【摘要】 目的研究本室构建的可溶性PD-1(sPD-1)真核表达质粒pPD-1A的抑瘤机理。方法用转染了pPD-1A的BHK细胞分泌产物去封闭树突状细胞上的PD-1配体(PD-L),并用MTT法检测树突状细胞(DC)对小鼠脾细胞的增殖激活作用。BALB/c小鼠接种H22肝癌细胞后次日开始接受质粒pPD-1A的注射。用RT-PCR法分析小鼠脾细胞的细胞因子和共刺激分子的mRNA表达水平,用流式细胞术测定肿瘤内T淋巴细胞的数量。结果在淋巴细胞活化的早期sPD-1对IL-10-DC激活淋巴细胞的作用有一定的促进,但作用并不十分显著(P>0.05)。注射了质粒pPD-1A的小鼠产生了较强的抗瘤免疫反应,H22肝癌细胞的生长明显受到抑制(P<0.01)。脾脏淋巴细胞的4-1BB、B7.1、IFN-γ和TNF-α表达均上调,以IFN-γ的增加最明显;OX40、IL-10表达下调。肿瘤内TIL数量明显增多(75.86%)。结论sPD-1通过对PD-L的封闭,不仅增加了肿瘤内部CD3+T细胞的数量,而且可以调节细胞因子和共刺激分子的表达,从而促进抗瘤免疫。更多还原

【Abstract】 Objective To study the mechanism of a eukaryotic expression plasmid (pPD-1A) on antitumor immunity. Methods sPD-1 (soluble PD-1) expressed by pPD-1A-transfected BHK cells was used to block PD-L on DC in vitro. Activation of lymphocytes stimulated by dendritic cells was measured with MTT colorimetry. Mice inoculated with murine hepatoma(H22 cells) were treated by intramuscular injection of pPD-1A. The mRNA expression was analyzed with RT-PCR. T lymphocytes in the tumor were detected by Flow cytometry. Results After treatment with sPD-1, activation of lymphocytes stimulated by DC in vitro improved partly was at early phase of activation(P>0.05). The growth of H22 cells was significantly inhibited after pPD-1A administration(P< 0.01 ). The mRNA expression of 4-1BB, B7.1, IFN-γ and TNF-α of lymphocytes was upregulated and that of OX40 and IL-10 was downregulated. CD3+ T lymphocytes in tumor injected with pPD-1A increased significantly( 75.86% ). Conclusion Blockade of PD-L by sPD-1 results in not only the increase of T cells in the tumor, but also the regulation of both cytokines and costimulatory molecules in splenocytes, so that antitumor immunity was improved.更多还原