载siRNA磁性脂质体体内靶向抑制假体周围炎性微环境内破骨细胞RANK/RANKL途径的研究

The balance between bone resorption and formation keeps the dynamic integrity of the whole bone. Such balance is determined by the interaction between osteoclast and osteoblast. In artificial arthroplasty field, polyethylene particles would induce local osteoclast activation and cause periprothetic osteolysis. This phenomenon is called "particles disease". Nowadays, the approaches for treatment and prevention of osteolysis are divided into two groups: 1, reducing the production of wear particles; 2, blocking the osteolysis process and restoring the bone anabolic balance. Various methods have been introduced to solve this problem, such as bisphosphonates. However, bisphosphonates may lead into some complications, such as over-inhibition of bone metabolism(frozen bone). Lone-time usage of bisphosphonate, especially over 5 years, is not recommended in clinical practice. . In the year of 2008, NFATc1 specific siRNA was reported to inhibit mouse osteoclast activation. SiRNA can effectively bind with special mRNA and silence special genes and take effects in an extremely small amount. More importantly, it would not completely silence osteoclast, keeping the function of osteoclast in certain low level and avoiding over-inhibition. . The immunoliposome technique is another important immune-targeted technique. It packages drugs or monoclonal antibodies with liposome. These drugs or antibodies would be released into the targeted cells and affect their functions. If magnetic material is introduced into such liposomes, the magnetic immunoliposome could be guided by the direction of magnetic field to the targeted organs, which is of great importance in clinical practice. This technique has been matured recently and it is now broadly applied. Additional, our research team successfully produced spherical hydroxyapatite particles with the diameter of 20-nm(20HA) in our previous research. Our 20HA has the advantage to induce maturation and differentiation of stromal cells and osteoblast, and regulate mineral process of the cell. This paper is regarded by China Science and Technology Information Institute to be one of the 100 best study papers in China. In our program, our research aims to mix iron oxide, pore-forming agent and 20HA together to form a new material. This material could be sprayed over the prosthesis and forming a magnetic 20HA coating. Such magnetic coating would attract the magnetic liposome aggregated around the prosthesis. This could effectively reduce the systemic side effects of the liposome. . The RANK/RANKL signal approach is the main known mechanism to activate osteoclast. Our study aims to mix anti-RANK antibody and liposomes together, and uses siRNA loaded magnetic liposome technique to make targeted inhibition on periprosthetic osteoclast. We consider, this would significantly inhibit local osteoclast activity and reduce the periprosthetic osteolysis.

正常的骨代谢有赖于骨吸收和骨形成之间的相互平衡。在人工关节领域，由聚乙烯磨损微粒为首介导的生物学反应可以在局部大量激活以破骨细胞为主的巨噬细胞系统，从而在人工关节部件周围造成大量的骨溶解，这也是假体远期失败的最主要的原因，也称之为"微粒病"或又称为"假体周围炎性微环境相关性骨溶解"。RANK/RANKL途径是目前已知引起破骨细胞激活的主要信号途径。本研究拟将RANK单克隆抗体与脂质体结合形成免疫脂质体，并使用载siRNA磁性脂质体技术靶向抑制假体周围破骨细胞激活的RANK/RANKL途径来治疗假体周围骨溶解疾病。本方法存在以下优点：第一， siRNA不破坏目标细胞的DNA结构，没有潜在的致突变性等风险。第二，靶向定位可以使siRNA抑制破骨细胞更有针对性，减少全身反应。第三,siRNA的研制技术目前已经基本成熟。本研究具有良好的研究基础，足以完成该项研究。

正常的骨代谢有赖于骨吸收和骨形成之间的相互平衡。在人工关节领域，由聚乙烯磨损微粒为首介导的生物学反应可以在局部大量激活以破骨细胞为主的巨噬细胞系统，从而在人工关节部件周围造成大量的骨溶解，这也是假体远期失败的最主要的原因，也称之为“微粒病”或又称为“假体周围炎性微环境相关性骨溶解”。研究结果显示微粒病中导致骨溶解的具体机制非常复杂，RANK/RANKL 途径是目前已知引起破骨细胞激活的主要信号途径。目前的免疫脂质体已具有制备工艺简便，无毒、无免疫原性及可被生物膜利用的特点，它携带、保护及释放药物的能力高于单克隆抗体,是现阶段抗体靶向治疗的研究热点，但在抗骨质疏松领域特别是抑制破骨细胞功能方面，尚未见到报导。. 在本次研究中，我们建立了成骨细胞、破骨细胞离体磨损颗粒骨溶解复合体模型的建立并进行鉴定，用siRNA抑制RANK对成骨细胞、破骨细胞离体磨损颗粒骨溶解复合体模型，观察其对细胞功能的影响并探索机制，建立兔股骨远端颗粒诱导骨溶解模型的建立，用载siRNA 脂质体靶向治疗预防股骨远端骨溶解发生。. 通过我们的研究发现，RANK siRNA可以通过明显抑制OPG/RANK/RANKL信号通路，抑制体外成骨细胞、破骨细胞离体磨损颗粒骨溶解复合体模型中破骨细胞的形成， Raw264.7细胞增值，促进Raw264.7细胞和破骨细胞凋亡以及迁移，促进细胞的碱性磷酸酶(ALP)和抑制 抗酒石酸酸性磷酸酶 (TRAP)的分泌，从而降低体外假体松动的发生。我们也通过兔关节腔注射RANK siRNA脂质体，成功的预防了假体松动的发生。. 我们的研究成过的抗骨丢失性能可以广泛的用于骨质疏松和各种骨溶解症的治疗，并且在人工关节领域可以预防和治疗微粒病的发生，解决松动失败的问题，延长假体寿命，因此具有非常重要的临床应用价值和前景，并具有巨大的社会意义和经济价值。