rAAV介导CTLA4-FasL融合基因对类风湿性关节炎的基因治疗研究
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摘要
类风湿性关节炎（Rheumatoid arthritis）是一种以关节滑膜炎为特征的慢性自身免疫性疾病，主要侵犯关节腔内的滑膜组织，引起滑膜的过度增殖，甚至侵犯软骨造成骨破坏，致畸率很高，严重影响患者的生存质量和预期寿命，一直以来都是世界范围内研究热点。
类风湿性关节炎的病理特征为关节腔内滑膜细胞的异常增殖及炎症细胞的浸润；其发病机制不明确，致病环节及致病因素十分复杂，给研究工作带来很多困难。目前研究表明，治疗方案可分为传统中药、西药、手术及生物治疗。基因治疗可采取局部基因转移方法，在病变关节局部达到理想的蛋白质浓度，降低了正常组织和器官与外源药物接触所造成的危险。
成纤维样滑膜细胞（FLSs）会显著促进类风湿性关节炎的发病。通过引进凋亡诱导物FasL抑制关节内成纤维样滑膜细胞的增殖，为RA的治疗提供了一种有效的方法。而T淋巴细胞是一种对类风湿关节炎的发病机理非常重要的炎症细胞，所以针对T淋巴细胞生物治疗也可以提供一个治疗类风湿性关节炎较有效率的方法。细胞毒T细胞相关抗原4（CTLA4）可以抑制自身反应性T细胞的活化，并使其进入无能；而FasL则可以上调Fas抗原调节的凋亡信号，从而诱导滑膜细胞凋亡，抑制滑膜炎症。CTLA4-FasL融合基因整合了CTLA4和FasL的胞外区基因，一方面通过CTLA4功能域抑制自身反应性T细胞激活；另一方面，对逃脱抑制的活化细胞，通过FasL功能域诱导其凋亡从而实现多途径抑制和清除自身反应性T细胞，成为具有治疗RA治疗潜能分子。
在本研究中，我们探讨CTLA4-FasL蛋白对RA动物模型的治疗效果，及其在细胞模型中抑制炎性成纤维样滑膜细胞增殖和炎症的作用。
我们首先构建了CTLA4-FasL融合基因，并通过表达载体表达后进行纯化并验证其生物活性。然后构建了重组病毒载体pAAV.CTLA4-FasL，对其进行病毒包装并初纯化。利用热灭活结核杆菌H37Ra菌株、矿物油免疫Lewis大鼠建立了佐剂诱导性关节炎（AIA）动物模型，并证明其具有关节炎的临床症状和典型的组织学特征。随后通过关节腔注射重组病毒rAAV.CTLA4-FasL的方法，以增强的绿色荧光蛋白(rAAV.EGFP)作为对照，将CTLA4-FasL由重组腺相关病毒载体递送到AIA大鼠关节中，通过观察并收集临床表现和病理组织学等方面数据，与对照组相比，实验组表现出明显的治疗效果，同时我们还发现通过治疗也能显著降低AIA关节中关键性炎性细胞因子的水平，而从各个方面证明重组病毒rAAV.CTLA4-FasL能够对AIA大鼠的关节炎症起到显著的缓解和治疗作用。并通过实验证明CTLA4-FasL蛋白与FLSs共孵育，可以明显诱导FLSs的凋亡，进一步从细胞模型上证明了CTLA4-FasL蛋白对RA的治疗作用。有以上结果我们得出结论：由AAV载体介导的CTLA4-FasL基因递送明显抑制RA的动物模型的关节炎症，表明通过局部关节递送CTLA4-FasL治疗RA具有潜在的应用前景。
Rheumatoid arthritis (RA) is a chronic, autoimmune disease, which is characterized by jointsynovitis. It mainly affects the joint cavity of the synovial tissue and even results in bonedestruction of cartilage. The high rate of teratogenicity affects the quality of patients’life.Rheumatoid arthritis is characterized by intra-articular pathology of the abnormal proliferation ofsynovial cells, and inflammatory cell infiltration.
Fibroblast-like synoviocytes (FLSs) contribute significantly to the pathogenesis of RA.Through introducing apoptosis inducer FasL to suppress the proliferation of arthritic FLSs mayprovide an efficient approach for treatment of RA. T lymphocytes are key inflammatory cellscontributing significantly to the pathogenesis of RA. Biological treatments targeting Tlymphocytes may provide an efficient approach for treatment of RA. CTLA4–FasL, a fusionproduct of extracellular domains of CTLA4(which can induce T cell anergy through blockingcostimulatory signal) and FasL(which may upregulate Fas-mediated apoptosis in inflammatorysynoviocytes), integrating two inhibitory elements against T cells into one molecule, might be adesirable derivative of engineered soluble FasL or CTLA4and have therapeutic potential in RA.In present study, we investigated the possible effect of CTLA4-FasL protein on suppressing theproliferation of inflammatory FLSs and inflammation in experimental model of RA. The purifiedCTLA4-FasL protein exerted a significant proliferation-inhibition activity to activated arthriticFLSs through both unbounded free and membrane-anchorage manners. Recombinantadeno-associated virus (rAAV) vectors encoding rat CTLA4–FasL fusion gene(rAAV.CTLA4–FasL) or enhanced green fluorescent protein (rAAV.EGFP) were injectedintraarticularly into both ankle joints after immunization. The ankles were monitored by measuresof clinical, histological and inflammatory cytokines’ changes. Treatment usingrAAV.CTLA4–FasL resulted in a significant suppression of AIA compared with rAAV.EGFP control, as reflected in the mainly clinical signs including articular index, ankle joint thickness andpaw swelling and typically histological characters of arthritic joints including synovial hyperplasia,inflammatory cells infiltration and cartilage degradation. Treatment with rAAV.CTLA4–FasL alsosignificantly decreased the levels of key proinflammatory cytokines in AIA joints. Moreover, localproductions of transgene mRNA and protein of CTLA4–FasL were found in injected joints.Treatment with rAAV.CTLA4-FasL significantly decreased the levels of key proinflammatorycytokines in AIA joints. Our observations indicate that CTLA4-FasL protein represents asignificantly suppressive effect on inflammatory FLSs’ proliferation and CTLA4-FasL genetransfer profoundly suppressesAIA, implicating potential application for treatment of RA by localjoint delivery of CTLA4-FasL.
类风湿性关节炎的病理特征为关节腔内滑膜细胞的异常增殖及炎症细胞的浸润；其发病机制不明确，致病环节及致病因素十分复杂，给研究工作带来很多困难。目前研究表明，治疗方案可分为传统中药、西药、手术及生物治疗。基因治疗可采取局部基因转移方法，在病变关节局部达到理想的蛋白质浓度，降低了正常组织和器官与外源药物接触所造成的危险。
成纤维样滑膜细胞（FLSs）会显著促进类风湿性关节炎的发病。通过引进凋亡诱导物FasL抑制关节内成纤维样滑膜细胞的增殖，为RA的治疗提供了一种有效的方法。而T淋巴细胞是一种对类风湿关节炎的发病机理非常重要的炎症细胞，所以针对T淋巴细胞生物治疗也可以提供一个治疗类风湿性关节炎较有效率的方法。细胞毒T细胞相关抗原4（CTLA4）可以抑制自身反应性T细胞的活化，并使其进入无能；而FasL则可以上调Fas抗原调节的凋亡信号，从而诱导滑膜细胞凋亡，抑制滑膜炎症。CTLA4-FasL融合基因整合了CTLA4和FasL的胞外区基因，一方面通过CTLA4功能域抑制自身反应性T细胞激活；另一方面，对逃脱抑制的活化细胞，通过FasL功能域诱导其凋亡从而实现多途径抑制和清除自身反应性T细胞，成为具有治疗RA治疗潜能分子。
在本研究中，我们探讨CTLA4-FasL蛋白对RA动物模型的治疗效果，及其在细胞模型中抑制炎性成纤维样滑膜细胞增殖和炎症的作用。
我们首先构建了CTLA4-FasL融合基因，并通过表达载体表达后进行纯化并验证其生物活性。然后构建了重组病毒载体pAAV.CTLA4-FasL，对其进行病毒包装并初纯化。利用热灭活结核杆菌H37Ra菌株、矿物油免疫Lewis大鼠建立了佐剂诱导性关节炎（AIA）动物模型，并证明其具有关节炎的临床症状和典型的组织学特征。随后通过关节腔注射重组病毒rAAV.CTLA4-FasL的方法，以增强的绿色荧光蛋白(rAAV.EGFP)作为对照，将CTLA4-FasL由重组腺相关病毒载体递送到AIA大鼠关节中，通过观察并收集临床表现和病理组织学等方面数据，与对照组相比，实验组表现出明显的治疗效果，同时我们还发现通过治疗也能显著降低AIA关节中关键性炎性细胞因子的水平，而从各个方面证明重组病毒rAAV.CTLA4-FasL能够对AIA大鼠的关节炎症起到显著的缓解和治疗作用。并通过实验证明CTLA4-FasL蛋白与FLSs共孵育，可以明显诱导FLSs的凋亡，进一步从细胞模型上证明了CTLA4-FasL蛋白对RA的治疗作用。有以上结果我们得出结论：由AAV载体介导的CTLA4-FasL基因递送明显抑制RA的动物模型的关节炎症，表明通过局部关节递送CTLA4-FasL治疗RA具有潜在的应用前景。
Rheumatoid arthritis (RA) is a chronic, autoimmune disease, which is characterized by jointsynovitis. It mainly affects the joint cavity of the synovial tissue and even results in bonedestruction of cartilage. The high rate of teratogenicity affects the quality of patients’life.Rheumatoid arthritis is characterized by intra-articular pathology of the abnormal proliferation ofsynovial cells, and inflammatory cell infiltration.
Fibroblast-like synoviocytes (FLSs) contribute significantly to the pathogenesis of RA.Through introducing apoptosis inducer FasL to suppress the proliferation of arthritic FLSs mayprovide an efficient approach for treatment of RA. T lymphocytes are key inflammatory cellscontributing significantly to the pathogenesis of RA. Biological treatments targeting Tlymphocytes may provide an efficient approach for treatment of RA. CTLA4–FasL, a fusionproduct of extracellular domains of CTLA4(which can induce T cell anergy through blockingcostimulatory signal) and FasL(which may upregulate Fas-mediated apoptosis in inflammatorysynoviocytes), integrating two inhibitory elements against T cells into one molecule, might be adesirable derivative of engineered soluble FasL or CTLA4and have therapeutic potential in RA.In present study, we investigated the possible effect of CTLA4-FasL protein on suppressing theproliferation of inflammatory FLSs and inflammation in experimental model of RA. The purifiedCTLA4-FasL protein exerted a significant proliferation-inhibition activity to activated arthriticFLSs through both unbounded free and membrane-anchorage manners. Recombinantadeno-associated virus (rAAV) vectors encoding rat CTLA4–FasL fusion gene(rAAV.CTLA4–FasL) or enhanced green fluorescent protein (rAAV.EGFP) were injectedintraarticularly into both ankle joints after immunization. The ankles were monitored by measuresof clinical, histological and inflammatory cytokines’ changes. Treatment usingrAAV.CTLA4–FasL resulted in a significant suppression of AIA compared with rAAV.EGFP control, as reflected in the mainly clinical signs including articular index, ankle joint thickness andpaw swelling and typically histological characters of arthritic joints including synovial hyperplasia,inflammatory cells infiltration and cartilage degradation. Treatment with rAAV.CTLA4–FasL alsosignificantly decreased the levels of key proinflammatory cytokines in AIA joints. Moreover, localproductions of transgene mRNA and protein of CTLA4–FasL were found in injected joints.Treatment with rAAV.CTLA4-FasL significantly decreased the levels of key proinflammatorycytokines in AIA joints. Our observations indicate that CTLA4-FasL protein represents asignificantly suppressive effect on inflammatory FLSs’ proliferation and CTLA4-FasL genetransfer profoundly suppressesAIA, implicating potential application for treatment of RA by localjoint delivery of CTLA4-FasL.
引文
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[2].莫选荣罗心静类风湿性关节炎发病机制的研究进展医学临床研究J Clin Res, Feb.2007,Vol24, l2
[3].Feldmann M, An dreakos E, Smith C, et al.Is NF2JB a usefμl therapeutic target in rheumatoidarthritis [J] Ann RheumDis,2002,61(suppl.2): ii132ii18.
[4].陈桂敏代林刚贺金类风湿性关节炎相关机制研究进展海南医学院学报2007.13(3)Journal of Hainan Medical College232-235
[5].张义浜,刘志敏,熊凌霜.类风湿性关节炎发病机制及其治疗方法研究进展[J].细胞与分子免疫学杂志,2005,21(suppl):s88-94
[6].王吉波,潘琳.类风湿关节炎病因及其发病机制.山东医药,2002,42(18).
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