APPLICATION OF TRISTETRAPROLIN TO GENE THERAPY FOR RHEUMATOID ARTHRITIS

背景：TNF-α在类风湿关节炎（RA）的免疫发病机制中起重要作用。TNF-α的表达在体内受到一系列调控机制的严格调控。这些包括转录、翻译和翻译后调节。Tristetraprolin（TTP）是近年来发现的一种RNA结合蛋白，可降解TNF-α信使RNA。它与TNF-α mRNA的3 '非翻译区结合，加速转录本的降解。本研究探讨类风湿关节炎患者滑膜组织中TTP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反应（RT-PCR）、免疫印迹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。使用ABI Prism 7900定量信使RNA。结果：6例胃癌组织中5例检测到TTP mRNA ...更多信息 使用RT-PCR从RA患者的滑膜组织样品中检测。使用Western印迹法在所有滑膜组织样品中检测到TTP蛋白。免疫组化结果显示TTP在RA患者滑膜组织衬里层及衬里下层微血管周围均有表达。OA患者的滑膜组织不表达TTP。制备人和小鼠TTP的表达质粒并转染COS 7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 cDNA沿着四环素应答元件导入腺病毒（人TTP腺病毒）中。当用人TTP腺病毒和四环素控制的反式激活病毒共感染COS 7细胞时，诱导了人TTP的强表达。结论：TTP在RA患者的滑膜组织中有强表达，而在OA患者的滑膜组织中无表达。制备了能诱导人TTP表达的腺病毒。人TTP腺病毒基因治疗实验性关节炎的效果有待进一步研究。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