中文摘要：

目的：设计、合成Foxp3 siRNA序列,并体外干扰肝癌患者调节性T细胞（regulatory T cells,Treg）,评价Foxp3 siRNA干扰后对Treg表型及抑制功能的影响以及是否能上调肝癌患者抗肿瘤免疫应答的能力。方法：设计并化学合成Foxp3 siRNA,将Foxp3 siRNA通过脂质体转染的方式体外转染Treg。通过实时定量PCR以及流式细胞术分析Foxp3 siRNA对Treg中Foxp3表达的抑制效能,并通过流式细胞术评价Foxp3 siRNA干扰后对Treg表面分子CD127、CTLA-4、GITR的表达以及对CD4＋CD25-T细胞的抑制作用。通过酶联免疫斑点法（enzyme-linked im-munospot assay,Elispot）以及五聚体（pentamer）分析法评价Foxp3 siRNA干扰肝癌患者Treg后对经肿瘤特异性抗原NY-ESO-1b诱导的肿瘤特异性免疫应答的影响。结果：通过Foxp3 siRNA的转染可实现对Treg Foxp3表达的部分沉默,并且上调Treg表面CD127分子的表达,下调CTLA-4和GITR的表达。肝癌患者Treg Foxp3表达的沉默可下调Foxp3＋Treg对CD4＋CD25-T效应性细胞增殖抑制的能力。通过五聚体法及Elispot法检测发现,Foxp3 siRNA以及siRNA-control转染组NY-ESO-1157-165特异性的细胞毒T细胞（cytotoxic T lymphocyte,CTL）的数量分别为0.21%±0.17%和0.57%±0.39%（P〈0.05）,与siRNA-control进行干扰的肝癌患者组相比,Foxp3 siRNA干扰后的Treg与特异性CTL共培养后,肿瘤特异性CTL数量及分泌的干扰素（interferon-γ,IFN-γ）的数量显著上调（132±55vs27±11,P〈0.05）。结论：通过Foxp3 siRNA对于肝癌患者Treg中Foxp3表达的沉默,可在体外干扰肝癌患者Treg免疫抑制功能,继而增强肝癌患者经肿瘤抗原诱导的CTL抗肿瘤免疫应答。

英文摘要：

Objective ： To design the suitable sequence siRNA of Foxp3 to interfere the function of regu- latory T cells（Treg） , and to evaluate whether the suppression of Treg could enhance the anti-tumor im- mune response in hepatocellular carcinoma patients or not. Methods： Foxp3-specifie siRNAs by chemi- cal synthesis were delivered into regulatory T cells. The inhibition efficiencies of Foxp3-specific siRNAs were evaluated by real-time PCR and fluorescently stained for intracellular Foxp3 and analyzed using mul- tiparameter FCM. The Foxp3＋ Treg subpopulation was selectively analyzed for surface expression levels of CD127, CTLA-4 and GITR. The suppression of Treg to CD4 ＋ CD25 - T cells or anti-tumor specific CD8＋ T-cell responses induced by tumor specific antigen （NY-ESO-lb） was evaluated by CFSE, Elispot and Pentamer analysis. Results： A subpopulation of Tregs with reduced levels of Foxp3 mRNA and protein mediated by siRNA was CD127 up-regulation and CTLA4 or GITR down-regulation compared with those in Foxp3 high Tregs. Knockdown of intracellular Foxp3 in Treg reduced suppression of Foxp3 ＋ Treg to pro- liferative capacity of CD4 ＋ CD25 - T cells. IFN-γ released of NY-ESO-l-specific CD8 ＋ T cells from the group of Tregs with Foxp3 specific siRNA transfeeted was increased as compared with the group of Treg with non-specific control siRNA transfected（ 132 ± 55 vs 27 ± 11, P 〈 0.05 ）. Frequency of NY-ESO-lb- specific CD8 ＋ T cells by Pentamer analysis from the group of Tregs with Foxp3 specific siRNA transfected was increased as compared with the group of Treg with non-specific control siRNA transfected （0. 21% ± 0. 17% vs 0. 57% ±0.39% ,P 〈0. 05）. Conclusion： Knockdown of intracellular Foxp3 in Treg media- ted by siRNAs can inhibit the suppression of Foxp3＋ Treg and enhance the anti-tumor immune response in hepatocellular carcinoma patients in vitro.