摘要目的 观察氟在单层细胞培养系统即二维(2D)培养体系和成纤维细胞(FB)胶原凝胶系统即三维(3D)培养体系中对小鼠FB血管内皮生长因子(VEGF)mRNA及蛋白表达的影响,探讨VEGF表达改变对FB成骨功能的作用.方法 在2D、3D培养体系中,FB按染氟剂量不同分为0(对照)、0.0001、0.0010、0.1000、1.0000、10.0000、20.0000mg/L组,染氟培养48h后,应用RT-PCR法、酶联免疫吸附法(ELISA)和免疫组化方法(IHC)检测VEGF mRNA和蛋白的表达.结果 3D培养体系中,0.1000 mg/L组VEGF mRNA表达(1.08±0.09)高于对照组(0.93 ±0.02,P<0.05).2D培养体系中,0.1000、1.0000、10.0000 mg/L组VEGF蛋白表达(0.19±0.02、0.26±0.01、0.32±0.01)高于对照组(0.14±0.01,P均<0.05);3D培养体系中,0.1000、1.0000 mg/L组VEGF蛋白表达(0.59±0.06、0.52±0.03)高于对照组(0.37±0.05,P均<0.01).2D培养体系中,0.0010mg/L组VEGF蛋白阳性表达细胞数(0.45±0.05)高于对照组(0.36±0.03,P<0.05);3D培养体系中,0.0010、0.1000、1.0000 mg/L组VEGF蛋白阳性表达细胞数(0.62 ±0.04、0.70±0.06、0.65 ±0.07)高于对照组(0.44±0.04,P<0.05或<0.01).结论 2D和3D培养体系中FB VEGF mRNA和蛋白,提示VEGF在氟化物刺激FB成骨功能增强方面具有重要作用.

abstractsObjective To observe the expression of vascular endothelial growth factor(VEGF) mRNA and protein in fluoride(F~-) treated fibroblast(FB) of mice in planar(2D) and FBs populated collagen lattice(3D) culture systems and to further explore the effects of VEGF on the osteogenic action of FB. Methods FB were divided into 0 (control group), 0.0001,0.0010,0.1000,1.0000,10.0000 and 20.0000 mg/L groups(F~-). The levels of VEGF mRNA and protein at 48 h were measured by using RT-PCR, ELISA and immunohistochemistry (IHC) methods. Results The expression of VEGF mRNA increased obviously in group of 0.1000 mg/L(1.08 ± 0.09) in 3D FB compared with the control group(0.93 ± 0.02, all P < 0.05). Fluoride increased the content of VEGF protein obviously in groups of 0.1000,1.0000,10.0000 mg/L(0.19 ± 0.02, 0.26 ± 0.01 and 0.32 ± 0.01 ), higher than that in 2D FB culture supematant in the control group(0.14 ± 0.01, all P < 0.05) ; and in groups of 0.1000, 1.0000 rag/L(0.59 ± 0.06 and 0.52 ± 0.03) it was higher than that in 3D FB culture supematant in the control group(0.37 ± 0.05, all P< 0.01 ). The IHC results showed that the VEGF positive staining cells increased significantly in group of 0.001 mg/L (0.45 ± 0.05) in 2D FB when it was compared with control group(0.36 ± 0.03, P< 0.05); and in groups of 0.0010, 0.1000, 1.0000 rag/L(0.62 ± 0.04,0.70 ± 0.06 and 0.65 ± 0.07) are it was higher than that in 3D FB control group (0.44 ± 0.04, P < 0.05 or < 0.01 ). Conclusions The higher expression of VEGF mRNA and protein in 2D and 3D FB induced by fluoride may play an important role in stimulating the osteogenesis ability in FB.