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
[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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