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目的 探讨腺病毒载体介导EB病毒 (EBV)潜伏期膜蛋白 2A(LMP2A)基因转染树突状细胞 (DC)对其功能的影响。方法 通过同源重组法构建带有EBV LMP2A基因的重组腺病毒Ad5 LMP2A ,用不同感染滴度 (MOI)的Ad5 LMP2A转染成熟的DC ,用流式细胞术 (FACS)检测DC的LMP 2A蛋白表达细胞百分率及用台盼蓝染色计数DC死亡百分率 ,选择最佳MOI;用最佳MOI的Ad5 LMP2A转染成熟的DC ,FACS检测转染前后DC表面分子CD1a、CD83、CD40、CD80及HLA DR的变化 ;并用3 H TdR掺入法检测转染前后DC刺激同种淋巴细胞增殖能力及荧光定量PCR检测表达IL 1 2P40mRNA等功能的改变。结果 MOI 2 0 0为Ad5 LMP2A转染DC的最佳滴度 ,此时约 80 %的DC表达LMP2A蛋白及 92 %以上细胞为活细胞。Ad5 LMP2A转染成熟DC前后对细胞表面共刺激分子及特征性表面标志无影响 ;转染后的DC仍具有较强的刺激同种异体淋巴细胞增殖能力和表达IL 1 2P40mRNA的功能。结论 腺病毒载体能高效介导EBVLMP2A基因在DC中表达 ,Ad5 LMP2A转染成熟DC对其功能无明显影响 ,便于进一步用于EBV相关肿瘤的免疫治疗
Objective To investigate the effect of dendritic cells (DCs) transfected with the Epstein - Barr virus (EBV) latent membrane protein 2A (LMP2A) gene on the function of adenovirus vector. Methods Recombinant adenovirus Ad5 LMP2A with EBV LMP2A gene was constructed by homologous recombination. Mature DCs were transfected with Ad5 LMP2A with different infection titer (MOI). Flow cytometry (FACS) was used to detect the expression of LMP 2A protein The percentage of cells expressing DC and counting the percentage of DC death by trypan blue staining were used to select the optimal MOI. Mature DCs were transfected with Ad5 LMP2A with the best MOI. The surface molecules CD1a, CD83, CD40, CD80 and HLA DR The changes of proliferation of allogeneic lymphocytes stimulated with DC before and after transfection were detected by 3 H TdR incorporation and the changes of IL 1 2P40 mRNA expression by fluorescence quantitative PCR. Results MOI 200 was the best titer of DCs transfected with Ad5 LMP2A. About 80% of the DCs expressed LMP2A protein and 92% of the cells were viable cells. The transfection of DCs with Ad5 LMP2A had no effect on the cell surface costimulatory molecules and characteristic surface markers before and after transfection of mature DCs. The transfected DC still had strong ability of stimulating the proliferation of allogeneic lymphocytes and expressing IL-12P40 mRNA. CONCLUSION: The adenoviral vector can efficiently mediate the expression of EBVLMP2A gene in DC. Ad5 LMP2A transfected with mature DC has no obvious effect on its function and is suitable for further immunotherapy of EBV-related tumors