DEC1对成骨细胞分化的调控及其在药物抗骨质疏松的机制及意义

Based on the following data we had gotten: (1) The differentiated embryonic chondrocytes 1 (DEC1) expression significantly decreased in both two types of osteoporotic mice induced by bilateral ovariectomy and glucocorticoid. (2) 17-estradiol (E2) and icariin treatment can reverse the decrease of DEC1 expression and prevent the bilateral ovariectomy and glucocorticoid induced osteoporosis respectively. (3) Dexamethasone (DEX) decreases the osteogenesis capacity such as alkaline phosphatase (ALP) activity and mineralization along with decreasing the DEC1 expression in the osteoblasts (Saos-2). On the contrary, E2 increases the osteogenesis along with increasing the DEC1 expression in the osteoblasts. (4) The overexpression of DEC1 alone increases the ALP activity and mineralization synchronously, and it not only partially reverses the decrease of ALP activity induced by DEX, but almost abolishes the decrease of mineralized nodules induced by DEX. Therefore, it is hypothesized that DEC1 probably plays a critical role in the osteogenic differentiation of osteoblasts. Form the whole, the cell and molecule levels, this study will investigate the regulation and mechanism of DEC1 in the osteogenesis of osteoblasts, and the mechanism and significance of DEC1 in the anti-osteoporosis effects produced by anti-osteoporosis drugs using knockout DEC1 mice, the overexpression and knockdown of DEC1 in the osteoblasts, primary cultured mouse osteoblasts and mesenchymal stem cells. Expected outcome will reveal the regulation and mechanism of DEC1 in the osteogenesis of osteoblasts, and the mechanism and significance of DEC1 in the anti-osteoporosis effects produced by anti-osteoporosis drugs. This study not only expands the DEC1 in the research field of bone metabolism academically, but also provides a molecular target therapy and the anti-osteoporosis drugs development based on DEC1.

申请者基于前期两种骨质疏松小鼠模型（双侧卵巢切除、大剂量皮质激素）DEC1表达明显减少；雌激素和淫羊藿苷分别处理两种骨质疏松小鼠模型后，均能改善小鼠骨质疏松症状，同时亦可明显逆转DEC1表达降低的作用；大剂量地塞米松（>0.5-1μM）所致细胞骨质疏松模型，其成骨能力明显下降，同时伴DEC1明显降低；过度表达DEC1(能)取消地塞米松的所致成骨细胞成骨能力降低作用。因此，提出DEC1在成骨细胞成骨分化过程中可能发挥重要调控作用的假设。本项目应用DEC1基因敲除小鼠、原代培养成骨细胞和骨髓间充质干细胞为研究模型，运用分子克隆技术、现代分子生物学等方法，从整体、细胞和分子水平，研究并阐明DEC1对成骨细胞成骨分化中的调控作用和分子机制以及DEC1在药物抗骨质疏松作用中的作用机制和意义。预期研究结果不仅拓展DEC1在骨代谢的研究领域，且为预防和治疗骨质疏松药物的研发提供新的靶标。

基于前期两种骨质疏松小鼠模型（双侧卵巢切除、大剂量皮质激素）DEC1表达明显减少；雌激素和淫羊藿苷分别处理两种骨质疏松小鼠模型后，均能改善小鼠骨质疏松症状，同时亦可明显逆转DEC1表达降低的作用；大剂量地塞米松所致细胞骨质疏松模型，其成骨能力明显下降，同时伴DEC1明显降低；过度表达DEC1取消地塞米松的所致成骨细胞成骨能力降低作用。因此，提出DEC1在成骨细胞成骨分化过程中可能发挥重要调控作用的假设。本项目应用DEC1基因敲除小鼠、SaoS-2 细胞株和原代培养骨髓间充质干细胞诱导成骨细胞为研究模型，运用分子克隆技术、现代分子生物学等方法，从整体、细胞和分子水平，研究并阐明DEC1对成骨细胞成骨分化中的调控作用和分子机制以及DEC1在药物抗骨质疏松作用中的作用机制和意义。结果发现：（1）地塞米松（DEX）抑制而淫羊藿苷（ICA）促进SaoS-2 细胞成骨细胞分化，其机制通过降低或上调SaoS-2 细胞DEC1表达；（2）在体和离体实验发现：ICA可拮抗有糖皮质激素诱导的骨质疏松表型以及成骨分化能力降低，其机制主要是DEC1通过PI3K/Akt/GSK3b/β-catenin通路，最终导致参与成骨分化关键因子Runx2上调，促进成骨形成；（3）观察1月龄、6月龄两种基因型（DEC1+/+、DEC1-/-）小鼠的骨质变化，结果发现：与同龄同窝小鼠比较，6月龄DEC1-/-小鼠相比出现明显骨质疏松表型；进一步研究发现DEC1-/-小鼠骨组织中DKK1明显增加，而PI3K/Akt/GSK3b/β-catenin通路上的信号蛋白表达明显降低。5月龄DEC1-/-小鼠腹腔给予LiCl治疗4周可挽救6月龄骨质疏松表型。（4）原代培养源于两种基因型（DEC1+/+、DEC1-/-）小鼠的骨髓间充质干细胞(BMSCs)进行成骨分化诱导发现：源于DEC1-KO小鼠骨髓间充质干细胞成骨分化能力明显低于源于WT小鼠；敲减源于WT小鼠骨髓间充质干细胞的DEC1后,其诱导成骨分化能力也明显降低，而将DEC1导入源于DEC1-KO小鼠骨髓间充质干细胞后，其诱导成骨分化能力明显高于DEC1-KO小鼠。研究提示：DEC1促进成骨细胞分化及功能，为预防和治疗骨质疏松药物的研发提供潜在靶标。