FoxO3α在失重性骨质丢失中作用机制的研究

FoxOs family plays an important role in the development of osteoporosis, FoxO3a was found to promote osteoblast differentiation and proliferation, and inhibit osteoclastogenesis and differentitation.We have found that microgravity induced osteopenia,which are co-related with changes of osteoblast and osteoclast. Since the role of FoxO3a in the process of microgravity induced down-regulation of activity of osteoblast and changes of osteoclast activity has not been reported, we designed this research. First, by using hindlimb unloaded rats, level of micro-structural and morphological changes in weight bearing bones was measured, and then change of FoxO3a and its related ROS pathway, apoptosis was evaluated, so to initially confirm the role and its way of FoxO3a in simulated weightlessness induced bone loss. Second, rotating clinostat was used to simulate weightlessness treatment, to further explore the molecular mechanism of action of FoxO3a.Changes of MC3T3-E1 cell and raw264.7 cell in rotating clinostat and in MC3T3-E1-raw264.7 co-culture system were observed. FoxO3a changes and factors of ROS signaling pathway and apoptosis related factors after siRNA silence FoxO3a expression were examined. With these researches, we can further reveal the molecular mechanism of action of FoxO3a in osteopenia induced by weightless.

FoxO家族在骨质疏松的发生发展过程中发挥着重要的调控作用，已发现FoxO3a可促进成骨细胞分化增殖、抑制破骨细胞生成分化。我们前期研究发现模拟失重引起骨质丢失，OPG/RANKL比值下降，提示成骨细胞和破骨细胞的功能变化是引起此现象的重要原因。但FoxO3a在失重环境下对成骨细胞和破骨细胞的作用仍不清楚。本课题以此为切入点，重点观察后肢去负荷大鼠承重骨中FoxO3a及其相关的ROS通路、细胞凋亡的改变，初步证实FoxO3a在失重性骨质丧失中的作用及其途径。为进一步探究FoxO3a 的分子作用机制，采用回转器模拟失重处理MC3T3-E1、raw264.7细胞，并在两者的共育体系中分别观察FoxO3a及其下游相关因子的表达变化，并观察siRNA法沉默FoxO3a表达后的相关因子变化，为揭示FoxO3a在失重性骨质丢失中的作用机制提供实验依据，对长期航天失重引起的骨质丢失的解决提供新的思路。

本研究拟探讨模拟失重条件下FoxO3a对成骨细胞细胞凋亡的影响及其作用机制，为进一步探究揭示FoxO3a在失重性骨质丢失中的作用机制提供实验依据。本课题组在四年的研究工作中较好的达到了预期目标。完成了以下工作：1）模拟失重环境可促进成骨细胞凋亡，抑制细胞中FoxO3a的表达；2） FoxO3a表达下降可导致成骨细胞凋亡增多。过表达FoxO3a可以部分逆转模拟失重导致的成骨细胞凋亡增加；3）FoxO3a通过抑制ROS表达进而对成骨细胞凋亡进行调控；4）FoxO3a是miR-132-3p的靶基因。模拟失重可通过miR-132-3p/FoxO3a/ROS通路促进成骨细胞凋亡；5）（AspSerSer）6-脂质体输送系统可以将miRNA-132-3p的抑制剂antagomir-132靶向输送至骨形成区，特异性降低小鼠股骨骨组织中miR-132-3p表达，有效地缓解尾吊模拟失重引起的骨形成能力降低、骨小梁微观结构退化和骨骼力学性能降低。研究结果明确了FoxO3α在模拟失重下成骨细胞凋亡变化中作用的分子机制，验证了靶向基因药物对模拟失重导致的骨质丢失的防护效果。从而丰富了失重性骨质丢失发生机制的研究理论，为防护失重性骨质丢失提供了新的思路。