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目的:构建携带人巨细胞病毒(hCMV)UL144基因的重组腺病毒,研究UL144基因修饰的树突状细胞(dendritic cell,DC)的功能变化。方法:从hCMV-DNA阳性患者外周血提取cDNA,采用PCR技术扩增出UL144基因。采用AdEasy系统构建携带hCMVUL144基因的重组腺病毒载体pAd-UL144,转染HEK293细胞后包装产生重组腺病毒Ad-UL144。通过RT-PCR检测目的基因的表达。将此重组腺病毒转染小鼠骨髓来源的DC,采用流式细胞术检测DC表面分子,采用ELISA技术分析DC培养上清中的细胞因子,采用3H-TdR掺入法检测UL144基因修饰的DC激活T细胞增殖的能力。结果:成功将UL144基因片段克隆至载体上,并经HEK293细胞包装出病毒颗粒,经测定病毒滴度为3×1010pfu/ml。流式细胞术检测显示,UL144基因修饰的DC表面分子CD80、CD86和I-Ad表达水平低于对照组(P<0.01)。ELISA分析表明,UL144基因修饰的DC分泌TNF-α、IL-6和IL-1β的能力减弱(P<0.05)。UL144基因修饰的DC介导的T细胞增殖功能明显低于DC对照组(P<0.01)。结论:UL144基因修饰的DC具有维持相对未成熟状态的能力,并减弱了对抗原特异性T效应细胞的刺激功能。
Objective: To construct the recombinant adenovirus harboring human cytomegalovirus (hCMV) UL144 gene and to study the function of dendritic cell (DC) modified by UL144 gene. Methods: cDNA was extracted from the peripheral blood of hCMV-DNA positive patients and the UL144 gene was amplified by PCR. Recombinant adenoviral vector pAd-UL144 carrying hCMVUL144 gene was constructed by AdEasy system and transfected into HEK293 cells to produce recombinant adenovirus Ad-UL144. The expression of the target gene was detected by RT-PCR. The recombinant adenovirus was transfected into mouse bone marrow-derived DCs, DC surface molecules were detected by flow cytometry, and the cytokines in DC culture supernatants were analyzed by ELISA. The UL144 gene-modified DCs were detected by 3H-TdR incorporation The ability to activate T cell proliferation. Results: The UL144 gene fragment was successfully cloned into the vector and packaged into HEK293 cells. The virus titer was 3 × 1010pfu / ml. Flow cytometry showed that the expression of CD14, CD86 and I-Ad on DCs modified by UL144 gene was lower than that of the control group (P <0.01). ELISA analysis showed that the ability of DCs modified by UL144 gene to secrete TNF-α, IL-6 and IL-1β was weakened (P <0.05). UL144 gene modified DC mediated T cell proliferation was significantly lower than the DC control group (P <0.01). CONCLUSIONS: UL144 gene-modified DCs have the ability to maintain relatively immature state and attenuate the stimulation of antigen-specific T-effector cells.