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

【背景】树突状细胞是一种能够有效激活抗原特异性T细胞的专业抗原提呈细胞。为了有效诱导树突状细胞CTL，研究了多种方法来加载抗原。最近的报道表明，通过电穿孔将抗原编码或肿瘤来源的RNA转染树突状细胞可诱导强烈的免疫反应。然而，电穿孔转染rna的最佳条件仍存在争议。【目的】研究树突状细胞RNA电穿孔的不同条件，确定最佳表达条件。【材料与方法】采用pTNT/EGFP和pGEM4Z/EGFP/A64体外转录的EGFP RNA，以及pTNT/lacZ和pGEM4Z/lacZ/A64体外转录的lacZ RNA。将体外转录的RNA转染到C57BL/6小鼠骨髓源性树突状细胞中。在200μl Opti-MEM （Invitrogen）溶液中，用RNA电穿孔5 × 10e6细胞，形成0.2 cm的缺口曲线。分别在200-lOOOV、150-3000μs、1-5个脉冲和0-50μg范围内检测电压、脉冲长度、脉冲数和RNA量等多种条件。同时检测了0K-432和LPS对转染树突状细胞EGFP表达的影响。【结果】体外转录EGFP和lacZ RNA电穿孔树突状细胞，流式细胞术和X-gal染色显示EGFP和13 -半乳糖苷酶的表达。加帽RNA比添加β珠蛋白先导序列的RNA和未加帽RNA更好地表达EGFP。在300V、500μs、1脉冲条件下，电穿孔效率最高，且对细胞损伤较小。转染25μg的EGFP RNA后，DC细胞的EGFP表达达到平台期。EGFP在电穿孔后12小时检测到表达，在电穿孔后24小时达到表达高峰。LPS和OK-432抑制EGFP的表达。【结论】我们证明了目的RNA可以通过电穿孔有效地转染和表达。只有在经过充分检验的特定条件下。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