APPLICATION OF TRISTETRAPROLIN TO GENE THERAPY FOR RHEUMATOID ARTHRITIS

背景：肿瘤坏死因子-α在类风湿关节炎(RA)的免疫发病机制中起核心作用。在体内，肿瘤坏死因子-α的表达受到一系列调控机制的严格调控。这些包括转录调控、翻译调控和翻译后调控。雷公藤红素(Tristetraprolin，TTP)是新近发现的一种负责降解肿瘤坏死因子-α信使核糖核酸的核糖核酸结合蛋白。它与肿瘤坏死因子-α基因的3‘非翻译区结合，加速转录本的降解。本研究探讨了TTP在RA患者滑膜组织中的表达，并制备了诱导表达TTP的腺病毒。方法：采用逆转录聚合酶链式反应(RT-PCR)、Western blotting和免疫组织化学方法检测类风湿关节炎(RA)和骨关节炎(OA)患者滑膜组织中TTP的表达。采用体外连接法制备腺病毒。信使RNA用ABI Prism 7900定量。结果：6例…中有5例检测到TTP基因RT-PCR检测RA患者滑膜组织标本。免疫印迹法检测所有滑膜组织中TTP蛋白的表达。免疫组织化学显示TTP在RA患者滑膜组织衬里层及滑膜下层微血管周围均有表达。骨性关节炎患者的滑膜组织不表达TTP。制备了人和小鼠TTP的表达载体，并将其导入COS7细胞。这些质粒成功地诱导了人或小鼠TTP在这些细胞中的表达。将人TTP基因与四环素反应元件一起导入腺病毒(人TTP腺病毒)。人TTP腺病毒与四环素控制的反式激活病毒共感染COS7细胞后，可诱导强表达人TTP。结论：TTP在RA患者滑膜组织中呈强阳性表达，而在OA患者滑膜组织中不表达。制备了可诱导表达人TTP的腺病毒。人TTP腺病毒基因治疗对实验性关节炎的疗效有待进一步研究。较少

Background : TNF-α plays a central role in the immunopathogenesis of rheumatoid arthritis (RA). Expression of TNF-α is tightly regulated by a series of regulatory mechanisms in vivo. These include transcriptional, translational and post-translational regulation. Tristetraprolin (TTP) is a recently described RNA binding protein which is responsible for degradation of TNF-α messenger RNA (mRNA). It binds to 3'-untranslated region of TNF-α mRNA and accelerates degradation of the transcript. In this study, we explored the expression of TTP in synovial tissue from patients with RA and prepared adenovirus for inducible expression of TTP. Methods : Expression of TTP in synovial tissue of patients with RA or osteoarthritis (OA) was evaluated using reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry. Adenovirus was made using in vitro ligation method. Messenger RNA was quantified using ABI Prism 7900. Results : TTP mRNA was detected in 5 out of 6 … More synovial tissue samples from patients with RA using RT-PCR. TTP protein was detected in all synovial tissue samples using Western blotting. Immunohistochemistry revealed expression of TTP in the lining layer of synovial tissue as well as around micro-vessels in the sublining region of synovial tissue from patients with RA. Synovial tissue from patients with OA did not express TTP. The expression plasmids for human and mice TTP was prepared and transfected into COS7 cells. These plasmid successfully induced expression of human or mice TTP in these cells. Human TTP cDNA along with tetracycline responsive element was introduced into adenovirus (human TTP adenovirus). When COS7 cells were co-infected with human TTP adenovirus and tetracycline-controlled transactivator virus, strong expression of human TTP was induced. Conclusion : TTP is strongly expressed in synovial tissue from patients with RA, but not from OA. Adenovirus which can induce expression of human TTP was prepared. Investigation of the effects of human TTP adenovirus gene therapy against experimental arthritides should be determined in future. Less