人工合成的siRNA转染外周血活化T淋巴细胞方法学研究

观察人工合成siRNA与脂质体DMRIE-C Reagent的复合物对人外周血活化T淋巴细胞转染效果。人外周血标本中分离的外周血单个核细胞用抗原和IL-2刺激扩增培养，产生活化的T淋巴细胞，用不同配比浓度的荧光标记的siRNA—FAM与脂质体的复合物转染活化后的T淋巴细胞，分别采用转染一次和连续转染两次的方法，并在转染后4h，分别取样本，通过流式细胞测定技术，比较两种方法的转染效率，并筛选出最佳转染效率剂量配比。活化T细胞用转染一次和两次的方法后，平均转染阳性率分别为（12．5±0．64）％和（50．37±6．77）％。采用连续两次转染的方法进行转染，转染效率要明显高于一次转染法。

The methodology for transfection of human peripheral blood activated T lymphocytes with synthetic siRNA.

The effect of siRNA/DMERI-C Reagent complex on transfection of human activated T lymphocytes from peripherial blood was investigated. The peripheral blood mononuclear cells （PBMC） was isolated from blood by Ficoll-hypaque method, and treated with IL-2 and antigen to produce activated T cells. Different finial concentration of siRNA-FAM was transfectated into activated T cells by DMERI-C Reagent for one or two times . 4h later, the sample was analgzed by FACS Calibur. The result showed the percentages of siRNA-FAM in activated T cells were （ 12. 5 ± 0. 64） % and （50. 37 ± 6. 77 % at average （ n = 4） for transfecting once and twice respectively. Use the method transfection by two times can induce the siRNA into activated T cells more successful than that by one times.