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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。
Objective To study the anti-tumor mechanism of soluble PD-1 (sPD-1) eukaryotic expression plasmid pPD-1A constructed in our laboratory. Methods PD-1 ligand (PD-L) secreted by BHK cells transfected with pPD-1A was blocked by dendritic cells. The proliferation of dendritic cells (DCs) was detected by MTT assay Activation. BALB / c mice were vaccinated with H22 hepatoma cells the next day after receiving plasmid pPD-1A injection. The expression of cytokines and costimulatory molecules in mouse spleen cells was analyzed by RT-PCR. The number of T lymphocytes in the tumor was determined by flow cytometry. Results The effect of sPD-1 on the activation of lymphocytes by IL-10-DC was promoted in the early stage of lymphocyte activation, but the effect was not significant (P> 0.05). Mice injected with plasmid pPD-1A produced a strong anti-tumor immune response, H22 hepatocellular carcinoma cells were significantly inhibited (P <0.01). The expression of 4-1BB, B7.1, IFN-γ and TNF-α in splenic lymphocytes were up-regulated, the most obvious increase was IFN-γ and the expressions of OX40 and IL-10 were down-regulated. The number of tumor TIL increased significantly (75.86%). Conclusion Blocking PD-L by sPD-1 not only increases the number of CD3 + T cells in the tumor, but also regulates the expression of cytokines and costimulatory molecules to promote anti-tumor immunity.