Cancer treatment by non-myeloablative hematopoietic stem cell transplant with Flt3L gene transfection.

[背景]树突状细胞（dendriticcells，DC）是一种专职的抗原提呈细胞，能有效激活抗原特异性T细胞。已经研究了将抗原负载在树突状细胞上以有效诱导CTL的各种策略。最近的报道表明，使用电穿孔用抗原编码或肿瘤衍生的RNA转染的树突状细胞可以诱导强烈的免疫应答。然而，电穿孔RNA转染的最佳条件仍然存在争议。[Aim]我们研究了树突状细胞的RNA电穿孔的各种条件，以确定表达的最佳条件。【材料与方法】以pTNT/EGFP和pGEM 4 Z/EGFP/A64体外转录的EGFP RNA和pTNT/lacZ和pGEM 4 Z/lacZ/A64体外转录的lacZ RNA为研究对象。将体外转录的RNA转染C57 BL/6小鼠骨髓来源的树突状细胞中。将200μl Opti-MEM（Invitrogen）中的5x 10 e6个细胞在0.2-cm间隙的细胞池中用RNA电穿孔。 ...更多信息 tte。在200- 1000 V、150-3000μs、1- 5次脉冲和0-50μg RNA量范围内分别考察了电压、脉冲长度、脉冲数和RNA量的多个条件。还检测了OK-432或LPS对转染的树突状细胞中EGFP表达的影响。【结果】流式细胞术和X-gal染色结果显示，体外转染EGFP和lacZ RNA的树突状细胞表达EGFP和13 -半乳糖苷酶。加帽的RNA比加β珠蛋白前导序列的RNA或未加帽的RNA更能表达EGFP。在300 V、500μs、1次脉冲条件下，电穿孔效率最高，细胞损伤较小。在用25μ g加帽的EGFP RNA转染的DC中，EGFP表达达到平台。电穿孔后12小时可检测到EGFP的表达，24小时达到高峰。LPS和OK-432促进EGFP表达。【结论】电穿孔法可以有效地转染和表达目的RNA。只有在经过严格审查的具体条件下。少

[Background] Dendritic cells are professional antigen presenting cells that can efficiently activate antigen specific T cells. Various strategies have been investigated to load antigen on dendritic cell for efficient CTL induction. Recent reports have shown that strong immune responses can be induced by dendritic cells transfected with antigen-coding or tumor-derived RNA using electroporation. However, the optimal condition of RNA-transfection with electroporation is still controversial. [Aim] We examined various conditions of RNA electroporation for dendritic cells to determine the optimal conditions for expression. [Materials and Methods] We used EGFP RNA transcribed in vitro from pTNT/EGFP and pGEM4Z/EGFP/A64, and lacZ RNA transcribed in vitro from pTNT/lacZ and pGEM4Z/lacZ/A64. In vitro transcribed RNA was transfected into day-7 bone marrow-derived dendritic cells of C57BL/6 mice with an. 5x10e6 cells in 200μl Opti-MEM (Invitrogen) were electroporated with RNA in 0.2-cm gapped cuve … More tte. Multiple conditions of voltage, pulse length, number of pulse and RNA amount were examined ranging between 200-lOOOV, 150-3000μs, 1-5pulses and 0-50μg, respectively. Effect of 0K-432 or LPS on EGFP expression in transfected dendritic cell was also examined.[Results] Flow cytometry and X-gal staining showed the expression of EGFP and 13 -galactosidase in dendritic cells electroporated with EGFP and lacZ RNA transcribed in vitro, respectively. Capped RNA better expressed EGFP than by RNA added β globin leader sequence or by uncapped RNA. The best efficiency with minor cell damage of electroporation was obtained with a condition at 300V, 500μs and one pulse. EGFP expression reached a plateau in DC transfected with 25μg-capped EGFP RNA. EGFP expression was detected in 12hr after electroporation, and was at its peak at 24hr after electroporation. LPS and OK-432 argued EGFP expression. [Conclusion] We demonstrated that RNA of interest could be efficiently transfected and expressed with electroporation. only with well-examined specific conditions. Less