【摘要】：研究背景： 類風(fēng)濕性關(guān)節(jié)炎(rheumatoid arthritis,RA)是人類最常見的自身免疫病之一,主要表現(xiàn)為慢性炎性骨病。其基本病理改變是滑膜炎,并可引起原發(fā)和繼發(fā)的全身和/或局部骨代謝變化,包括骨和軟骨的侵蝕、破壞以及系統(tǒng)性骨丟失。盡管目前存在很多抗風(fēng)濕類藥物,如非甾類抗炎藥(NSAIDs).四環(huán)素類抗生素、腎上腺糖皮質(zhì)激素、甲氨蝶呤(MTX)等,其中新近開發(fā)研究的也為數(shù)不少,包括TNF-a阻斷劑、特定炎性因子的中和抗體(IL-6、17)以及其他頗有應(yīng)用前景的靶標(biāo)分子治療,例如RANK-RANKL信號軸、Wnt信號通路[1]。但是探索類風(fēng)濕致病機(jī)理,并且依此研發(fā)相關(guān)新藥仍是醫(yī)學(xué)界的熱點(diǎn)。 骨組織的正常代謝有賴于破骨細(xì)胞(osteoclast)和成骨細(xì)胞(osteoblast)的動態(tài)平衡,這也是維持正常的骨重建過程的基礎(chǔ)[2、3]。成骨細(xì)胞與破骨細(xì)胞的失衡,即破骨細(xì)胞比例增高被認(rèn)為是類風(fēng)濕性關(guān)節(jié)炎患者骨關(guān)節(jié)損害的重要因素。成骨細(xì)胞與破骨細(xì)胞之間的平衡是受一個重要的信號傳導(dǎo)體系,即RANKL-RANK-OPG信號通路來調(diào)控的,OPG作為RANKL的可溶性假受體競爭性抑制其與破骨前體細(xì)胞或成熟破骨細(xì)胞膜受體RANK結(jié)合。三者之間相互作用構(gòu)成破骨細(xì)胞的分化和活性調(diào)節(jié)系統(tǒng)。主流觀點(diǎn)認(rèn)為,破骨細(xì)胞的分化和活性受到RANKL/OPG的比例的影響,而RANKL蛋白的比例升高是導(dǎo)致類風(fēng)濕性關(guān)節(jié)炎骨侵蝕的原因,例如幾乎所有的骨吸收調(diào)節(jié)因子如IL-1.IL-6.IL-11.IL-17.TNF-α.PTH,都是通過使RANKL表達(dá)上調(diào)[4、5、6]發(fā)揮作用。 圍繞RANKL-RANK-OPG言號傳導(dǎo)系統(tǒng),理論上可以通過以下幾種方法對類風(fēng)濕性關(guān)節(jié)炎進(jìn)行治療： 1.過量表達(dá)OPG或者應(yīng)用外源性O(shè)PG阻斷RANKL的信號傳導(dǎo)： 2.阻斷破骨細(xì)胞膜RANK蛋白后續(xù)的信號傳導(dǎo)(即破骨細(xì)胞內(nèi)的信號傳導(dǎo)),從而抑制破骨細(xì)胞分化； 3.通過抑制其它因子(如TNF-α和IL-17),抑制RANKL的水平； 4.應(yīng)用抗RANKL抗體阻斷其與RANK的結(jié)合[7、8、9]。 目前,由Amgen公司研發(fā)的AMG162 (denosumab) [10],是完全人源化的單克隆抗體,為IgG2免疫球蛋白業(yè)型,對十RANKL分子高親和力和高特異性；藥代動力學(xué)與劑量呈非線性關(guān)系,與其他完全人源性單克隆抗體相似；治療效果迅速、持久,在抑制破骨細(xì)胞骨質(zhì)吸收上效果可逆；與二膦酸鹽相比,骨密度增加明顯,在絕經(jīng)后婦女骨質(zhì)疏松治療中,降低了椎體骨折、非椎體骨折、以及骨盆骨折的風(fēng)險,觀察到的副作用與安慰劑組無差異,療效甚至療效優(yōu)于OPG和福善美[11]。2010年5月28日,歐盟委員會已批準(zhǔn)denosumab上市,用于絕經(jīng)后婦女骨質(zhì)疏松癥以及前列腺癌患者激素抑制相關(guān)骨丟失的治療,以降低患者骨折的風(fēng)險；6月2日,獲美國FDA批準(zhǔn)。2010年11月18日,FDA批準(zhǔn)denosumab用于預(yù)防實(shí)體瘤骨轉(zhuǎn)移患者的骨相關(guān)事件。已在歐盟27個成員國以及挪威、冰島、列支敦士登獲得批準(zhǔn),是第一個和唯一獲批的特異性靶向RANK配體的藥物。具有：方便給藥(每六個月給藥一次),效果明顯的優(yōu)勢。最常見不良反應(yīng)為泌尿道感染,上呼吸道感染,坐骨神經(jīng)痛,白內(nèi)障,便秘,皮疹和肢體疼痛。 然而,應(yīng)用單克隆抗體治療疾病這種治療方式仍存在著一些缺點(diǎn),例如容易發(fā)生免疫反應(yīng),不易到達(dá)靶細(xì)胞,價格昂貴等。為了克服這些缺陷,利用基因工程技術(shù)制備的人工抗體開始被開發(fā)并應(yīng)用。其中具有代表性的單鏈抗體(Single chain antibody, scFv或single chain antigen binding protein, SCA)具有以下優(yōu)點(diǎn)：①分子小,免疫原性低,用于人體不易產(chǎn)生抗異種蛋白反應(yīng)；②容易進(jìn)入實(shí)體瘤周圍的微循環(huán)；③血循環(huán)和全身廓清快,半衰期短,腎臟蓄積很少；④無Fc段,不易與具有Fc受體的非靶細(xì)胞結(jié)合；⑤易于基因操作和基因工程大量生產(chǎn)[12、13]。此外,單鏈抗體還可以與毒素、前體藥物轉(zhuǎn)化酶、放射性同位素、細(xì)胞因子等效應(yīng)分子構(gòu)建成多種雙功能抗體分子,并且單鏈抗體也是構(gòu)建雙特異性抗體的理想元件。 基于抗RANKL抗體在類風(fēng)濕性關(guān)節(jié)炎治療中的潛在價值,本研究嘗試生產(chǎn)并檢驗(yàn)抗RANKL單克隆抗體對類風(fēng)濕性關(guān)節(jié)炎的治療作用,并探索新的細(xì)胞內(nèi)效應(yīng)分子,希望尋找到一個有效的治療類風(fēng)濕性關(guān)節(jié)炎的新藥物。 主要研究內(nèi)容： 本研究探索抗RANKL單克隆抗體治療類風(fēng)濕疾病的作用,基本研究方案為利用基因克隆、雜交瘤技術(shù)、親和純化、免疫熒光分析、免疫組化技術(shù)、血清和關(guān)節(jié)液指標(biāo)檢測、動物局部情況觀察、大體標(biāo)本檢測、標(biāo)本組織學(xué)檢測等多項(xiàng)實(shí)驗(yàn)技術(shù),完成以下工作： 1.抗大鼠RANKL的單克隆抗體的制備、發(fā)酵和提純； 2.抗RANKL單克隆抗體對大鼠類風(fēng)濕模型的治療作用； 3.積極進(jìn)行細(xì)胞基因調(diào)控水平的探索性研究。 重要研究結(jié)果： 1.單克隆抗RANKL抗體的生產(chǎn),選取12C12、15C1、15H6三株單抗。 2.動物模型的建立：(1)佐劑加牛膠原低溫乳化,采用玻璃注射器反復(fù)機(jī)械運(yùn)動(最佳次數(shù)為推拉注射器1000次)；(2)大鼠尾部多點(diǎn)皮內(nèi)注射；(3)初次注射后1周加強(qiáng),初次注射后13～15天發(fā)病,一月左右腫脹消退,關(guān)節(jié)變形僵硬。 3.抗體治療預(yù)試驗(yàn)結(jié)果：建模5只Wistar大鼠,12C12單抗治療3只(3mg,單次腹腔注射,PBS為溶劑),其中2只大鼠后肢腫脹消退較早,余1只無明顯差異。未觀察到明顯的毒副作用。不同時間點(diǎn)采血,提取血清,使用ELISA法檢測抗體代謝半衰期。實(shí)驗(yàn)完畢后解剖動物尸體,除四肢關(guān)節(jié)結(jié)構(gòu)改變外,未見明顯其他臟器損害。 4.動物試驗(yàn)結(jié)果：設(shè)立5組,正常組、陽性藥物(地塞米松)組、藥物溶劑(PBS)組、小鼠源性IgG組、抗RANKL單克隆抗體干預(yù)治療組；從形態(tài)學(xué)觀察,治療效果為：陽性藥物組抗RANKL單克隆抗體干預(yù)治療組藥物溶劑(PBS)組小鼠源性IgG組。 5. RANKL細(xì)胞表達(dá)的基因調(diào)控,以及在炎癥和調(diào)往相關(guān)信號通路中作用的探索研究,e2fl, yap, mstl的明顯調(diào)控作用,原代OB細(xì)胞中結(jié)果重復(fù)性不好。 關(guān)鍵數(shù)據(jù)及其科學(xué)意義： 1.通過對RANKL分子相關(guān)文獻(xiàn)分析,針對炎性因子結(jié)合區(qū)域——膜外片斷合成多肽鏈,并且利用雜交瘤技術(shù),自行研制單克隆抗體,進(jìn)行體外試驗(yàn),選取3株特異性較好的抗體進(jìn)行預(yù)實(shí)驗(yàn)以及動物實(shí)驗(yàn)。 2.就大鼠關(guān)節(jié)炎動物模型摸索有效的建模方法,以保證最高的建模成功率：其中包括：完全弗氏佐劑、不完全弗氏佐劑、牛膠原蛋白的乳化方法、注射方法、注射劑量。 3.動物實(shí)驗(yàn)：(1)預(yù)試驗(yàn)中使用ELISA法檢測抗體代謝半衰期,誘導(dǎo)RAW263.7細(xì)胞分化,少量動物建模治療觀察發(fā)病及治療的時間點(diǎn)；(2)正式試驗(yàn)中,嚴(yán)格設(shè)立對照,對于建模后動物采取陽性藥物、藥物溶劑、小鼠源性IgG、抗RANKL單克隆抗體干預(yù)治療,收集一般情況、四肢形態(tài)功能、骨密度、血清檢測炎性因子、組織學(xué)切片、免疫熒光檢測,收集相關(guān)實(shí)驗(yàn)數(shù)據(jù)。 4. RANKL分子的細(xì)胞內(nèi)表達(dá)調(diào)控,以及在炎癥和凋亡相關(guān)信號通路中作用的探索研究,e2f1, yap, mstl等基因調(diào)控上游分子對于RANKL分子的作用明顯。
[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.
【學(xué)位授予單位】：中國人民解放軍軍醫(yī)進(jìn)修學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R593.22;R-332

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