骨细胞程序性坏死通过上调RANKL促发绝经后骨质疏松发生的机制研究

Postmenopausal osteoporosis (PMOP) seriously damage to health benefits for older women. The imbalance between bone formation and bone resorption is considered a key pathological mechanism of osteoporosis. Therefore, it has great significance for preventing and treating PMOP that explore the mechanism of the imbalance between bone formation and bone resorption. In our earlier studies, we found that osteocyte necroptosis might be a main cause osteoporosis in OVX rats and it might correlate with increased RANKL protein expresstion. However, the role of osteocyte necroptosis in the imbalance between bone formation and bone resorption of PMOP patients and its mechanism has remained unclear. In this study, osteocytes from PMOP patients will be investigated to understand whether necroptosis is an important type of osteocyte death using western blot, laser confocal microscopy and transmission election microscopy. In addition, we explore whether necroptotic osteocytes can lead to the imbalance of RANKL/RANK/OPG system via up-regulation of RANKL protein with different intervening therapies，which cause increasing bone resorption and may be a critical regulation mechanism in osteoporosis. This will also be test in rat model in vivo. We hope what we get from this study will answer how necroptotic osteocytes tip the balance toward increased bone resorption coupled with decreased formation and the progression of osteoporosis. It will be a novel insight for the preventing and treating of postmenopausal osteoporosis.

绝经后骨质疏松症（PMOP）严重危害老年女性身体健康。骨形成和骨吸收偶联失衡是导致PMOP的主要机制，因此探讨骨形成和骨吸收偶联失衡的机制对防治PMOP意义重大。前期我们发现骨细胞程序性坏死可能是卵巢切除大鼠骨质疏松的重要原因，且该过程可能与骨细胞RANKL表达上调相关。但骨细胞程序性坏死在PMOP患者骨形成和骨吸收偶联失衡中的作用及其机制尚不清楚。本项目在前期工作基础上以PMOP患者为研究对象，采用western blot、激光共聚焦和电镜等技术，明确程序性坏死是PMOP发病过程中骨细胞死亡的主要方式。采用不同干预措施探讨骨细胞程序性坏死是否是通过上调RANKL使RANKL/ RANK/OPG系统失衡而导致骨吸收大于骨形成。并在动物模型上加以验证。以期回答骨细胞程序性坏死是如何引起骨形成和骨吸收偶联失衡，导致PMOP这一科学问题，可能为防治PMOP提供新的研究靶点和切入点。

绝经后骨质疏松症（PMOP）严重危害老年女性身体健康。探讨PMOP的发生机制对于PMOP的防治具有重要意义。课题组的前期研究表明，骨细胞程序性坏死是PMOP发生的重要原因，但程序性坏死的骨细胞如何促进PMOP的发生，目前尚不清楚。本研究项目首先以不同阶段的PMOP患者为研究对象，通过透射电镜、免疫荧光等技术检测了程序性坏死和RANK/RANKL/OPG系统的多种信号分子，证明PMOP发生过程中骨细胞RIP1、RIP3、MLKL、RANKL分子表达上调而OPG表达减少，其结果表明PMOP发生过程中骨细胞死亡的主要方式可能是程序性坏死；并且PMOP的发生可能与RANKL表达的上调以及RANK/RANKL/OPG系统的失衡有关。其次，我们构建了卵巢切除（OVX）大鼠动物模型，并使用E2、zVAD、Nec-1s对模型进行干预；通过Micro-CT检测OVX大鼠骨组织微观结构变化，提示OVX大鼠骨组织发生了骨质疏松；使用免疫组化、Western blot技术比较不同干预组程序性坏死标志物及RANKL的表达情况，结果提示骨细胞死亡过程中RIP3、MLKL等程序性坏死标志物表达上调且伴有RANKL的表达上调。最后，应用TNF-α诱导MLO-Y4细胞构建程序性坏死细胞模型，通过Western blot检测到程序性坏死的MLO-Y4细胞RANKL表达上升；通过不同干预状态下的MLO-Y4细胞与RAW 264.7细胞共培养，并使用TRAP染色验证了程序性坏死的MLO-Y4细胞能诱导破骨细胞的生成；且RIP1特异性抑制剂Nec-1s能抑制这些现象的发生。另外，我们用RANKL诱导RAW 264.7细胞，并通过TRAP染色验证了RANKL能促进破骨细胞分化。这些结果提示：程序性坏死的骨细胞可能通过促进RANKL表达促进了破骨细胞活化，这信号通路可能是PMOP发生的机制之一。该项目可为PMOP的防治提供新的切入点。

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