中文摘要：

目的旨在寻求一种能完全替代目前常规体外转录合成肿瘤相关抗原（TAA）mRNA的方法。方法构建4个质粒包括pmRNA荧光素（Luc）、pmRNA IRES-Luc、pmRNA鸡卵清蛋白（OVA）和pmRNA IRES-OVA，用Luc报告系统检测不同形式的Luc mRNA转染小鼠树突状细胞（DC）后的蛋白表达水平；以OVA作为靶抗原，以小鼠黑色素瘤肺转移作为动物模型，通过体内细胞毒性T淋巴细胞（CTL）杀伤实验及荷瘤实验，比较Capped-OVA mRNA和IRES-OVA mRNA修饰DC所诱发的抗原特异性细胞免疫反应。结果将IRES序列插入mRNA转录模板编码基因cDNA序列前，不影响体外转录合成相应mRNA的产量。不同形式Luc mRNA转染DC后8h，IRES-Luc mRNA较Capped-Luc mRNA表达的酶活性高1倍，而较Uncapped-Luc mRNA高20倍；IRES-Luc mRNA和Capped-Luc mRNA转染DC96h后仍能检测到Luc活性。用Capped-OVAmRNA和IRES-OVA mRNA修饰DC免疫小鼠1周后行体内CTL杀伤实验，结果示Capped-OVA mRNA／DC免疫组4hCTL杀伤率为（284-3）％，而IRES-OVA mRNA／DC免疫组4hCTL杀伤率为（32±4）％。荷瘤实验显示，Capped-OVA mRNA／DC和IRES-OVA mRNA／DC免疫小鼠后均能保护小鼠肺部免受黑色素瘤转移结节形成。结论含IRES序列的TAA mRNA可作为一种更经济、适用的方法，完全可以取代Cap化mRNA修饰DC，并能诱导与Cap化mRNA修饰DC同样有效的抗原特异性细胞免疫反应。

英文摘要：

Objective To develop a more economic and applicable substitute for modification of dendrtic cells （DCs） by capped mRNA in induction of anti-tumor immunity . Methods Four DNA plasmids as templates of mRNA in vitro transcription were constructed： pmRNA luciferase （Luc） , pmRNA internal ribosomal entry site （IRES） -Luc, pmRNA ovalbumin （ OVA）, and pmRNA IRES-OVA. The translational efficiency of uncapped-Luc, capped-Luc, or IRES-Luc mRNA in murine DCs was detected via a Luc reporter system. Nine C57BL/6 mice were divided into 3 equal groups to be injected intraperitoneally with DCs transfected with IRES-OVA or capped-OVA mRNA and untreated DCs respectively, 1 weeks later the mice were injected with splenocytes wrapped with the target peptide or control peptide labeled by CFSE, and 4 hours later the mice were killed and suspension of splenocytes was made to test the activity of cytotoxic lymphocytes （CTLs）. Another 30 mice were divided into 3 equal groups to undergo immunization by DCs as mentioned above, 1 week later mice melanoma cells of the line MO5 stably expressing OVA were injected the caudal vein, and 3 weeks later the mice were killed and their lungs were taken out to observe the metastasis of tumor, using OVA as a target antigen. Results Insertion of IRES into upstream of gene of interest in mRNA transcriptional templates didn＇t affect the yield of mRNA in vitro transcription. The level of Luc activity expressed by IRES-Luc mRNA in the DCs was 20 times higher than that by expressed by Uncapped Luc mRNA, and one time higher than that by Capped-Luc mRNA 8 h after transfection of DC. Expression of Luc could be detected up to 96 h after the transfection with IRES-Luc and Capped-Luc mRNA. Four hours after the injection of peptides the CTL activity of the mice immunized with DCs pulsed with Capped-OVA mRNA was （28±3） %, not significantly different from that of the mice immunized with DCs pulsed with IRES-OVA mRNA [ （ 32±4 ） %, P 〉 0.05 ]. Mata static melanoma nodules could be se