AAV介导的TNFR关节内基因治疗对血友病性关节病预防和治疗的实验研究

Adeno-Associated Virus Mediated Tumor Necrosis Factor Receptor for Prevention and Treatment of Hemophilic Arthropathy in Hemophilic Mice

血友病性关节病(HA)的发病机制与关节内炎性细胞因子过表达有关,肿瘤坏死因子α(TNFα)是最重要的炎性因子之一。可溶性肿瘤坏死因子受体(sTNFR)作为TNFα的受体可以特异性地与TNFα结合拮抗TNFα炎性效应。为探讨关节腔内持续表达sTNFR对HA关节的保护作用,将表达TNFR的质粒(pAAV-TNFR:Fc)包装为rAAV5-TNFR:Fc载体,注射到血友病小鼠膝关节内。结果显示炎症持续42 d后,关节内仍能表达TNFR:Fc,两个给药组中TNFα和白介素-1β(IL-1β)的表达不同程度地下降,关节病变均显著减轻,表明该关节内基因治疗对血友病性关节病有预防和治疗效果。

Hemophilia is an X chromosome-linked bleeding disorder disease caused by a lack of coagulation factor.Hemophilia patients suffer from spontaneous bleedings that may occur in different organs and tissues.Hemophilic arthropathy(HA)is the primary etiology leading to disability in hemophilia patients which is caused by the recurrent bleeding in the joints.Over expression of pro-inflammatory cytokines has been putatively recognized as one of the mechanisms.In the progression of HA,pro-inflammatory cytokines serve as signaling mediators,among which TNFαis one of the most important.As a receptor of TNFα,sTNFR(soluble tumor necrosis factor receptor)specifically binds to TNFαand antagonizes its pro-inflammtory effect.To investigate the therapeutic effect of local long-term TNFαexpression on HA,hemophilia B mice were intra-articularly injected with rAAV5-TNFR:Fc.In the present study,plasmid pAAV-TNFR:Fc was constructed and used for the package of rAAV5-TNFR:Fc.Transgene expression mediated by infection in vitro and in vivo were confirmed by western blot.Then hemophilia B mice divided into prophylaxis group and treatment group were intraarticularly injected with rAAV5-TNFR:Fc,and 6 weeks after HA was induced,joint tissues were collected for RNA extract to measure the mRNA expression level of TNFα、IL-1β、IL-6 and IL-10.The pathology changes of joints were also graded by histology and the scorings of synovitis arthritis,macrophage infiltration and neovascularization were obtained.The results suggested that,42 d after joints hemarthrosis induction,sTNFR expression in joints persisted.The pathological sequela in two groups were reduced to different degrees after rAAV5-TNFR:Fc local delivery and delivery as a prophylaxis showed better outcome than as a treatment.It was concluded that recombinant adeno-associated virus mediated soluble tumor necrosis factor receptor could be a therapeutic approach for HA treatment.