成纤维生长因子23在笼养蛋鸡骨质疏松发生中的作用研究

Cage layer osteoporosis (CLO), induced by calcium and phosphorus metabolic disturbance, is one of the serious diseases in laying hens. Fibroblast growth factor 23 (FGF23) which was discovered in 2000 has been defined as a hormone regulating calcium and phosphate homeostasis by binding to FGFRs under the help of its coreceptor Klotho in target tissues. Abnormalities of bone mineralization in mice were observed in accociation with FGF23 excess or deficiency, but its mechanism is still unclear. We have previously found that FGFRs and Klotho mRNA were expressed in osteoblasts and uterus of laying hen. The concentration of serum FGF23 in CLO hens was obviously higher than that of wild type, suggesting that FGF23 may play a vital role in CLO. The present study aims to determine the level or mRNA expression of FGF23, hormones or factors related to calcium and phospate metabolism in serum, duodenum, kidney, bone and uterus, as well as the change of bone bionomics in CLO and wild type laying hens by real-time PCR and micro-CT. Meanwhile, effect of FGF23 on differentiation of osteoblasts and osteoclasts, bone resorption and formation, communicated factors expression between osteoblast and osteoclast will be approached through real-time PCR and western blot in vitro. Finally, the regulative mechanisms of FGF23 in CLO will be elucidated in order to provide a theoretical foundation for CLO diagnosis and prevention in laying hens industry.

钙磷代谢紊乱导致的骨质疏松（CLO）是影响当前蛋鸡养殖的一种重要疾病。成纤维生长因子23（FGF23）是2000年发现的一种钙磷代谢调控因子，在Klotho辅助下与FGFRs结合而影响钙磷代谢，FGF23表达异常动物出现骨代谢紊乱症状，但机制不清楚。项目组已证明：破骨细胞和蛋壳腺存在FGFRs和Klotho表达；CLO蛋鸡FGF23水平显著高于健康鸡，提示FGF23在CLO发生中有重要作用。项目拟应用real-time PCR和micro-CT等方法检测健康和CLO蛋鸡FGF23以及血液-骨-肠-肾-骨-蛋壳腺中钙磷代谢相关激素/因子含量或mRNA表达及骨生物学特性的改变；通过细胞培养试验，应用western blot等方法检测FGF23对破骨/成骨细胞分化、骨吸收、骨形成以及两种细胞对话因子表达的影响，最终阐明FGF23在CLO发生中的作用及机制，为生产中CLO的诊断和预防提供理论依据。

项目探讨了FGF23在笼养蛋鸡骨质疏松（CLO）发生中的调控作用及其可能机制。发现：①CLO蛋鸡FGF23表达明显增加；血液Ca、P、1，25-(OH)2VD3和E2含量显著降低，而PTH、CT、ALP、PICP和TRAP含量明显升高；十二指肠VDR、CaBP-D28k、PMCA1b表达量显著上调，而NCX1和NaPi-Ⅱb表达量显著下调；肾脏TRPV5、TRPV6、NCX1和 NaPi-IIa表达量显著下调，PMCA1b、FGFRs和Klotho表达量上调；蛋壳腺钙CaBP-D28K、PMCA、CA2、VDR及OPN 表达水平均明显降低；成骨细胞钙磷代谢相关因子ALP、RANKL、OPG、M-CSF和V-ATPase a3 表达均显著降低，而破骨细胞CtsK、FGFRs、TRAP、SPHK1、BMPs的表达显著增高。结果表明，CLO蛋鸡钙磷代谢发生紊乱，表现为十二指肠、肾脏、蛋壳腺和骨钙磷代谢异常。②CLO蛋鸡腿骨指数和骨密度以及胫骨最大应力和三点弯曲载荷明显降低；胫骨中皮质骨和髓质骨发生溶解；胫骨皮质骨部分显著变薄，髓质骨部分骨小梁数量减少、体积变小。结果表明，CLO蛋鸡骨生物学特性的下降。③FGF23能够上调OC中FGFRs和Klotho 表达水平；能够提高OC骨吸收活性，并上调骨吸收相关因子Atp6i、CtsK、Trap和Mmp-9 表达水平；上调BMPs和Sphk1表达水平。结果表明，FGF23能够依赖FGFRs-Klotho途径促进OC分化成熟、提高OC骨吸收活性，并能够间接调控OB活动。④FGF23能上调OC与OB共培养下促OC分化/成熟的相关因子ATP6i、Cts K、TRAP、MMP-9、FGFRs、Klotho、PDGF-bb、SPHK1和BMPs相对表达量，下调抑制OC分化/成熟的相关因子OPG和OCIL相对表达量，促进骨吸收；能够增强OC与OB共培养下促OB分化的相关因子PDGF-bb、SPHK1和BMPs表达，促进OB分化；抑制矿化期OB的矿化和骨形成。结果表明，FGF23可通过调节成骨细胞和破骨细胞之间的“对话因子”促进OB和OC共存下破骨细胞分化、成熟及其介导骨吸收，抑制OB矿化和骨形成。综上，本研究表明，FGF23表达上调与CLO发生密切相关，其机制与FGF23升高导致的钙磷代谢紊乱有关，尤其是骨形成能力减弱和骨吸收能力增强有关。