lnc-A0介导DC-STAMP/NFATc1负反馈环路调控破骨细胞前体在TEB修复中的作用机制研究

To better elucidate the detailed mechanism of bone remodeling in Tissue Engineering Bone (TEB) is considered to be one of the main issues before large-scale clinical application. Osteoclasts (OCs) are crucial players during bone remodeling, however, the mechanism of osteoclastic function in TEB-mediated bone repair remains unclear. Particularly, the role of cell-cell fusion among osteoclast precursors (OCPs) in bone remodeling is rarely studied. Therefore, our group focused on the role of OCPs fusion as part of the mechanism in TEB-mediated bone remodeling. We previously confirmed that the key fusogenic protein DC-STAMP could down regulate its upstream molecule NFATc1 in turn. Recently, our LNCRNA microarray results indicated that lnc-AK077216 exspression was directly associated with NFATc1. Based on the evidence above, we hypothesized that lnc-AK077216 is involved in the negative feedback loop of “DC-STAMP/NFATc1” potentially playing a key role in osteoclastogenesis. To verify our hypothesis, we intend to verify this targeting relationship by dual luciferase gene report system and RIP-seq. Techniques such as ChIRP-seq will be adopted to certify the hypothesized feedback loop and to observe OCPs proliferation, differentiation and especially fusion process. Furthermore, lnc-AK077216 mediated loop will be tested during bone repairing by TEB in mice with bone defect. We deem our study will further illustrate the mechanism of cell-cell fusion of osteoclasts and their role in TEB-mediated bone remodeling, and thus provide novel strategies and methods in building more efficient TEB.

阐释组织工程骨（TEB）修复中的骨重建机制，是探索TEB临床应用的重要命题。破骨细胞（OC）作为骨重建的要素之一，其参与该过程的机理不详，尤其是OC前体融合的调控研究甚少，故以此切入探求TEB骨重塑机制极为必要。在证实关键融合分子DC-STAMP可下调其上游转录分子NFATc1的基础上，基于DC-STAMP为变量的lncRNA芯片结果提示lnc-AK077216 (lnc-A0)可靶向调控NFATc1,因此提出“lnc-A0介导DC-STAMP/NFATc1负反馈环路调控OC前体融合”假说。拟通过荧光素酶基因报告系统、RIP-seq等证实前述靶向关系；利用ChIRP-seq等技术证实该环路，并观察其对OC前体分化及融合的效应。利用小鼠TEB修复模型，在体观察lnc-A0对环路分子的调控,检测干预lnc-A0表达对骨重建的影响。本研究有利于阐明OC分化机理，为TEB的构建和修复拓展新思路

破骨细胞骨吸收与成骨细胞骨生成间的相互耦联，是TEB修复过程中骨重建的核心，在这一动态的过程中，OC对于确保骨重建的动力学必然具有不可替代的作用，但鲜见关于OC在TEB骨修复中的作用及调控机制研究。因此探求OC分化成熟，尤其是聚焦OC前体细胞融合机制，不仅为全面理解OC在TEB修复中的机制提供理论基础，也将为进一步进行优化种子细胞和相应生物材料设计提供科学依据，对于骨组织工程学新技术和新方法的发展也具有重要科学意义。LncRNA作为表观遗传调控的重要方式之一，在骨骼系统中也逐渐受到关注。LncRNA的作用机理为我们提供了一种全新的视角，用来进行阐释其调控NFATc1调节机制，尤其是在破骨细胞分化融合的分子机制方面，有望取得一些新的资料和发现。.课题组建立了破骨细胞体外诱导模型并送检了样本，检测了lncRNA、mRNA、miRNA及circRNA的表达谱。利用小鼠来源单核巨噬细胞系RAW264.7细胞，建立了稳定的体外诱导其向破骨细胞分化及融合模型；通过Real-time PCR技术，对RAW264.7细胞中lnc-AK077216干扰后诱导其向破骨细胞分化。证实lnc-AK077216在破骨细胞分化形成过程中发挥重要作用，从表型及破骨细胞标志性基因、蛋白质检测水平可得出lnc-AK07726能够正向调控破骨细胞分化，其作用机制为抑制了负向调控蛋白NIP45的表达来促进破骨细胞分化。另外以LncRNA-AK077216作为标记物筛选抑制破骨细胞分化的植物提取物（光色素）。研究结果有利于更好的阐明破骨细胞分化机理及其在骨相关疾病中的病理生理作用，为临床上TEB修复骨重建治疗手段提供新的思路和策略。.同时利用所建立多阶段破骨细胞分化模型，检测了HDAC2对破骨细胞融合的机制。还进行了基于负载转染microRNA质粒的石墨烯药物递送系统、自然提取物虫草素、花椒毒素、双氢青蒿素、蒜氨酸等对破骨细胞及前体细胞作用效应的相关研究。已发表SCI10篇，最高影响因子15.84，参加国内外会议报告10次。通过本课题的研究，毕业博士2名，硕士3名，在读博士研究生2名，硕士研究生3名。按国家自然基金财务规定执行，经费严格按预算使用。