腺病毒载体介导EB病毒潜伏期膜蛋白2A基因转染树突状细胞对其功能的影响

Functional effects of dendritic cells transfected with Epstein-Barr virus latent membrane protein 2A recombinant adenovirus

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 。

Objective To study the effects of dendritic cells(DC)transfected with recombinant adenovirus encoding Epstein Barr virus(EBV)latent membrane protein 2A(LMP2A)gene,and to provide evidence for further investigation on the therapeutic vaccine against EBV associated malignancies.Methods DCs were transfected with EBV LMP2A recombinant adenovirus(Ad5 LMP2A),which was generated by homologous recombination.The expression of LMP2A protein on mature DC transfected with Ad5 LMP2A at different multiplicity of infection(MOI)was analyzed by fluorescence activated cell sorting(FACS),and the dead cells were counted by trypan bule staining.The alteration of surface markers on mature DC including CD1a,CD83,CD40,CD80,HLA DR was detected by means of FACS before and after transfection.Meanwhile,the functions including stimulating allogenetic T cells reaction and expressing IL12 P40 mRNA on transfected DC were measured by methods of 3H thymidine uptake and fluorescenct semi quantitative polymerase chain reaction(PCR),respectively.Results About 80% mature DC expressed LMP2A protein and >92% cells were viable after transfection at the MOI of 200 No. significant changes in the surface markers and the cytomorphology of mature DCs were detected during the transfection.Transfected DC still have strong potential to stimulate the proliferation of allogenetic T cells and to express IL 12 P40 mRNA.Conclusion EBV LMP2A gene,which was carried by adenovirus vectors,could be transferred into DC with high efficiency. The function of mature DC was not affected significantly by the transfection of Ad5 LMP2A.