瘦素通过雌激素受体调控椎体和长骨不同生长代谢表型的机制研究

Endochondral ossification in the growth plate is the key step for bone growth. The bone structures and development of spine and limb are different. Children with abnormal leptin bioavailability are often associated with spinal scoliosis. However, most of these patients do not present limb disorders. Our previous study indicated that: the effects of leptin on the primary cultured chondrocytes differentiation of vertebral and tibial growth plate are different; and the effects of estrogen receptors antagonist on the metabolism of spine and limb growth plate are also different；leptin can regulate the expressions of ERα and ERβ during the endochondral ossification, and the effects are region specific and differentiation stage specific. Therefore，we presume that leptin could affect the region specific bone growth by means of estrogen receptors. However, the specific effects and the potential mechanism are not yet clear. In the present project, we intend to observe the different effects of leptin on the proliferation, differentiation and mineralization of spine and limb growth plate via ERα and ERβ at animal, organic and cellular-molecular levels. Comparing with the limb growth plate, we plane to study the specific interaction of leptin and PTHrP et al. in the spine growth plate after selective estrogen receptors modulation. By evaluating and contrasting with the limb growth plate, we also intend to study the action of the classic signaling pathways of leptin on the metabolism of spine growth plate via ERα and ERβ. These results of our study should be helpful to reveal the region-specific mechanism of leptin on the bone growth via estrogen receptors, and to enrich the theory of bone growth. The present researches will set up a theoretical foundation for prevention and etiological studies of the bone developmental disorders.

椎体和四肢长骨的骨结构不同，发育形式亦不同。瘦素异常儿童易出现脊柱畸形，但极少合并四肢异常。我们前期工作发现：瘦素对椎体和胫骨骺板软骨细胞增生分化作用不同；雌激素受体阻断对椎体和胫骨骺板代谢影响不同；瘦素能调控软骨内骨化中雌激素受体表达，且有椎体和长骨差异性和分化时期特异性。提示瘦素能通过雌激素受体调控部位差异性骨生长，但具体机制不明。本课题拟从动物、器官和细胞分子水平观察瘦素通过雌激素受体ERα和ERβ对椎体和长骨骺板增生、分化和矿化的不同影响；研究ERα和ERβ选择性调控，对瘦素在椎体骺板内与PTHrP（甲状旁腺激素相关肽）等重要因子之间，不同于长骨的，相互作用机制；探讨经典信号通路在瘦素通过雌激素受体调节椎体骺板生长代谢中，相对长骨的，特异性作用规律。结果有助于揭示瘦素通过雌激素受体调控部位差异性骨生长的机制，丰富骨生长发育理论，为骨发育异常等相关疾病防治和病因学研究奠定理论基础。

骨结构形成于骺板内的软骨内骨化，软骨内骨化是骺板软骨细胞有规律地进行增生、成熟、肥大性转化和矿化的过程。在瘦素基因缺陷小鼠(ob/ob)中，瘦素（Leptin, Lep）的缺失导致了脊柱椎体和四肢长骨的生长发育的差异。软骨内骨化是影响骨生长的重要过程，这一过程也受到雌激素及其受体(ER)信号通路的调控。本课题旨在探讨椎体和四肢长骨生长发育差异性中Lep和ER的交互作用。我们研究发现，不同ER的阻断对于青春期小鼠四肢和脊柱骨的生长代谢表型存在部位差异性，并且ER的调控作用主要通过Ihh/PTHrP信号轴实现。我们进一步运用C57BL/6小鼠和ob/ob小鼠对比研究，发现ob/ob小鼠股骨较对照小鼠长度缩短，而脊柱长度增长。ob/ob小鼠股骨和脊柱生长板软骨细胞增殖性蛋白和肥大性标记蛋白的表达模式也存在差异。ERα在ob/ob小鼠的脊柱和四肢长骨骺板内存在部位差异性表达，即ERα在脊柱骺板内高表达，而在长骨骺板内低表达。Spearmen分析显示脊柱骨和股骨长度与生长板中雌激素受体α的表达水平呈正相关。，基于X线和HE染色结果，我们还发现瘦素缺乏似乎扰乱了ER拮抗剂对纵骨生长的调节作用。这些结果提示ERα的部位特异性表达可能与ob/ob小鼠脊柱和四肢长骨的生长模式有密切关系，瘦素能够明确弱化ER的这一调节作用。研究结果有助于为骨发育异常等相关疾病的病因学研究奠定理论基础。