TRPV5/TRPV6钙离子通道对蛋鸡髓质骨形成及信号传导系统的影响

The unique medullary bone is formed in the marrow cavities of the long bones of mature female birds, and it serves as a labile source of Ca2+ for shell calcification. The transient receptor potential vanilloid receptor 5，6 (TRPV5, TRPV6) are considered to facilitate the entry of Ca2+ into the epithelial cells of osteoblast (OB) and osteoclast (OC) in bone, but the specific role and form mechanism of the medullary bone has not yet been studied in laying hen. Effects of TRPV5/6 on the growth, development, proliferation, differentiation and apoptosis of osteoblasts and osteoclsts and regulation of TRPV5/6 on genetic expression of the signaling system of OPG、RANKL、MAPK and sSmad, and so on were in vitro investigated with construction of the recombined plasmid TRPV5/6 small interfering RNA（siRNA）in this project. In addition, The gene silence effect of TRPV5/6 siRNA on the expression of genes including TRPV5/6, the signal pathway of OPG, RANKL, MAPKs and sSmads, bone metabolic markers, morphometry of medullary bone, and laying performance is determined in vivo. The objective of this study is able to reveal the action approach and molecular mechanism of TRPV5/6 calcium channel and their regulation of signal transduction systems on the medullary bone formation. Moreover, this project may play an important role in providing the theoretical evidence for etiology and pathology of calcium metabolism disorders of osteoporosis and joint diseases in layers.

髓质骨是雌禽所特有的结构，其主要功能是为蛋壳钙化提供钙源。新型钙离子通道TRPV5和TRPV6是成骨细胞膜和破骨细胞膜上调控钙离子转运的重要通道，但该蛋白在维持髓质骨形成与重建的作用途径和分子机制仍不详。本项目通过构建TRPV5/6 siRNA重组表达质粒，研究TRPV5/6对成骨细胞和破骨细胞、发育、增殖、分化及凋亡的影响，探讨TRPV5/6对OPG、RANKL、MAPKs及sSmads等信号系统基因表达的调控作用。另外，通过体内试验，研究TRPV5/6 siRNA质粒对未性成熟和高产蛋鸡髓质骨OPG、RANKL、MAPKs及Smads等信号通路基因的表达、骨代谢标志物、骨形态计量学及蛋鸡生产性能的影响等，揭示蛋鸡髓质骨形成TRPV5/6钙离子通道及其调控信号传导系统的作用途径及分子机理，为蛋鸡骨质疏松症症病因学、病理学等研究提供理论依据。

髓质骨是雌禽在性成熟时出现的独特的骨骼类型，与蛋鸡体内钙、磷代谢，骨骼代谢以及蛋壳形成均具有重要关联。通过成骨细胞和破骨细胞数量增减以实现髓质骨的重建与吸收。TRPV5/TRPV6（Transient receptor potential vanilloid receptor）是存在于成骨细胞和破骨细胞中的对钙离子有高度选择性的钙离子蛋白通道。通过对钙离子跨细胞转运以调控细胞生物功能、维持机体钙离子浓度及正常骨骼代谢。鉴于TRPV5/TRPV6在钙离子转运及骨代谢中的重要调控作用，本项目旨在构建鸡TRPV6 RNA干扰质粒；通过脂质体介导转染鸡成骨细胞，干扰TRPV6基因表达，观察沉默TRPV6基因对成骨细胞增殖、分化、钙离子转运相关基因、OPG和RANKL基因表达及凋亡的影响；体外试验：成功构建了小鼠TRPV6过表达慢病毒载体；通过慢病毒转染小鼠破骨细胞，观察TRPV6基因过表达对破骨细胞钙离子转运相关基因及细胞凋亡的影响；体内试验，通过给高产蛋鸡肌肉注射TRPV6 RNA 干扰质粒，探讨TRPV6干扰质粒在小肠内分布及沉默效果、对十二指肠和空肠钙转运相关基因表达以及对蛋鸡生化指标和骨骼代谢的影响。结论：成功构建了鸡TRPV6 RNA干扰质粒，其转染成骨细胞后能够抑制成骨细胞增殖分化，促进成骨细胞凋亡，导致钙离子转运相关基因、OPG和RANKL基因表达改变；成功构建了小鼠破骨细胞TRPV6过表达慢病毒载体，转染后导致破骨细胞钙转运相关基因表达及凋亡率改变；体内试验证实：TRPV6干扰质粒抑制蛋鸡十二指肠和空肠TRPV6表达，导致PTH上升，钙转运相关基因表达改变。这些将为进一步研究骨骼生物学功能和骨代谢提供有价值的试验依据和参考。