重组腺病毒Ad-PD-L1转染供体小鼠树突状细胞对受体小鼠淋巴细胞激活的影响

Effect of recombinant adenovirus Ad-PD-L1 transfection of donor mouse dendritic cells on the activation of receptor mouse lymphocytes

目的:观察重组腺病毒载体Ad-PD-L1转染供体C57BL/6(H-2b)小鼠树突状细胞(DC)对受体DBA/2(H-2d)小鼠淋巴细胞增殖和激活的影响.方法:构建腺病毒穿梭质粒pShuttle-GFP-CMV(-)-PD-L1和腺病毒骨架质粒pAdxsi-GFP-CMV-PD-L1,对重组腺病毒Ad-PD-L1进行包装、扩增和纯化.分离培养供体C57BL/6小鼠的DC,分为3组:腺病毒载体Ad-PD-L1转染组(A组),空载体转染组(B组)和对照组(C组).Western blot检测转染后各组细胞PD-L1的表达.分离受体DBA/2小鼠的淋巴细胞,用羧基荧光素二醋酸琥珀酰亚胺酯(CFSE)标记,将供体C57BL/6小鼠的DC和受体DBA/2小鼠淋巴细胞混合培养,流式细胞仪观察淋巴细胞的增殖情况.结果:酶切及测序证实含PD-L1的重组腺病毒载体Ad-PD-L1构建成功.转染供体C57BL/6小鼠的DC后其PD-L1的表达升高37%(P<0.05),将转染了PD-L1的DC与受体DBA/2小鼠的淋巴细胞进行共培养.与对照组相比,PD-L1表达升高后,明显地抑制了淋巴细胞的增殖和活化.受体DBA/2小鼠的淋巴细胞增殖降低41%(P<0.01).结论:转染供体C57BL/6小鼠DC后通过共刺激通路PD-1/PD-L1抑制了受体DBA/2小鼠淋巴细胞的增殖和激活.

AIM： To investigate the effect of recombinant adenovirus Ad-PD-L1 transfection of donor mouse C57BL/6 （H-2^b） dendritic cells on the activation of receptor mouse DBA/2（H-2^d） lymphocytes. METHODS： pShuttle-GFP-CMV（-） plasmid and adenovirus bone plasmid pAdxsi-GFP-CMV- PD-L1 were constructed. The recombinant adenovirus Ad-PD-L1 was packed, amplied and purified. Dendritic cells derived from donor mouse C57BL/6 bone marrow were isolated and cultured. The cells ware divided into 3 groups. Group A and Group B were transfected with recombinant adenovirus Ad-PD-L1-GFP and Ad-GFP respectively, and Group C served as blank control group. The expression of PD-L1 was monitored by GFP fluorescence in the infected cells and identified by Western blot. The lymphocytes of receptor mouse DBA/2 were isolated, and then labeled by carboxyfluorescein succinimidylester （CFSE）. After co-cultured with the dendritic cells in the three groups, flow cytometry was used to observe the proliferation and activation of lymphocytes from DBA/2 mice. RESULTS： Enzyme digestion and sequencing confirmed the successful construction of recombinant adenovirus Ad-PD-L1. After transfection with Ad-PD-L1, PD-L1 expression of dendritic cells was verified by Western blot. The expres- sion of PD-L1 in the dendritic cells infected with Ad-PD-L1 was increased by 37% （P 〈 0.05）. After co-cultured with the lymphocytes from DBA/2 mice, the proliferation and activation of receptor lymphocytes were suppressed remarkably, and the proliferation rate was decreased by 41% as compared with that in the control group. CONCLUSION： Transfection of donor mouse dendritic cells with recombinant adenovirus suppresses the proliferation and activation of lymphocytes from receptor mice through PD-1/ PD-L1 co-stimulatory pathway.