Takahashi Masuhiro, Narita Miwako, Ayres Flavio, Satoh Naoko, Abe Takashi, Yanao Toshio, Furukawa Tatsuo, Toba Ken, Hirohashi Takeshi, Aizawa Yoshifusa
School of Health Sciences, Faculty of Medicine, Niigata University Graduate School of Medical and Dental Sciences, 2-746 Asahimachi, Niigata 951-8518, Japan.
Med Oncol. 2003;20(4):335-48. doi: 10.1385/MO:20:4:335.
The present study was designed for identifying the protein synthesis in cytoplasm of dendritic cells transfected with in vitro transcribed mRNA and cellular total RNA extracted from tumor cells. Dendritic cells were generated from cord blood-CD34+ cells by culture with GM-CSF, SCF, and TNF-alpha, or from peripheral blood adherent cells or CD14+ cells by culture with GM-CSF and IL-4. Dendritic cells were transfected with in vitro transcribed EGFP mRNA or cellular total RNA, which was isolated from EGFP expressing K562, by electroporation using a square-wave pulse. Optimal in vitro transcribed EGFP mRNA transfection efficiency (>90%) was observed in a single electroporation of 1.75 kV/cm (electric field strength) with a pulse width of 250 micros. Although the intensity of EGFP expression in dendritic cells transfected with cellular total RNA was less compared with that in dendritic cells transfected with in vitro transcribed EGFP mRNA, a definite cytoplasmic synthesis of EGFP was demonstrated in dendritic cells transfected with cellular total RNA. The visual identification of cytoplasmic expression of cellular total RNA in dendritic cells revealed that electroporation of tumor cell-derived RNA could be a useful tool to load dendritic cells with tumor antigens for establishing an efficient dendritic cell-based tumor immunotherapy.
本研究旨在鉴定用体外转录的mRNA和从肿瘤细胞中提取的细胞总RNA转染的树突状细胞胞质中的蛋白质合成情况。树突状细胞由脐血-CD34+细胞通过与GM-CSF、SCF和TNF-α培养产生，或由外周血贴壁细胞或CD14+细胞通过与GM-CSF和IL-4培养产生。树突状细胞通过使用方波脉冲电穿孔，用体外转录的EGFP mRNA或从表达EGFP的K562中分离的细胞总RNA进行转染。在1.75 kV/cm（电场强度）、脉冲宽度为250微秒的单次电穿孔中观察到最佳的体外转录EGFP mRNA转染效率（>90%）。虽然与用体外转录的EGFP mRNA转染的树突状细胞相比，用细胞总RNA转染的树突状细胞中EGFP表达强度较低，但在用细胞总RNA转染的树突状细胞中证实了EGFP的明确胞质合成。对树突状细胞中细胞总RNA胞质表达的视觉鉴定表明，肿瘤细胞来源RNA的电穿孔可能是一种有用的工具，用于将肿瘤抗原加载到树突状细胞中，以建立基于树突状细胞的高效肿瘤免疫疗法。
