利用hepcidin基因敲除小鼠研究"铁过载"对绝经后骨质疏松的影响及其机制

The causes of postmenopausal osteoporosis are not completely clear. Estrogen deficiency is generally considered as the most important risk factors. Recently, reports showed that iron overload was another potential risk factor independent of estrogen deficiency. Our previous studies confirmed the association of iron overload with postmenopausal osteoporosis. To explore the action mechanism, we designed this project. Pilot studies: First, bone density at proximal femur of hepcidin knockout mouse was lower than wild type mouse. Second, Hepcidin increases intracellular Ca2+ of osteoblast hFOB1.19 through L-type Ca2+ channels. Third, the expression of ferroportin 1 presents in cultured hFOB 1.19 cells. Aims: First, investigate the changes of bone mineral density and bone microstructure of hepcidin knockout mice model. Second, analyze the effect of iron overload on bone mineral density and elucidate its mechanism. Methods: Fifty hepcidin knockout mice will be equally divided in to 5 groups, and 10 wild type mice was brought into the wild type control group. Three groups of hepcidin knockout mice will be respectively treated with deferoxamine 30mg/kg/day, 100mg/kg/day and 200mg/kg/day from 20 weeks to 28 weeks. Another group of hepcidin knockout mice will be treated with distilled water. The residual two groups will not be treated. Some data will be collected and analyzed, including bone mineral density, bone microstructure, biomechanical property of bone, bone mineral content, bone formation markers, bone resorption biomarkers, serum iron level, serum calcium level, serum magnesium level, serum ferroprotein level and iron content of bone and liver. Osteoblasts and osteoclasts from bone marrow of knockout and wild mice will be co-cultured. Activity of osteoblasts and osteoclasts will be detected. The data will be analyzed using analysis of variance. Expected results and significance: The effect of iron overload on bone mineral density will be systematically elucidated. This study helps to reveal the pathogenesis of postmenopausal osteoporosis. This study will provide more information to develop new drugs and therapeutic strategies for treatments of postmenopausal osteoporosis.

我们既往研究提示："铁过载"与绝经后骨质疏松密切相关。在已完成腹腔注射枸橼酸铁铵建立铁过载小鼠模型并发现其双侧股骨中段和第四腰椎骨密度明显低于对照组的工作基础上，利用基因敲除技术，建立hepcidin基因敲除小鼠（标准铁过载模型）及野生型小鼠原代成骨细胞与破骨细胞共培养体系，通过在体（动物）和离体（细胞）实验结合并且关联验证的实验设计方案，研究"铁过载"对绝经后骨质疏松的影响及其作用机制。预实验结果显示：hepcidin基因敲除小鼠组织铁过载明显，股骨远端骨密度下降，骨小梁稀疏。本项目将进一步完善在体实验，结合离体实验，运用MicroCT三维重建、生物力学测试、骨组织形态学检测及分子生物学方法等多重技术和先进检测手段，来检测hepcidin基因敲除小鼠骨微结构、骨密度、铁代谢指标等的异常变化，明确铁过载对绝经后骨质疏松的影响及其作用机制，为现有的绝经后骨质疏松症防治方法提供有益补充。

我们既往研究提示：“铁过载”与绝经后骨质疏松密切相关。在已完成腹腔注射枸橼酸铁铵建立铁过载小鼠模型并发现其双侧股骨中段和第四腰椎骨密度明显低于对照组的工作基础上，本研究通过敲除小鼠的hepcidin基因，建立遗传背景一致的铁过载小鼠模型，通过检测hepc-/-小鼠及hepc+/+小鼠的铁代谢指标，观察比较两组小鼠的肝脏铁、骨组织铁、血清铁及血清铁蛋白的含量，明确hepc-/-小鼠存在严重的铁过载；通过检测两组小鼠的MicroCT及骨转换指标，明确hepc-/-小鼠股骨远端骨密度下降，骨小梁稀疏，骨量下降，且骨形成标志物骨钙素水平明显偏低，提示铁过载通过抑制成骨细胞途径诱发骨质疏松。提取hepc-/-小鼠及hepc+/+小鼠的原代成骨细胞及破骨细胞，分离培养，发现hepc-/-小鼠的成骨细胞碱性磷酸酶（ALP）和I型胶原的表达量均低于对照组，进一步研究发现铁过载抑制了BMP/samd信号通道，导致BMP2、BMP6以及Smad1水平下降，从而导致磷酸化的Smad1水平下降，最终Runx2和osteorix的表达水平下降，抑制了成骨细胞的分化和功能，骨形成能力下降，骨密度下降。我们应用不同浓度的外源性hepcidin干预hepc-/-小鼠及hepc+/+小鼠的原代成骨细胞，发现铁过载对小鼠成骨细胞的增殖无明显抑制或促进作用，但可以提高BMP2、BMP6及Smad1的表达量，解除hepcidin基因敲除对BMP/Smad信号通路的抑制，从而提高骨形成能力。