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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 DC 96 h 后仍能检测到 Luc 活性。用 Capped-OVA mRNA 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4 h CTL 杀伤率为(28±3)%,而 IRES-OVA mRNA/DC 免疫组4 h CTL 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,完伞可以取代 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。
The purpose is to find a way to completely replace the current conventional in vitro transcription of tumor associated antigen (TAA) mRNA. Methods Four plasmids including pmRNA IRES-Luc, pmRNA chicken ovalbumin (OVA) and pmRNA IRES-OVA were constructed. Luc reporter was used to detect different forms of Luc mRNA transfected mouse dendritic cells (DC). Using OVA as target antigen and murine melanoma lung metastasis as an animal model, the cytotoxic T lymphocyte (CTL) killing experiments and tumor-bearing experiments were performed in vivo. Capped-OVA mRNA and IRES -OVA mRNA modified DC induced antigen-specific cellular immune response. Results The insertion of IRES sequence into cDNA sequence of mRNA transcription template did not affect the yield of corresponding mRNA synthesized in vitro. At 8h after transfection with different forms of Luc mRNA, the enzyme activity of IRES-Luc mRNA was 1-fold higher than that of Capped-Luc mRNA, and 20-fold higher than that of Uncapped-Luc mRNA. The IRES-Luc mRNA and Capped-Luc mRNA were transfected into DC 96 Luc activity was still detected after h. One week after the mice were immunized with DCs modified with Capped-OVA mRNA and IRES-OVA mRNA, the cytotoxicity of CTL in mice immunized with Capped-OVA mRNA / DC for 4 h was (28 ± 3)%, while the IRES-OVA mRNA The killing rate of CTL at 4 h after immunization with DC was (32 ± 4)%. Tumor-bearing experiments showed that mice immunized with Capped-OVA mRNA / DC and IRES-OVA mRNA / DC could protect mouse lung from melanoma metastasis nodules. Conclusions TAA mRNA containing IRES sequence can be used as a more economical and suitable method. Umbrella can substitute Cap-like mRNA to modify DC and induce antigen-specific cellular immune response which is as effective as Cap-mRNA modified DC.