靶向抑制flot-2对破骨细胞生成和骨破坏的影响及分子免疫机制研究

Flot-2 elevates in multiple tumors which can destroy bone including breast cancer et al., while the effects of flot-2 on osteoclasts and bone destruction are still lacking. In the recent study, we observed that flot-2 expression increased in pre-osteoclastic cells stimulated by RANKL, LPS, culture supernatant of tumor cells during osteoclastogenesis, and flot-2 siRNA transfection which reduced flot-2 expression could inhibit the osteoclast formation obviously. Then in this project, we further value the effects of flot-2 siRNA on osteoclast formation and bone absorption activated with several different stimulators by inhibiting flot-2 gene expression in vitro. In order to elucidate the mechanisms of flot-2 siRNA on osteoclastogenesis, osteoclast cytoskeleton and the relative fusion protein, cell apoptosis were firstly analyzed; in addition, signal pathways such as SLP76-PLCγ-NFATc1, AKT-NF-kB et al., upstream signals and signal protein complex were also studied after flot-2 siRNA treatment in pre-osteoclastic cells in vitro. Finally, the effects of flot-2 inhibition on osteoclast related bone destruction in mice in vivo were evaluated by flot-2 siRNA injection or by using flot-2 deficiency mice. This project will find the relationship between flot-2 and osteoclast, bone destruction，therapy which targeting flot-2 might become new strategy to treat bone destruction especially induced by tumor cells, flot-2 might become a new target for valuing the effects of drugs to treat bone lysis diseases.

flot-2在乳腺癌等多个骨侵蚀肿瘤表达升高，但其对破骨细胞影响未见报道。我们发现，flot-2在RANKL、LPS、肿瘤上清激活的破骨前体细胞中显著升高，flot-2siRNA可直接抑制破骨细胞生成。本课题进一步研究flot-2siRNA对不同刺激诱导的破骨细胞生成、骨吸收影响；并探讨其是否通过影响破骨细胞骨架及相关融合、凋亡发挥作用，以及其对SLP76-PLCγ-NFATc1、AKT-NF-kB等信号通路的作用，对破骨细胞分化过程上游信号蛋白及其复合体的影响，分析其抑制破骨细胞生成的分子机制；并利用siRNA体内抑制flot-2表达或flot-2基因敲除鼠，整体评价其降低或缺失对不同破骨细胞活化造成小鼠骨破坏的影响。本课题将明确flot-2与破骨细胞和相关骨破坏的关联，探讨靶向flot-2是否可能成为治疗骨破坏、尤其是肿瘤细胞介导骨破坏的新策略，为抗骨破坏药物研发提供新的潜在作用靶点。

Flot2是一个重要的脂筏蛋白, 在多种肿瘤表达升高。我们前期研究发现，它在多种破骨细胞生成及骨质疏松疾病中表达显著上调，项目进一步探讨Flot2与破骨细胞分化的关联，探讨靶向抑制Flot2对破骨细胞生成和相关骨破坏疾病的影响。结果表明：（一）Flot2可正向调控破骨细胞生成，靶向抑制Flot2能够在体内外抑制人和小鼠破骨细胞生成和病理性骨质丢失。1. 发现靶向抑制Flot2后，体外能降低小鼠破骨前体巨噬细胞的破骨细胞生成和骨吸收活性：利用整体全敲、体外Flot2-siRNA、Flot2-siADV靶向抑制小鼠破骨前体细胞Flot2表达后，破骨前体巨噬细胞RAW264.7 和BMM细胞RANKL诱导的破骨细胞生成和骨吸收功能显著下降。 2. Flot2基因敲除小鼠整体病理性骨破坏减轻：Flot2全敲后小鼠生长发育等正常，且对健康小鼠的骨质无显著影响；但能显著抑制卵巢切除、LPS、肿瘤细胞诱导的体内病理性骨破坏。3. 小鼠胫骨注射Flot2-siAAV，体内能够显著降低小鼠乳腺癌4T1细胞诱导的骨丢失。4. homoFlot-2-siRNA靶向抑制人Flot-2表达后，能显著降低骨质疏松病人及乳腺癌骨转移病人破骨前体CD14+巨噬细胞破骨细胞生成及骨破坏。 （二）研究 了阐明了Flot2影响破骨细胞分化的信号分子机制。1. 研究发现Flot2缺失可显著抑制破骨前体巨噬细胞Ca2+、NF-κB-ROS-MAPK信号通路，抑制Plcγ2而非c-src磷酸化、抑制NFATc1活化入核，降低破骨细胞骨架和融合蛋白表达，抑制破骨细胞特异基因的表达。2. 进一步机制研究发现RANKL在破骨前体巨噬细胞可诱导Flot2-Plcγ2复合物生成，Flot-2敲除后可抑制该复合物的形成，这可能影响脂笩内信号传递，从而影响Plcγ2磷酸化，最终抑制关键核转录因子NFATc1，从而抑制破骨细胞分化和功能。 （三）项目申报Flot2抑制剂抗破骨细胞分化授权专利1项，并已获得授权；已发表相关SCI论文6篇，中文论文1篇。研究发现了新的破骨细胞分化和功能相关分子Flot2，发现了骨破坏疾病病理性靶点Flot2和特异性siRNA抑制剂，为破骨细胞相关骨破坏疾病如骨质疏松、骨转移肿瘤骨破坏治疗提供了新思路和策略。