腺病毒介导的T-bet基因转染诱导Th1型淋巴细胞分化

Adenovirus-mediated T-bet gene transfers into lymphocyte to induce type-1 T-helper differentiation

目的:构建含有T细胞转录因子T-bet(T-boxexpressedinTcells)的重组腺病毒Ad.T-bet,并诱导Th1型淋巴细胞分化。方法:采用RT-PCR的方法从健康人外周血淋巴细胞的总RNA中克隆T-bet基因,亚克隆入腺病毒穿梭载体成为pAdtrack-CMV.T-bet,与腺病毒基因组载体pAdeasy-1同源重组为pAd.T-bet,经PacI酶切后转染293细胞,用Westernblotting和RT-PCR的方法鉴定Ad.T-bet;联合腺病毒和脂质体以感染倍数(m.o.i)5000感染活化的淋巴细胞,用ELISA法检测培养液中IFN-γ含量。结果:采用RT-PCR方法从健康人外周血淋巴细胞中扩增出T-bet基因,构建出重组腺病毒Ad.T-bet,Westernblotting和RT-PCR均检测出细胞内T-bet蛋白表达;联合Ad.T-bet和脂质体感染淋巴细胞诱导Th1型细胞因子IFN-γ持续高表达。结论:重组腺病毒Ad.T-bet有效诱导Th1型细胞分化,有望成为改变肿瘤患者免疫抑制状态的有效方法。

AIM: To construct recombinant adenovirus containing transcription factor T-bet (T-box expressed in T cells),and induce type-1 T-helper differentiation of lymphocytes. METHODS: T-bet gene was cloned from total RNA of lymphocyte stimulated with IFN-γ with RT-PCR methods,then subcloned into transfer vector pAdtrack-CMV in BgIII/SaII sites. The new transfer vector pAdtrack-CMV. T-bet was digested with Pme I,subsequently cotransformed into BJ5183 cells with adenoviral backbone plasmid pAdEasy-1. The resultant plasmid pAd. T-bet was linearized by Pac I and transfected into 293 cells with liposome LIPOFECTAMINE 2000 for producing Ad.T-bet. The recombined adenovirus Ad.T-bet was identified through RT-PCR and Western blotting methods. Lymphocytes purified from patients suffering from liver cancer was infected with liposome and Ad.T-bet with multiplicity of infection (m.o.i) 5000,and the concentration of IFN-γ in culture media was evaluated with ELISA methods. RESULTS: T-bet gene was successfully cloned from lymphocytes and incorporated into recombinant adenovirus Ad.T-bet. Lymphocytes infected with Ad. T-bet constantly and strongly secreted Th1 cytokine IFN-γ. CONCLUSION: Recombinant adenovirus Ad.T-bet effectively induces type-1 T-helper differentiation,which is a promising method for restoration of patients' immune reaction against cancer.