氨基葡萄糖对蛋鸡骨骼代谢和钙代谢的调控机理研究

Glucosamine is a naturally occurring sugar that is synthesized by virtually all cells with various physiological properties,including antibacterial,immunomodulator and osteoarthritis treatment.Our initial results showed that the egg shell thickness of layers fed diet with glucosmine was higher than those of the control group,egg shell also contained more calcium than the control. Hens also gained more calcium and higher ratio of trabecular bone in tibia.Those results strongly hinted glucosamine have putative roles in bone metabolsim, but the pathway invovled is yet unwared.Bone is continuously and dynamically remodeled through two coupled processes:degradation of existing bone through the resorption activity of osteoclasts and deposition of new bone through the matrix formation activity of osteoblast.Regulation of recruitment,proliferation,and differentiation of osteoclasts and osteoblast is critical for the maintenance of bone mass.Under many pathologic conditons including osteoporosis,osteosclerosis,the amount of bone removed by osteoclast and deposited by osteoblasts is unbalanced.osteoclasts are the principal resorptive cells of bone. OPG/RANKL/RANK, a cell-cell contact signal pathway is essential for the bone formation and remolding directed by nutrients,hormome and cytokines.This project aim to study effects of glucosamine on regulation of bone formation and remolding in layer hens in vitro and in vivo.800 laying hens with same age, similar laying rate will be individed into 4 groups at 18 week, 25 week and 59 week age,respectively. Hens fed with 0%, 0.4%, 0.6% and 0.8% GA diet. Laying performance, egg shell qualities of different laying stage, and bone biocheimical markers (serum calcium,phosphorus, ALP, TRAP, estrogen,) will be determined by feeding trial to evaluated the effects Glucosamine. At 0, 30,60 day, 6 layers /groups be slaughtered,groum radiographic density, cortical width, tibia cortical width and bone volume will be tested with electron microscope or radioimmunoassay. Furthermore,OPG,RANKL and RANK in the bone amplified with Real Time-PCR to judge the contribution and the expression in different groups.Osteocytes,osteoblast and osteoclasts be cultured in cell medium,then,the influcences of glucosamine on bone absorption,proliferation,opoptosis and function were evaluated by MIT,flow cytometry,and TRAP stain. Those study will help us to elucidate the effects of Glucosamine on skeletal metaboslim. The present investigations will led us to understand of the mechanisms of gluocsamine involved in bone metabolism better.And it will help us to applicate the potential oraganic feed additive on layer farming.

氨基葡萄糖（GA）可提高蛋壳厚度及钙含量、骨钙量及骨小梁比例，强烈提示GA参与骨骼及钙代谢的调节，但其途径未知。项目拟以0.4%、0.6%、0.8%GA饲粮饲喂开产、高峰和末期蛋鸡，考察GA对各阶段产蛋性能、蛋壳质量的影响，检测血液骨代谢相关理化指标、骨矿含量、骨组织代谢标志物、骨密度、骨显微和超微结构等骨质性状，以探索GA作用规律；测定钙消化率及小肠钙调蛋白表达差异，以明确GA是否参与钙消化吸收的调节；因OPG/RANKL/RANK信号途径为介导营养、激素和细胞因子调节骨骼形成和重建所必须。故实验还将利用RT-PCR探讨GA对OPG、RANKL和RANK基因表达差异的影响，再以体外细胞培养研究骨细胞、成骨细胞和破骨细胞增殖、分化、凋亡、细胞分泌功能及钙浓度的变化，以阐明GA对骨骼细胞和钙代谢的调节机理。以期为有效防治蛋鸡骨质疏松症，提高蛋壳质量，开辟新的绿色饲料添加剂提供理论和技术支撑。

骨质疏松症（OCL)是严重威胁蛋鸡养殖业健康发展的代谢性疾病之一，造成了巨大损失。目前尚未有效防治OCL的营养措施。前期研究发现 GlcN可提高骨钙含量和蛋壳厚度，及调节RNAKL活性，提示GlcN介导OPG-RANK-RANKL信号系统，该系统在骨生成和骨吸收等过程发挥重要作用。为阐明GlcN在蛋鸡中的作用规律及其机制，本项目重点开展(1) GlcN对3个生产阶段蛋鸡产蛋性能、蛋壳质量、骨质性状和血液生化指标的影响、(2) GlcN对蛋鸡饲粮钙吸收及小肠钙结合蛋白表达的调节、(3) GlcN对蛋鸡骨代谢相关基因表达的影响、（4）在细胞水平验证GlcN对骨骼细胞的中骨代谢基因表达的作用和（5）GlcN对蛋壳钙沉积的调节等方面研究。结果发现：GlcN促进蛋鸡采食，提高开产、高峰上升期和后期蛋鸡产蛋率，减少异常蛋率，增加蛋壳钙和磷，增大蛋壳厚度，各阶段适宜GlcN水平为0.6%；GlcN上调Cabp-D28k表达，提高钙吸收率11.42%；GlcN增加血钙浓度，促进E2和CT分泌，降低IL-6、PGE2和TRAP，抑制炎症因子分泌，不同阶段呈现出效应时间差异；3个阶段荧光定量分析显示，GlcN上调蛋鸡胫骨中OPG基因表达，抑制RANKL、RANK、 CTK和IL-6基因表达，降低RANKL/OPG比值，减少破骨细胞活性以及骨基质降解，增强骨小梁连续性，增加皮质骨面积和厚度，骨密度，增强骨骼抵抗外界力量破坏能力，GlcN对骨骼代谢的调节也因生产阶段和骨骼的不同而呈现出差异；GlcN刺激子宫OPN、OC-116、OCX-32及OCX-36的表达，增加蛋壳腺钙分泌，促进蛋壳钙的沉积，增加蛋壳钙的含量最终蛋壳比例和蛋壳厚度增加，增大蛋壳强度。项目实施过程中，培养硕士研究生5名，完成硕士学位论文4篇和博士论文1篇(含2020年参加答辩2篇)，撰写发表科研论文11篇(含待发表7篇)。