miR-135依赖破骨细胞外泌体干扰牙槽骨代谢稳态的通讯分子机制研究

It is known that periodontitis is associated with imbalance between bone resorption and remodeling during the process of alveolar bone metabolism destruction. Paracrine products derived from osteoclast bone resorption can regulate bone formation and osteoblast differentiation. However, the mechanism of specific bio-communication substances is still unknown. Our preliminary study first found that the exosome derived from osteoclast resorption (mOC-exo) is a pivotal communicational substance, and it could significantly regulate the JAK2/STAT3 signaling pathway of osteoblasts as well as osteoblast differentiation. We further discovered that miR-135 delivered by the exosome mOC-exo could shuttle into osteoblasts, whereas its specific communicational mechanism is unknown. Based on the above findings, this project will investigate the biological functions of mOC-exo that affects osteoblasts differentiation and consequently disrupts the absorption and formation of alveolar bone using CRISPR/Cas9 technology, dual luciferase reporter gene detection etc. Moreover，we will testify the site of interaction between miR-135 and the target gene JAK2, and focus on the molecular mechanism of miR-135 inhibiting transcriptional activity of RUNX2 and regulating RANKL and OPG cascades. The expected data may elucidate the role of mOC-exo in the process of the destruction of alveolar bone metabolism, and it may provide a potential intervention target for periodontal disease treatment, which has important scientific theoretical significance and potential application value.

已知，牙周炎致牙槽骨代谢破坏过程中存在骨吸收和重建间稳态失调现象，且破骨细胞骨吸收过程中旁分泌产物具有调控骨形成/成骨细胞分化作用，但具体生物通讯物质及机制至今未明。新近,我们率先发现破骨细胞来源外泌体（mOC-exo）可调控成骨细胞JAK2/STAT3信号传导抑制成骨细胞分化；且mOC-exo递送miR-135可穿梭进入成骨样细胞，但调控机制不详。本项目在此研究基础上拟应用CRISPR/Cas9技术、双荧光素酶报告基因检测等手段探究mOC-exo影响成骨细胞分化而扰乱牙槽骨吸收与形成间失衡的生物学功能；鉴定mOC-exo递送miR-135与成骨细胞JAK2可能存在的结合位点，重点关注miR-135沉默RUNX2转录活性，调控RANKL、OPG级联反应的分子机制，诠释mOC-exo在牙槽骨代谢稳态破坏过程中所扮演角色，有望为牙周病治疗提供可能的干预靶点，具有重要科学理论意义和潜在应用价值。

已知牙周炎的发生发展中可能存在破骨细胞-成骨细胞间信息交流的动态平衡失调，我们推测破骨细胞来源外泌体可递送miR-135、miR-5134等信息物质并通过与靶基因JAK2结合沉默其表达，阻断成骨细胞JAK2/STAT3信号通路的活化，干扰RUNX2的转录活性，影响破骨细胞-成骨细胞间的平衡状态。在体外实验中发现miR-30、miR-135等在成骨细胞中表达明显下调，而在成熟破骨细胞表达显著上调；同时也发现成熟破骨细胞外泌体可抑制成骨细胞的增殖活性，表明成熟破骨细胞外泌体可对成骨细胞增殖分化的能力产生负向调控作用。经破骨细胞外泌体测序发现：与对照组相比，miR-5134-5p在诱导组上清外泌体中表达上调；验证后发现破骨细胞外泌体干预后的成骨细胞内miR-5134表达上调；进一步发现miR-5134-mimic可抑制成骨细胞增殖分化，而miR-5134-inhibtor起相反作用，提示分化破骨细胞外泌体来源的miR-5134可抑制成骨细胞分化。在体内构建实验性小鼠牙周炎模型，发现注射破骨细胞外泌体组牙周组织形态破坏，大量炎症细胞浸润，IL-10、TNF-αmRNA表达上调，CEJ-ABC距离明显增加，bv/tv显著降低，表明破骨细胞外泌体可加速牙周炎的发生发展。.我们发现成骨细胞来源外泌体同样在成骨细胞-破骨细胞之间的动态平衡中起到重要作用，可递送lnc-MALAT1等信息物质并通过与破骨细胞内miR-124结合并沉默其表达，从而经NFATc1信号通路促进破骨细胞分化。在体外实验中发现成骨细胞外泌体抑制破骨细胞的增殖活性，且其负向调控作用呈浓度依赖性。成骨细胞外泌体与RANKL共同诱导破骨细胞后破骨细胞分化相关指标上调，同时TRAP染色示成熟破骨细胞数量比单纯 RANKL 诱导组显著增多,表明成骨细胞分泌的外泌体可促进破骨前体细胞向破骨细胞分化。对其间涉及的信号分子进行研究，双荧光素酶活性结果表明lnc-MALAT1与miR-124、miR-124与NFATc1均存在特异结合位点；进一步发现miR-124-mimic可抑制破骨分化NFATc1的表达，而miR-124-inhibitor则起相反作用，TRAP染色也显示一致结果，提示miR-124可以抑制破骨细胞分化，表明成骨细胞分泌的外泌体可促进破骨细胞分化。