在"颗粒病"的发病机制和防治中泛素蛋白酶体系统通过调控NFκB信号通路所发挥的作用

Implants of mental and alloy have been widely used, but due to stress shielding and wear particles, it have been reported that implants causing aseptic inflammation, bone resorption, implant loss and other problems in long-term clinical studies. The term ‘particle disease’ was coined by Dr William Harris to stress the importance of particles generated by prosthesis for induction of host response. In order to deal with these problems, scientists invented a variety of alternative and improved materials, but the problem of‘particle disease’is always existing. For example, historically, ultrahigh molecular weight polythylene (UHMWPE) was the most commonly used implant materials, and recently, it was replaced by more wear-resistant highly cross-linked polyethylene with only negligible wear rates in comparison with conventional UHMWPE. However, the total number of polymer particles can achieve an order of hundreds of trillions, even when the loss of material is in the order of tenths of millimeters... On the one hand, nuclear factor-kappaB ligand (NF-kB) path-way plays a vital way in the process of induce ‘particle disease’. After stimulation by wear partiles, NF-kB signaling path-way was actived in cells of the monocyte/macrophage lineage, polymorphonuclear leukocytes, fibroblasts, osteoblasts, and other cells, who increase the transcription of proinflammatory substances, including tumor necrosis factor-α(TNFα), IL-1, IL-6, IL-8, receptor activator of nuclear factor-kappaB ligand (RANKL), macrophage chemotactic protein-1, and others. All of these resulted in causing the ‘particle disease’. On the other hand, ubiquitin E3 ligase, beta-TRCP and the de-ubiquitin enzyme, cylindromatosis (CYLD), are the positive and negative regulator of NF-kB, respectively. IkBa is the inhibitor of NF-kB, and bet-TRCP induces the degradation of IkBa. What’s more, it is discovered that all the IkBs were degraded by the trigger of beta-TRCP. And CYLD by de-ubiquitin TRAF2 or TRAF6, inhibited the activation of NF-kB path-way under the stimulation of TNFαand other inflammation mediators... Our pre-experiment results showed that beta-TRCP, CYLD and proteasome inhibitor all played important role in the osteoclastgenesis. And in this project, with biochemical, cell and animal experiment, we will reveal the effect of Ubiquitin Ptroteasome System and de-ubiquitin enzyme CYLD on the pathological mechanisms, prevention and treatment of ‘particle disease’. Meanwhile, we will investigate the effect of proteasome inhibitor on the treatment of ‘particle disease’ from multiple perspectives. These vital results will offer the basic evidences for the clinical application of proteasome inhibitor.

金属植入物已在骨科等领域得到广泛应用，但是由于应力集中，磨屑产生等造成骨组织的无菌性炎症反应，骨吸收，种植体脱落等问题。科研人员发明了各种替代和改良的材料，但是始终难以解决磨屑引起“颗粒病”的问题。磨屑通过激活巨噬细胞的NF-kB信号通路，造成纤维蛋白沉积、无菌性炎症和骨组织吸收。泛素连接酶beta-TRCP和去泛素化酶cylindromatosis(CYLD)分别是 NF-kB通路的激活因子和负向调节因子。CYLD是NF-kB通路的负向调节因子，通过去泛素化肿瘤坏死因子受体相关因子2、6（TRAF2，TRAF6）等, 阻断TNFα等对NF-kB的激活。本课题从分子水平、细胞水平和动物体水平，揭示泛素蛋白酶体系统和去泛素化酶CYLD在“颗粒病”致病机理和修复中的重要作用。同时通过多角度的研究为蛋白酶体抑制剂治疗“颗粒病”的科研成果转化提供理论依据，具有重要的学术意义和临床实用价值。

体内植入物已在骨科等领域得到广泛应用，但磨屑产生等造成无菌性炎症反应、骨吸收，是种植体脱落等最常见的原因。本项从分子、细胞、动物水平，从巨噬细胞、成骨细胞、牙周膜细胞三种组织来源系统的研究了泛素蛋白酶体系统通过NFƙB通路在不同材料、不同粒径颗粒的“颗粒病”中的调控作用，揭示蛋白酶体抑制剂在“颗粒病”防治中的作用。根据项目申请书和任务书的目标设定发现如下：巨噬细胞对颗粒有吞噬作用和BTZ的抑制作用。PCR、流式细胞仪、WB检测，在“颗粒病”的发病过程中不同颗粒引起TNF-ɑ， IL-1b，β-TrCP，CYLD，IL-6等NFKB通路中关键蛋白的变化。钛纳米颗粒、微米颗粒，碳纳米管多壁、单臂炎症反应较大，Al2O3纳米、微米颗粒较弱。Al-n可降低Ti-u材料炎症反应，β-TrCP，L-6的表达随着Ti-u颗粒浓度增加而增加，随着Al-n 增加降低；CYLD刚好相反。基因沉默β-TrCP,可抑制NFkB信号通路的活性及巨噬细胞向破骨细胞分化。不同颗粒（特别是碳纳米管多壁材料）可引起人牙周干细胞（hPDLsc）的干性改变，如细胞克隆形成受抑制。颗粒刺激可明显引起hPDLsc炎症因子的表达。蛋白酶体抑制剂Bortezomib (BTZ)可降低LPS引起的hPDLsc炎症因子TNF-a, IL-1b, IL-6的表达。BTZ可抑制破骨细胞的分化。基因沉默了成骨细胞MG-63中的β-TRCP。WB显示β-TrCP,IL-6的表达随着Ti-u颗粒浓度的增加而增加，随着Al-n 增加β-TrCP表达降低。1nM以下的BTZ对人牙周膜细胞无明显毒性，同时具有炎症抑制作用，抑制“颗粒病”的细胞凋亡等。“颗粒病”动物实验，Micro CT、组织染色、免疫组化结果显示Ti-u,Al-n,P-s对颅骨有一定的破坏，而多壁碳纳米管最为严重。Al-n对Ti-u的骨质破坏有一定的逆转作用。同时BTZ可抑制剂材料的炎症反应。BTZ可抑制LPS和结扎引起的大鼠牙周组织炎症因子表达和牙槽骨吸收。延伸课题： TiO2颗粒对PEEK生物活性的改善、氧化物的探针研究在“颗粒病”的发病过程中的检测应用。以第一标注方式发表口腔领域1区杂志SCI论文1篇、中文文章2篇，国内国际会议交流多次（另外有数篇SCI文章正在发表中），培养博士毕业1名，硕士在研3名，参与申请发明专利1项。