Di Ianni M, Di Florio S, Venditti G, Falzetti F, Mannoni P, Martelli M F, Tabilio A
Department of Clinical and Experimental Medicine, Perugia University, Italy.
J Hematother Stem Cell Res. 1999 Dec;8(6):645-52. doi: 10.1089/152581699319812.
In this study, we assessed the efficiency of T lymphocyte transduction with a retroviral vector carrying the herpes simplex virus thymidine kinase (HSV-tk) and neomycin phosphotransferase (neo) genes by four different protocols: standard supernatant infection, supernatant infection plus centrifugation steps, supernatant infection on fibronectin fragment-coated wells, and cocultivation. After retrovirus-mediated gene transfer of tk-neo in PHA/IL-2-stimulated primary T lymphocytes and G418 selection, T cells retained their proliferative activity, alloresponsiveness, ability to produce and to respond to IL-2, and ganciclovir (gcv)-specific sensitivity. When the four different transduction techniques were compared, no significant differences were seen in terms of cellular viability, proliferation capacity, and immunophenotyping. tk gene expression was the same in all transduced selected populations, as indicated by gcv sensitivity. Transduction efficiency was evaluated by semiquantitative PCR. Using the standard supernatant infection method, the rate of infection was extremely low (<5%). After adding the centrifugation step or performing supernatant infection on fibronectin fragment-coated wells, PCR analysis showed a 30%-40% rate of transduced cells. After infection by cocultivation, the rate of transduced cells was 30%-40%. These results demonstrate that supernatant infection plus centrifugation, supernatant infection on fibronectin fragment-coated wells, and cocultivation methods provide equivalent rates of transduced cells. The lack of reproducibility and safety indicates that cocultivation is not suitable for clinical studies. In our view, supernatant infection plus centrifugation is easier to perform than using fibronectin fragments, and it is currently the optimal method for clinical studies when large quantities of T lymphocytes are being processed.
在本研究中，我们通过四种不同方案评估了携带单纯疱疹病毒胸苷激酶（HSV - tk）和新霉素磷酸转移酶（neo）基因的逆转录病毒载体对T淋巴细胞的转导效率：标准上清液感染、上清液感染加离心步骤、在纤连蛋白片段包被的孔中进行上清液感染以及共培养。在经PHA/IL - 2刺激的原代T淋巴细胞中进行逆转录病毒介导的tk - neo基因转移并经G418筛选后，T细胞保留了其增殖活性、同种异体反应性、产生和对IL - 2作出反应的能力以及对更昔洛韦（gcv）的特异性敏感性。当比较这四种不同的转导技术时，在细胞活力、增殖能力和免疫表型分析方面未观察到显著差异。如gcv敏感性所示，所有转导的选定群体中tk基因表达相同。通过半定量PCR评估转导效率。使用标准上清液感染方法，感染率极低（<5%）。添加离心步骤或在纤连蛋白片段包被的孔中进行上清液感染后，PCR分析显示转导细胞率为30% - 40%。通过共培养感染后，转导细胞率为30% - 40%。这些结果表明，上清液感染加离心、在纤连蛋白片段包被的孔中进行上清液感染以及共培养方法提供了相当的转导细胞率。缺乏可重复性和安全性表明共培养不适用于临床研究。在我们看来，上清液感染加离心比使用纤连蛋白片段更容易操作，并且当处理大量T淋巴细胞时，它目前是临床研究的最佳方法。
