Wang Wen-Wen, Huang Ren-Wei, Hu Yuan, Li Xu-Dong, Wang Dong-Ning, He Yi, Liu Jia-Jun
Department of Hematology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, 510630, P.R. China.
Ai Zheng. 2009 Jun;28(6):602-6.
Specific immunological effect mediated by T lymphocytes plays an important role in treating chronic myelocytic leukemia (CML). Dendritic cells (DCs)-based immunotherapy has become popular in treating tumors. This study was to construct DC vaccines by transducing with replication-defective recombinant adenoviruses expressing bcr/abl fusion gene of CML, observe the lethal effects of specific cytotoxic T lymphocytes (CTLs) triggered by genetically modified DC vaccines expressing bcr/abl fusion gene against K562 cells in vitro.
DNA fragment of bcr/abl fusion gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) to construct a recombinant adenovirus vector and produce recombinant adenoviruses. DCs were induced from peripheral blood monocytes in vitro, and transfected with recombinant adenoviruses or pulsed with peptide to induce specific CTLs. The lethal effect of CTLs against leukemic K562 cells in vitro was observed.
We successfully constructed the replication-defective recombinant adenoviral vector expressing bcr/abl fusion gene. The recombinant adenoviruses we produced had a high virus titer of 2.0 x 10(10) pfu/mL. Transfection efficiency of DCs in vitro was 50%-60%. DC vaccines expressing bcr/abl fusion gene were successfully prepared and used to induce specific CTLs. With effector:target cell ratios of 40:1 and 20:1, the killing rates of K562 cells by CTLs were (47.6+/-4.7)% and (47.5+/-1.6)% in genetically modified DCs group, (25.8+/-4.4)% and (24.6+/-6.3)% in peptide-pulsed DCs group, and were (5.7+/-1.3)% and (4.5+/-1.6)% in control DCs group. The differences between every two groups were significant (all P<0.05).
Genetically modified DC vaccine expressing bcr/abl fusion gene has a stronger contribution than peptide-pulsed DCs in triggering specific CTLs against K562 cells.
T淋巴细胞介导的特异性免疫效应在慢性髓细胞白血病（CML）治疗中发挥重要作用。基于树突状细胞（DCs）的免疫疗法在肿瘤治疗中已受到广泛关注。本研究旨在构建携带CML的bcr/abl融合基因的复制缺陷型重组腺病毒转导的DC疫苗，观察表达bcr/abl融合基因的基因修饰DC疫苗在体外引发的特异性细胞毒性T淋巴细胞（CTLs）对K562细胞的杀伤作用。
采用逆转录-聚合酶链反应（RT-PCR）扩增bcr/abl融合基因的DNA片段，构建重组腺病毒载体并制备重组腺病毒。体外诱导外周血单核细胞为DCs，用重组腺病毒转染或用肽脉冲诱导特异性CTLs。观察CTLs在体外对白血病K562细胞的杀伤作用。
成功构建了携带bcr/abl融合基因的复制缺陷型重组腺病毒载体，所制备的重组腺病毒病毒滴度高达2.0×10(10) pfu/mL。DCs体外转染效率为50%-60%。成功制备了表达bcr/abl融合基因的DC疫苗并用于诱导特异性CTLs。在效应细胞与靶细胞比例为40:1和20:1时，基因修饰DCs组中CTLs对K562细胞的杀伤率分别为(47.6±4.7)%和(47.5±1.6)%，肽脉冲DCs组分别为(25.8±4.4)%和(24.6±6.3)%，对照DCs组分别为(5.7±1.3)%和(4.5±1.6)%。两两比较差异均有统计学意义（均P<0.05）。
表达bcr/abl融合基因的基因修饰DC疫苗在引发针对K562细胞的特异性CTLs方面比肽脉冲DCs具有更强的作用。
