AAV-mediated expression of p65shRNA and bone morphogenetic protein 4 synergistically enhances chondrocyte regeneration  

腺相关病毒介导p65shRNA和骨形态发生蛋白4协同表达促进软骨细胞的再生

作　　者：Yu Yangyi Song Zhuoyue Lian Qiang Ding Kang Li Guangheng 余洋溢;宋卓悦;廉强;丁康;李广恒(深圳市肌肉骨骼组织重建与功能恢复重点实验室,深圳市人民医院(暨南大学第二临床医学院,南方科技大学第一附属医院)骨科,成人关节重建与运动医学科,广东省深圳市518020;郑州市骨科医院骨科,河南省郑州市450000;深圳市平乐骨科医院骨科,广东省深圳市518000)

机构地区：[1]Shenzhen Key Laboratory of Musculoskeletal Tissue Reconstruction and Function Restoration,Division of Adult Joint Reconstruction and Sports Medicine,Department of Orthopedic Surgery,Shenzhen People’s Hospital(The Second Clinical Medical College Jinan University,The First Affiliated Hospital,Southern University of Science and Technology),Shenzhen 518020,Guangdong Province,China [2]Department of Orthopedics,Zhengzhou Orthopedics Hospital,Zhengzhou 450000,Henan Province,China [3]Department of Orthopedics,Shenzhen Pingle Orthopedics Hospital,Shenzhen 518000,Guangdong Province,China

出　　处：《中国组织工程研究》2025年第17期3537-3547,共11页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金面上项目(81472136),项目负责人:李广恒。

摘　　要：BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II 背景:近年来,腺相关病毒(Adeno-associated virus,AAV)基因治疗已被证明是治疗骨关节炎的可靠和安全的方法。然而,鉴于骨关节炎发病机制的复杂性,单一基因操作治疗骨关节炎可能不能产生令人满意的效果。先前的研究表明,核转录因子κB可以促进骨关节炎软骨细胞中的炎症通路,而骨形态发生蛋白4可以促进软骨再生。目的:利用一种可以特异性靶向核转录因子κB的p65短发夹RNA(p65shRNA)与骨形态发生蛋白4一起治疗骨关节炎。方法:制备包含AAV-p65-shRNA和AAV-骨形态发生蛋白4的病毒颗粒,通过转染AAV-p65-shRNA或AAV-骨形态发生蛋白4进入细胞,评估其抑制软骨细胞炎症和促进软骨形成的效果,并进行体内和体外实验。实验按干预方式分为5组:PBS组、骨关节炎组、AAV-骨形态发生蛋白4组、AAV-p65shRNA组、骨形态发生蛋白4-p65shRNA 1∶1组,然后分别于术后4,12,24周采集标本。采集关节组织后进行番红O和阿利新蓝染色。通过免疫荧光染色检测关节腔内注射病毒颗粒对软骨修复的影响。进一步研究两种转染病毒颗粒的最佳比例,以提高混合细胞在体内的软骨形成潜能。结果与结论:AAV-p65shRNA和AAV-骨形态发生蛋白4联合应用对软骨再生和骨关节炎治疗有协同作用。以1∶1的比例转染AAV-p65shRNA和AAV-骨形态发生蛋白4的混合细胞产生最多的细胞外基质合成(P<0.05)。体内实验结果也显示两种病毒的组合对骨关节炎软骨的再生潜力是所有组中最高的(P<0.05)。上述结果证实,当两种病毒的比例相同时,联合疗法的效果最佳。减少p65shRNA或骨形态发生蛋白4转染细胞会导致胶原蛋白Ⅱ合成减少。p65shRNA抑制炎症和骨形态发生蛋白4促进再生对治疗骨关节炎同样重要。实验结果为同时抑制软骨炎症和促进软骨修复治疗早期骨关节炎提供了一种新策略。

关 键 词：OSTEOARTHRITIS adeno-associated virus bone morphogenetic protein 4 p65-short hairpin RNA gene therapy short hairpin RNA transforming growth factor-β1 extracellular matrix articular cartilage chondrocytes. 

分 类 号：R459.9[医药卫生—治疗学] R363[医药卫生—临床医学] R364