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抗RANKL单克隆抗体在大鼠类风湿性关节炎模型中的应用及RANKL转录调控的探索性研究

发布时间:2018-09-13 13:38
【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。
[Abstract]:Research background:
Rheumatoid arthritis (RA) is one of the most common autoimmune diseases in humans, characterized by chronic inflammatory osteopathy. Its basic pathological changes are synovitis and can cause primary and secondary systemic and/or local bone metabolism changes, including bone and cartilage erosion, destruction and systemic bone loss. Many anti-rheumatic drugs, such as non-steroidal anti-inflammatory drugs (NSAIDs), tetracycline antibiotics, adrenocorticosteroids, methotrexate (MTX), have been developed and studied recently, including TNF-a blockers, neutralizing antibodies against specific inflammatory factors (IL-6, 17) and other promising target molecular therapies, such as RANK-RANKL. Signal axis, Wnt signaling pathway [1]. However, exploring the pathogenesis of rheumatoid arthritis and developing new drugs based on it are still hot spots in the medical field.
The normal metabolism of bone tissue depends on the dynamic balance of osteoclast and osteoblast, which is also the basis of maintaining normal bone remodeling process [2,3].The imbalance between osteoblasts and osteoclasts, i.e. the increase of osteoclast ratio, is considered to be an important factor in the bone and joint damage in patients with rheumatoid arthritis. The balance between cells and osteoclasts is regulated by an important signal transduction pathway, the RANKL-RANK-OPG signaling pathway. OPG, as a soluble pseudoreceptor of RANKL, competently inhibits its binding to osteoclast precursor cells or mature osteoclast membrane receptor RANK. The interaction between the three constitutes a regulatory system for osteoclast differentiation and activity. The mainstream view is that the differentiation and activity of osteoclasts are affected by the ratio of RANKL/OPG, and the increased ratio of RANKL protein is the cause of bone erosion in rheumatoid arthritis. For example, almost all bone resorption regulators, such as IL-1.IL-6.IL-11.IL-17.TNF-alpha.PTH, play a role by up-regulating the expression of RANKL [4,5,6].
Around the RANKL-RANK-OPG signal transduction system, rheumatoid arthritis can be treated theoretically by the following methods:
1. overexpression of OPG or the use of exogenous OPG to block RANKL signaling:
2. Blocking the subsequent signal transduction of RANK protein in osteoclast membrane (i.e. signal transduction in osteoclasts), thereby inhibiting osteoclast differentiation;
3. inhibit RANKL levels by inhibiting other factors, such as TNF- alpha and IL-17.
4. use anti RANKL antibody to block the binding of RANK to [7,8,9]..
At present, AMG162 (denosumab) [10], developed by Amgen Company, is a fully humanized monoclonal antibody with high affinity and specificity to 10 RANKL molecules. Its pharmacokinetics is nonlinear with dosage and is similar to that of other completely human monoclonal antibodies. Bone mineral density increased significantly compared with bisphosphonate, which reduced the risk of vertebral fracture, non-vertebral fracture and pelvic fracture in postmenopausal women with osteoporosis. Denosumab has been approved by the Commission for the treatment of osteoporosis in postmenopausal women and hormone-suppressor-associated bone loss in prostate cancer patients to reduce the risk of fracture; on June 2, it was approved by the FDA. On November 18, 2010, the FDA approved denosumab for the prevention of bone-related events in patients with bone metastases from solid tumors. Approved by Norway, Iceland and Liechtenstein, it is the first and only approved drug specifically targeting RANK ligands. It has the advantage of being easy to administer (once every six months) and of being effective. The most common adverse reactions are urinary tract infections, upper respiratory tract infections, sciatica, cataracts, constipation, rashes and limbs. Body pain.
However, there are still some shortcomings in the application of monoclonal antibodies in the treatment of diseases, such as immune response, difficulty in reaching target cells, and high cost. Tibody, scFv or single chain antigen binding protein, SCA) has the following advantages: (1) small molecule, low immunogenicity, for the human body is not easy to produce anti-xenogeneic protein reaction; (2) easy to enter the microcirculation around solid tumors; (3) blood circulation and systemic clearance fast, short half-life, little renal accumulation; (4) no Fc segment, not easy to have Fc to accept; In addition, single-chain antibodies can also be constructed into a variety of bifunctional antibody molecules with toxins, precursor drug converting enzymes, radioisotopes, cytokines and other reactive molecules, and single-chain antibodies are also ideal components for the construction of bispecific antibodies.
Based on the potential value of anti-RANKL antibody in the treatment of rheumatoid arthritis, this study tried to produce and test the therapeutic effect of anti-RANKL monoclonal antibody on rheumatoid arthritis, and explored new intracellular effector molecules, hoping to find an effective new drug for the treatment of rheumatoid arthritis.
Main research contents:
This study explored the effect of anti-RANKL monoclonal antibody in the treatment of rheumatoid diseases. The basic research programs were gene cloning, hybridoma technology, affinity purification, immunofluorescence analysis, immunohistochemistry, serum and joint fluid index detection, local animal observation, gross specimen detection, specimen histology and other experimental techniques. Work as follows:
1. preparation, fermentation and purification of monoclonal antibodies against rat RANKL.
2. the effect of anti RANKL monoclonal antibody on rat rheumatoid model.
3. actively explore the gene regulation level of cells.
Important findings:
1. monoclonal antibody RANKL production, select 12C12,15C1,15H6 three monoclonal antibody.
2. Establishment of animal model: (1) adjuvant and bovine collagen emulsified at low temperature, repeated mechanical movement with glass syringe (the best number is 1000 push-pull syringes); (2) multi-point intradermal injection in the tail of rats; (3) one week after the first injection to strengthen, 13-15 days after the first injection, swelling subsided, and joint deformation stiffness.
3. Pre-test results of antibody therapy: Five Wistar rats were modeled, and three rats were treated with 12C12 monoclonal antibody (3mg, single intraperitoneal injection, PBS as solvent). Among them, swelling of hind limbs disappeared earlier in two rats, and there was no significant difference in the other one. After dissection of the animal carcasses, no other visceral damage was observed except for the changes in the structure of the limbs.
4. Animal test results: set up 5 groups, normal group, positive drug (dexamethasone) group, drug solvent (PBS) group, mouse-derived IgG group, anti-RANKL monoclonal antibody intervention group; from the morphological observation, the therapeutic effect is: positive drug group anti-RANKL monoclonal antibody intervention group, drug solvent (PBS) group, mouse-derived IgG group.
5. Gene regulation of RANKL cell expression and its role in inflammation and signaling pathways. E2fl, yap and MSTL play an important role in regulating the expression of RANKL cells.
Key data and its scientific significance:
1. By analyzing the related literature of RANKL molecule, the peptide chain was synthesized from the extramembranous fragment of the inflammatory factor binding region, and the monoclonal antibody was developed by hybridoma technique. Three specific antibodies were selected for preliminary experiment and animal experiment.
2. To explore effective modeling methods for rat arthritis animal models to ensure the highest success rate of modeling: complete Freund's adjuvant, incomplete Freund's adjuvant, bovine collagen emulsification method, injection method, injection dose.
3. Animal experiment: (1) ELISA was used to detect the half-life of antibody metabolism, induce RAW263.7 cell differentiation, and a small number of animal models were used to observe the onset and treatment time points; (2) In the formal experiment, strict control was set up. Positive drugs, drug solvents, mouse-derived IgG, anti-RANKL monoclonal antibodies were used to intervene in the model animals. Treatment, collection of general conditions, limb morphology and function, bone mineral density, serum detection of inflammatory factors, histological sections, immunofluorescence detection, collection of relevant experimental data.
4. The regulation of intracellular expression of RANKL molecule and its role in inflammation and apoptosis-related signaling pathways. The upstream molecules of e2f1, yap, MSTL and other genes play an important role in the regulation of RANKL molecule.
【学位授予单位】:中国人民解放军军医进修学院
【学位级别】:博士
【学位授予年份】:2011
【分类号】:R593.22;R-332

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