ＜ 0.05),0.97 ± 0.08(t =0.093,0.109,0.108,all P＜ 0.05) ].A lot of mineralized nodules could be seen under light microscope in 1.00 and 10.00 mg/L groups.Conclusions The expression of fibronectin in bone of fluorosis rats and in vitro cultured osteoblasts are increased,and fluoride also promotes the mineralization nodules formation of osteoblasts.These results suggest that fibronectin may regulate the process of bone mineralization,and possibly play a role in the development of skeletal fluorosis.%目的 观察纤连蛋白(fibronectin)在氟中毒大鼠骨组织和体外培养成骨细胞中的表达,探讨纤连蛋白在氟骨症发生机制中的作用.方法 Wistar大鼠144只,雌雄各半,体质量约120 g,按体质量将大鼠分为4组:对照组(正常食料)、加氟(F-)组(正常食料+ 100 mg/L F-)、低钙偏食组(合成食料)、低钙偏食加氟组(合成食料+ 100 mg/L F-),每组36只,在喂养4、8个月时分别处死大鼠,取股骨组织固定、石蜡包埋；采用细胞培养的方法,体外培养乳鼠颅骨成骨细胞,按不同的染氟剂量分为4组:0(对照)、1、2、4 mg/L组,分别在培养47、72 h收集颅骨成骨细胞和培养上清.体外培养乳鼠颅骨成骨细胞,按不同的染氟剂量分为4组:0(对照)、0.01、1.00、10.00 mg/L组,在染氟培养的第2天加入矿化诱导液,继续培养3周后取出玻片、酒精固定.免疫组化(IHC)法检查纤连蛋白在大鼠股骨组织中的表达；原位杂交法测定大鼠股骨组织中纤连蛋白mRNA表达；酶联免疫吸附(ELISA)法测定颅骨成骨细胞培养液中纤连蛋白含量；RT-PCR法测定成骨细胞纤连蛋白mRNA表达；0.1％茜素红染色,光镜下观察颅骨成骨细胞矿化结节形成.结果 光镜下对照组和低钙偏食组大鼠股骨组织中纤连蛋白均有阳性表达,可见到棕黄色颗粒,但量较少；加氟组和低钙加氟组骨组织中可见到大量的纤连蛋白阳性表达的棕黄色颗粒.光镜下大鼠股骨组织中成骨细胞、骨细胞和骨髓内细胞均有纤连蛋白mRNA阳性表达,对照组和低钙偏食组纤连蛋白mRNA阳性表达的红色颗粒较少,加氟组和低钙偏食加氟组纤连蛋白mRNA阳性表达的红色颗粒较多.培养48 h,成骨细胞培养上清液中纤连蛋白均增加,4 mg/L组纤连蛋白(0.108±0.042)明显高于对照组(0.081±0.010,t=0.764,P＜0.05)；培养72 h,1、2、4 mg/L组纤连蛋白(0.089±0.010、0.087±0.012、0.098±0.023)明显高于对照组(0.070±0.014,t值分别为0.765、0.704、0.996,P均＜ 0.05).培养48和72 h,1、2、4 mg/L组成骨细胞纤连蛋白mRNA表达(0.61±0.06、0.77±0.07、0.77±0.07)和(1.61±0.14、2.54±0.20、2.75±0.22)均明显高于对照组[0.48±0.04(t值分别为0.111、0.182、0.182,P均＜ 0.05)和0.97±0.08(t值分别为0.093、0.109、0.108,P均＜0.05)].在1.00、10.00 mg/L组,光镜下成骨细胞中可见大量矿化结节形成.结论 纤连蛋白在氟中毒大鼠骨组织和体外培养成骨细胞中的表达均增加,染氟也促进成骨细胞形成矿化结节,提示纤连蛋白可能通过调节骨组织的矿化过程,在氟骨症发生机制中发挥作用.
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