结肠癌细胞总RNA电穿孔法体外转染成熟树突状细胞

目的：探讨结肠癌细胞总RNA电穿孔法转染成熟树突状细胞（mDCs）的转染效率及对树突状细胞(DCs）特征和功能的影响,为DCs核酸肿瘤疫苗的制备及其在临床应用奠定基础。方法： 用重组人粒细胞-巨噬细胞集落刺激因子（rhGM-CSF）、重组人白细胞介素4（rhIL-4）将人外周血单核细胞(PBMC)诱导成未成熟树突状细胞（imDCs），肿瘤细胞坏死因子-α（TNF-α）作用24 h刺激其成熟。实验设为imDCs组和mDCs组。Trizol法提取特异表达癌胚抗原(CEA)的结肠癌细胞SW480总RNA，电穿孔法将总RNA转染mDCs，设为RNA转染DCs组。流式细胞术检测各组细胞表面分子标志变化和转染DCs组的CEA蛋白表达情况。ELISA法检测各组细胞IL-12分泌水平。结果：rhGM-CSF、rhIL-4、TNF-α能将PBMC诱导成具有典型毛刺样突起形态学特征的DCs。imDCs组DCs表面CD1a表达呈阳性，CD80弱阳性，CD83阴性。mDCs组DCs表面CD1a、CD83、CD80表达均呈阳性。RNA转染DCs组DCs表面CD1a、CD83、CD80表达均呈阳性。总RNA转染mDCs后4 h检测到CEA蛋白表达。mDCs组、RNA转染DCs组IL-12分泌量与imDCs组比较均显著升高（P＜0.01)， 而mDCs组与RNA转染DCs组比较差异无显著性（P>0.05)。结论：结肠癌细胞总RNA可通过电穿孔法转染mDCs并表达CEA蛋白，电穿孔不改变mDCs的表面分子特征及功能。

Transfection of mature dentritic cells with whole RNA from colon adenocarcinoma cells by electroporation in vitro

Objective To investigate the protein expression in mature dendritic cells(mDCs) after transfection with total RNA from carcinoembryonic antigen(CEA)-expressing colon adenocarcinoma SW480 by electroporation and test whether electroporation can influence on the function and phenotype of DCs in order to lay a foundation for the preparation and clinical application of DCs nucleic acid tumor vaccine.Methods DCs were generated in vitro from human peripheral blood mononuclear cells(PBMC) in the presence of recombinaht human granulocyte monocyte colony stimulating factor(rhGM-CSF) and recombinant human interleukin-4 (rhIL-4),and mDCs were induced with tumor necrosis factor-α (TNF-α) after treated for 24 h.There were two groups in the experiment:mDCs group and immature DCs(imDCs) group.The total tumor RNA was extracted from SW480 by Trizol and was transfected into mDCs by electroporation.The phenotype of DCs and expression of CEA protein before and after electroporation were assessed by flow cytometry.The secretion of IL-12 in each group was investigated by ELISA.Results DCs with characteristic of morphological features were generated in vitro from PBMC by rhGM-CSF,rhIL-4 and TNF-α.After cultivated for 5 d,the cells gained the phenotype of imDCs,showed strong expression of CD1a,weak expression of CD80 and no expression of CD83.After maturation by TNF-α, the expressions of CD1α,CD80 and CD83 were positive and they were also positive in RNA transfection DCs group.The expression of CEA protein was meaured at 4 h after electroporation.The secretion levels of IL-12 in mDCs and RNA transfection DCs groups were significantly higher than that in imDCs group (P<0.01),but there was no significant difference between RNA transfection DCs group and mDCs group(P>0.05).Conclusion The total RNA from colon adenocarcinoma cells can be transfected into mDCs by electroporation and electroporation doesn't alter the phenotype and function of mDCs.