活性维生素D调控前成骨细胞增殖分化的作用机制研究

Vitamin D metabolism plays an essential role in the regulation of bone and mineral homeostasis. It regulates calcium and phosphorus levels and inhibits the synthesis and secretion of parathyroid hormone (PTH). Its active form, 1α, 25(OH)2D3, has been used for over 30 years to prevent and treat the complications of osteoporosis; the mechanism through which vitamin D regulates bone formation, however, is yet to be fully understood...Preosteoblasts may be strategic cells in the context of vitamin D-driven bone formation. Three preosteoblastic phenotypes have been described: endoplasmic reticulum (ER)-rich cells, ER-poor cells and undifferentiated cells. Preosteoblastic proliferation and differentiation are crucial phases of the process of bone formation. In previous studies, we have employed the ovariectomy (OVX) rat model to scrutinize the effects of a second-generation vitamin D analog, eldecalcitol, on bone formation. We verified that osteoclastic numbers and bone resorption parameters were significantly reduced in eldecalcitol-treated rats when compared to those from the OVX group. In addition, the preosteoblastic layer, with which osteoclastic precursors interact for mutual differentiation, was poorly developed in the eldecalcitol group, suggesting diminished cell-to-cell contact among precursors of the osteoblastic and osteoclastic lineages. A rather unexpected finding was that eldecalcitol was able to promote focal bone formation independently of bone resorption, a phenomenon known as bone mini-modeling. Altogether, these findings suggest that eldecalcitol stimulates preosteoblastic differentiation, thereby hindering cell-to-cell contact between preosteoblasts and osteoclastic precursors. The diminished interplay between these precursors may explain the lower osteoclastic numbers and the decrease in bone resorption after eldecalcitol treatment. Based on these histological findings, we conjectured that vitamin D participates in bone formation by affecting preosteoblastic proliferation and differentiation, and indirectly adjust osteoclastic formation. . .In order to elucidate the proposed hypothesis and to clarify the function of osteoclasts in the context of vitamin D treatment, we intend use in vivo and in vitro approaches, using immunohistochemical, ultrastructural and molecular biology techniques. Further clarification of the mechanism by which vitamin D regulates bone formation will provide new avenues of research and interesting cues for clinical applications and drug development.

活性维生素D被广泛应用在骨质疏松症引起的骨折防治方面，但其调控骨形成的确切机制仍然不是很清楚。在我们的前期研究中，除了观察到骨量和骨密度增加以及破骨细胞数量减少现象外，更重要的是，出现了前成骨细胞数量减少和mini-modeling现象（不依存于骨吸收的骨形成）。鉴于前成骨细胞包含有ER-rich cell、 ER-poor cell、 undifferentiated cell等不同亚型，本课题拟通过使用基因改变动物模型和细胞模型,研究活性维生素D调控前成骨细胞增殖分化的机制。计划采用免疫组织化学，免疫电镜等超微形态观察以及RT-PCR和Western blot等分子生物学技术，对c-fos-/-,RANKL-/-等基因敲除小鼠以及VDR转基因小鼠进行分析论证。活性维生素D调控骨形成机制的进一步阐明不仅会为临床应用提供有意义的科学依据，也会为新药开发提供新思路和新靶点。

针对活性维生素D影响骨形成的研究目标主要集中在成熟成骨细胞，以及破骨细胞与前成骨细胞之间也存在着相互影响的形态学位置关系的现状，课题组以本国家自然科学基金面上项目为支撑，从骨组织工程角度明确了活性维生素D在全身投药和局部给药两种不同方式下对前成骨细胞增殖分化、促进骨缺损愈合方面所发挥的作用。论证了活性维生素D在骨缺损愈合早期能够促进成骨细胞分化、抑制破骨细胞数量和活性；在骨缺损愈合晚期阶段能够促进骨质的矿化成熟。关于破骨细胞在活性维生素D调控前成骨细胞增殖分化过程中所扮演的角色，课题组发现活性维生素D的单纯应用抑制前成骨细胞的增殖，但破骨细胞培养上清的加入显示出促进前成骨细胞增殖的趋势。影响前成骨细胞分化方面，活性维生素D的单纯应用显示出抑制倾向，尽管随后抑制程度有所改善。破骨细胞在活性维生素D影响前成骨细胞分化方面没有体现出较为明显的调控作用。另一方面，在破骨细胞进行骨基质吸收破坏的前提下，虽然在初期活性维生素D仍然表现出抑制前成骨细胞增殖的趋势，但实验后期抑制趋势有所改变。这些实验结果说明骨基质破坏所释放出来的相关因子改变了单纯破骨细胞分泌物对活性维生素D调控前成骨细胞增殖的影响。影响成骨分化方面，破骨细胞骨质吸收破坏条件的增加，明显提高了活性维生素D促进前成骨细胞分化的程度，说明骨基质破坏所释放出来的相关因子在调控活性维生素D影响前成骨细胞分化方面发挥了重要作用。项目实施期间共发表相关SCI收录学术论文22篇，合计影响因子为55.187，所有文章均标注National Natural Science Foundation of China及项目资助编号No.81271965。培养包括博士后、博士研究生以及硕士研究生在内的青年科研人员15名。此外，以本国家自然科学基金面上项目为依托，与日本国立北海道大学合作，成功申报了国家自然科学基金国际（地区）合作交流项目（NSFC-JSPS，No.81311140173）。