【作者】 吕大伟；

【导师】 王岩； 袁增强；

【作者基本信息】 中国人民解放军军医进修学院 ， 外科学， 2011， 博士

【摘要】 研究背景：类风湿性关节炎(rheumatoid arthritis,RA)是人类最常见的自身免疫病之一,主要表现为慢性炎性骨病。其基本病理改变是滑膜炎,并可引起原发和继发的全身和/或局部骨代谢变化,包括骨和软骨的侵蚀、破坏以及系统性骨丢失。尽管目前存在很多抗风湿类药物,如非甾类抗炎药(NSAIDs).四环素类抗生素、肾上腺糖皮质激素、甲氨蝶呤(MTX)等,其中新近开发研究的也为数不少,包括TNF-a阻断剂、特定炎性因子的中和抗体(IL-6、17)以及其他颇有应用前景的靶标分子治疗,例如RANK-RANKL信号轴、Wnt信号通路[1]。但是探索类风湿致病机理,并且依此研发相关新药仍是医学界的热点。骨组织的正常代谢有赖于破骨细胞(osteoclast)和成骨细胞(osteoblast)的动态平衡,这也是维持正常的骨重建过程的基础[2、3]。成骨细胞与破骨细胞的失衡,即破骨细胞比例增高被认为是类风湿性关节炎患者骨关节损害的重要因素。成骨细胞与破骨细胞之间的平衡是受一个重要的信号传导体系,即RANKL-RANK-OPG信号通路来调控的,OPG作为RANKL的可溶性假受体竞争性抑制其与破骨前体细胞或成熟破骨细胞膜受体RANK结合。三者之间相互作用构成破骨细胞的分化和活性调节系统。主流观点认为,破骨细胞的分化和活性受到RANKL/OPG的比例的影响,而RANKL蛋白的比例升高是导致类风湿性关节炎骨侵蚀的原因,例如几乎所有的骨吸收调节因子如IL-1.IL-6.IL-11.IL-17.TNF-α.PTH,都是通过使RANKL表达上调[4、5、6]发挥作用。围绕RANKL-RANK-OPG言号传导系统,理论上可以通过以下几种方法对类风湿性关节炎进行治疗：1.过量表达OPG或者应用外源性OPG阻断RANKL的信号传导：2.阻断破骨细胞膜RANK蛋白后续的信号传导(即破骨细胞内的信号传导),从而抑制破骨细胞分化；3.通过抑制其它因子(如TNF-α和IL-17),抑制RANKL的水平；4.应用抗RANKL抗体阻断其与RANK的结合[7、8、9]。目前,由Amgen公司研发的AMG162 (denosumab) [10],是完全人源化的单克隆抗体,为IgG2免疫球蛋白业型,对十RANKL分子高亲和力和高特异性；药代动力学与剂量呈非线性关系,与其他完全人源性单克隆抗体相似；治疗效果迅速、持久,在抑制破骨细胞骨质吸收上效果可逆；与二膦酸盐相比,骨密度增加明显,在绝经后妇女骨质疏松治疗中,降低了椎体骨折、非椎体骨折、以及骨盆骨折的风险,观察到的副作用与安慰剂组无差异,疗效甚至疗效优于OPG和福善美[11]。2010年5月28日,欧盟委员会已批准denosumab上市,用于绝经后妇女骨质疏松症以及前列腺癌患者激素抑制相关骨丢失的治疗,以降低患者骨折的风险；6月2日,获美国FDA批准。2010年11月18日,FDA批准denosumab用于预防实体瘤骨转移患者的骨相关事件。已在欧盟27个成员国以及挪威、冰岛、列支敦士登获得批准,是第一个和唯一获批的特异性靶向RANK配体的药物。具有：方便给药(每六个月给药一次),效果明显的优势。最常见不良反应为泌尿道感染,上呼吸道感染,坐骨神经痛,白内障,便秘,皮疹和肢体疼痛。然而,应用单克隆抗体治疗疾病这种治疗方式仍存在着一些缺点,例如容易发生免疫反应,不易到达靶细胞,价格昂贵等。为了克服这些缺陷,利用基因工程技术制备的人工抗体开始被开发并应用。其中具有代表性的单链抗体(Single chain antibody, scFv或single chain antigen binding protein, SCA)具有以下优点：①分子小,免疫原性低,用于人体不易产生抗异种蛋白反应；②容易进入实体瘤周围的微循环；③血循环和全身廓清快,半衰期短,肾脏蓄积很少；④无Fc段,不易与具有Fc受体的非靶细胞结合；⑤易于基因操作和基因工程大量生产[12、13]。此外,单链抗体还可以与毒素、前体药物转化酶、放射性同位素、细胞因子等效应分子构建成多种双功能抗体分子,并且单链抗体也是构建双特异性抗体的理想元件。基于抗RANKL抗体在类风湿性关节炎治疗中的潜在价值,本研究尝试生产并检验抗RANKL单克隆抗体对类风湿性关节炎的治疗作用,并探索新的细胞内效应分子,希望寻找到一个有效的治疗类风湿性关节炎的新药物。主要研究内容：本研究探索抗RANKL单克隆抗体治疗类风湿疾病的作用,基本研究方案为利用基因克隆、杂交瘤技术、亲和纯化、免疫荧光分析、免疫组化技术、血清和关节液指标检测、动物局部情况观察、大体标本检测、标本组织学检测等多项实验技术,完成以下工作：1.抗大鼠RANKL的单克隆抗体的制备、发酵和提纯；2.抗RANKL单克隆抗体对大鼠类风湿模型的治疗作用；3.积极进行细胞基因调控水平的探索性研究。重要研究结果：1.单克隆抗RANKL抗体的生产,选取12C12、15C1、15H6三株单抗。2.动物模型的建立：(1)佐剂加牛胶原低温乳化,采用玻璃注射器反复机械运动(最佳次数为推拉注射器1000次)；(2)大鼠尾部多点皮内注射；(3)初次注射后1周加强,初次注射后13～15天发病,一月左右肿胀消退,关节变形僵硬。3.抗体治疗预试验结果：建模5只Wistar大鼠,12C12单抗治疗3只(3mg,单次腹腔注射,PBS为溶剂),其中2只大鼠后肢肿胀消退较早,余1只无明显差异。未观察到明显的毒副作用。不同时间点采血,提取血清,使用ELISA法检测抗体代谢半衰期。实验完毕后解剖动物尸体,除四肢关节结构改变外,未见明显其他脏器损害。4.动物试验结果：设立5组,正常组、阳性药物(地塞米松)组、药物溶剂(PBS)组、小鼠源性IgG组、抗RANKL单克隆抗体干预治疗组；从形态学观察,治疗效果为：阳性药物组>抗RANKL单克隆抗体干预治疗组>药物溶剂(PBS)组>小鼠源性IgG组。5. RANKL细胞表达的基因调控,以及在炎症和调往相关信号通路中作用的探索研究,e2fl, yap, mstl的明显调控作用,原代OB细胞中结果重复性不好。关键数据及其科学意义：1.通过对RANKL分子相关文献分析,针对炎性因子结合区域——膜外片断合成多肽链,并且利用杂交瘤技术,自行研制单克隆抗体,进行体外试验,选取3株特异性较好的抗体进行预实验以及动物实验。2.就大鼠关节炎动物模型摸索有效的建模方法,以保证最高的建模成功率：其中包括：完全弗氏佐剂、不完全弗氏佐剂、牛胶原蛋白的乳化方法、注射方法、注射剂量。3.动物实验：(1)预试验中使用ELISA法检测抗体代谢半衰期,诱导RAW263.7细胞分化,少量动物建模治疗观察发病及治疗的时间点；(2)正式试验中,严格设立对照,对于建模后动物采取阳性药物、药物溶剂、小鼠源性IgG、抗RANKL单克隆抗体干预治疗,收集一般情况、四肢形态功能、骨密度、血清检测炎性因子、组织学切片、免疫荧光检测,收集相关实验数据。4. RANKL分子的细胞内表达调控,以及在炎症和凋亡相关信号通路中作用的探索研究,e2f1, yap, mstl等基因调控上游分子对于RANKL分子的作用明显。更多还原

【Abstract】 Research Backgrounds:Rheumatoid arthritis (RA) is one of the most common autoimmune diseases, characterized by chronic inflammation on bone. The main symptoms include chronic synovitis on symmetrical joints and extra-articular changes. The synovitis can induce systemic and/or local bone loss primarily and secondarily in the form of bone and cartilage erosion, destruction and systemic bone loss. There are many drugs in the RA treatment, such as non-steroidal anti-inflammatory drugs (NSAIDs), tetracycline antibiotics, adrenal corticosteroids, methotrexate (MTX), and specific agents come into sight just these years, which cover a large part of recent research. These agents include: TNF-a blocking agents, inflammatory factors specific neutralizing antibodies (IL-6,17), and other quite promising therapeutic target molecules, such as:RANK-RANKL signaling axis, Wnt signaling pathway. A lot of effort is made to explore the pathogenesis of rheumatoid and develop new agents.The metabolic balance in the bone relies on the coordinated function of osteoclasts and osteoblasts, which forms the basis for the reconstruction process in this tissue. If the balance is broke down, there would appear diverse pathological changes. In the RA the increased proportion of osteoclasts in patients with rheumatoid arthritis is considered as an important factor in bone and joint damage. Osteoblasts and osteoclasts are modulated by an important signal transduction system:RANKL-RANK-OPG signaling pathway regulation. OPG as a soluble RANKL receptor competitively inhibits its binding to RANK on the membranes of osteoclast precursor cells or mature osteoclasts. The interactions between the three molecules modulate osteoclast differentiation and activation. Most professors hold the view that osteoclast differentiation and activation modulated by the RANKL/OPG ratio and the increase of RANKL lead to pathological changes in rheumatoid arthritis in the form of bone erosion. To conclude, almost all the factors prompting bone resorption such as IL-1, IL-6, IL-11, IL-17, TNF-α. PTH, carry out their function through up-regulating RANKL expression.Based on further understanding of RANKL-RANK-OPG signaling system, the following methods for treatment of rheumatoid arthritis may be advisable theoretically:1. Overexpression of OPG or application of exogenous OPG blocks the signal transduction; 2. Block the intracellular signal transduction in osteoclast, thereby inhibiting osteoclast differentiation; 3. Decrease RANKL levels by inhibiting other factors (such as TNF-a and IL-17); 4. Use anti-RANKL antibodies which inhibit downstream signaling of RANK. At present, AMG162 developed by the Amgen Corporation has been approved by FDA in June 2nd 2010, treating osteoporosis, which is fully human monoclonal antibody in the form of the IgG2 subtype immunoglobulin, with high elective affinity. The relationship between pharmacokinetics and dose is nonlinear, which is similar to the other fully human monoclonal antibody. During clinical trial the effects is fast and lasting, while the function of inhibiting osteoclast and bone resorption is reversible. In the treatment of postmenopausal osteoporosis, compared with bisphosphonates, bone density increased significantly, reducing incidence of vertebral fractures, non-vertebral fractures and pelvic fractures, and there were no additional side effects observed compared with the placebo group. Some studies show it is more effective than OPG and Fosamax.There are still some disadvantages in the treatment choice using monoclonal antibody, such as human anti-mouse antibody response, difficulty to reach the target cells and high expense and so on. To overcome these shortcomings, the use of genetic engineering technology to produce artificial antibodies began to be accepted by most researchers. The application of single-chain antibody (Single chain antibody, scFv or single chain antigen binding protein, SCA) has many advantages:①a small molecule, low immunogenicity, difficult to induce human anti-foreign protein reaction;②easy access to micro-circulation around the solid tumor;③fast blood circulation and systemic clearance, short half-life, less renal accumulation;④no Fc segment, reaction concerning the Fc segment is abolished;⑤easy process through genetic method and mass production using gene engineering technology. In addition single-chain antibodies also could combine with toxins, pro-drug converting enzyme, radioisotope, cytokine and other effecting molecules to construct a variety of bifunctional antibody molecules; meanwhile single chain antibody is also the ideal component to construct bispecific antibodies.Based on the background knowledge, we can get a common idea that anti-RANKL antibody used in the treatment of rheumatoid arthritis may have some potential value. We try to produce monoclonal antibody against RANKL and observe its effect on rheumatoid arthritis animal models (CIA rat model), in the hope to find a new effective agent to treat rheumatoid arthritis.Contents:This study resolved on the monoclonal antibodies against RANKL. The technology involved in this study listed as follows:gene cloning, hybridoma technology, affinity purification, immunofluorescence analysis, immunohistochemistry, ELISA testing inflammatory factor in serum and synovial fluid, general observation, tissue slice and many other experimental techniques.Complete the following tasks:1. Fermentation and purification of anti-RANKL monoclonal antibodies.2. Detecting monoclonal antibody and the RANKL protein binding.3. Anti-RANKL monoclonal antibody treatment on the model of rheumatoid arthritis.4. Actively exploring intracellular RANKL expression regulation.更多还原