椎体和长骨不同生长代谢表型中雌激素的特异性作用机制

There are different bone structures between vertebrae and limb long bones. Red marrow can be found in vertebral bodies of the adults. The growth phenotype is also region-specific. Spinal development disorders are often associated with abnormal estrogen receptors and estrogen level. However, most of these patients do not present limb disorders. Estrogen plays key roles during bone development. We previously showed that estrogen has local effects on bone growth through its receptors during development and adulthood. Estrogen also has an indirect effect on the bone growth by regulating the expression of leptin receptor. We also observed that the expression of estrogen receptor is region-specific in the growth plate. These findings indicated that estrogen may be involved in the region-specific bone growth. However, the mechanisms remain unclear. In the present project, we intend to observe the different effects of estrogen on the proliferation, differentiation and mineralization of spine and limb growth plate at animal, organic and cellular levels. We plan to study the region-specific effects of bone growth after selective estrogen receptors modulating. We also intend to observe the action of the classic signaling pathways of estrogen for the effect of estrogen on the region-specific bone growth and the influence of estrogen on other important signaling molecules such as leptin et al. These results of our study should be helpful to reveal the region-specific mechanism of estrogen on the bone growth, and to acknowledge and understand the growth, development and maturity of spine. The present researches will set up a theoretical foundation for prevention and etiological studies of the spinal developmental disorders.

椎体和四肢长骨的骨结构不同，成年椎体仍保留有红骨髓，两者生长发育表型也不同。脊柱发育异常多伴有雌激素及其受体异常，但四肢发育多正常。雌激素是骨生长的关键因子，我们前期研究提示整个发育期，雌激素都能直接作用于其受体影响骨生长，雌激素还可调控瘦素受体表达来间接影响骨生长。我们还发现骺板内雌激素受体表达呈部位差异性，提示部位差异性骨生长中，雌激素有特定作用，但具体机制不清楚。我们拟从动物整体、器官和细胞水平观察雌激素对椎体和长骨骺板增生、分化和矿化的不同影响；探讨雌激素受体α和β选择性调控对骨生长部位差异性的作用；观察在雌激素对骺板部位差异性作用，以及在雌激素对瘦素等重要信号分子的部位差异性影响中，经典信号通路的特定意义。研究结果将对雌激素在骨生长中的部位差异性影响机制有所揭示，有助于我们更好地认识和理解脊柱的生长、发育和成熟，同时为脊柱发育异常等相关疾病的防治和病因学研究奠定理论基础。

椎体和四肢长骨的骨结构不同，成年椎体仍保留有红骨髓，两者生长发育表型也不同。脊柱发育异常多伴有雌激素及其受体异常，但四肢发育多正常。雌激素是骨生长的关键因子，我们前期研究提示整个发育期，雌激素都能直接作用于其受体影响骨生长，雌激素还可调控瘦素受体表达来间接影响骨生长。本课题观察了不同浓度雌激素对新生小鼠长骨和椎体生长板细胞的作用，以及雌激素受体ERα 和 ERβ在不同部位生长板中的特异性作用。分离培养长骨和椎体骺板软骨细胞，然后选择不同浓度水平的17β-estradiol刺激，CCK-8检测细胞增殖，realtime-PCR和Western blotting检测II 型和 X型胶原的表达，Western blotting 和 immunofluorescence免疫荧光检测雌激素/无雌激素和雌激素受体特异性阻断剂联合刺激后椎体和四肢长骨骺板软骨细胞的反应。结果提示，在10-7M浓度时，雌激素刺激软骨细胞II型胶原的表达量最大。雌激素作用于软骨内骨化主要通过ERα受体通路，四肢长骨骺板软骨细胞对雌激素刺激产生II型胶原的敏感性高于椎体骺板软骨细胞。本研究还观察到骺板细胞内Ob-Rb分别和ERα、ERβ之间都有共表达现象。瘦素在不同细胞期对雌激素α受体和Ⅱ型胶原的调控作用并不相同, 软骨性分化期表现为上调ERα, 增加Ⅱ型胶原; 肥大性分化期则为下调ERα, 降低Ⅱ型胶原。软骨性分化期和肥大性分化期软骨细胞内瘦素均能上调ERβ, 下调ERα/ERβ比值和Ⅹ型胶原表达。瘦素刺激后Ⅱ型胶原的变化和ERα变化之间存在明确的线性正相关, Ⅹ型胶原变化和ERα/ERβ比值之间有显著的线性正相关。由此推测, 瘦素能够调控雌激素α和β受体的表达,还可能通过雌激素受体进一步影响Ⅱ型和Ⅹ型胶原表达。