“核转染”技术对树突状细胞功能的影响

The impaction of nuclefection to dendritic cell

目的:研究"核转染"方法对DC活率、细胞表面分子及细胞因子分泌水平的影响。方法:体外诱导单核细胞分化为DC后,通过"核转染"方法将p/IRES-EGFP质粒转染至细胞核内,同时设立未转染DC为对照组。应用台盼蓝染色计算转染后细胞的活率;荧光显微镜下观察转染效率;流式细胞术检测细胞表面分子CD80、CD83、CD86、HLA-DR及CD11c等的表达;ELISA法检测DC培养上清IL-12及TNFα-的分泌水平。结果:DC转染后随时间推移,细胞活率下降;荧光蛋白在转染后4小时即有表达,表达量逐渐增加,24小时转染效率为56%±12%,48小时转染效率为45%±15%;转染后细胞表面分子CD80、CD83及CD86表达随时间推移逐渐降低,CD11c及HLA-DR表达水平稳定;转染可诱导DC分泌IL-12及TNFα-水平增加。结论:"核转染"是一种有效的转染DC的方法,并可诱导DC分泌多种细胞因子。

Objective：To study the impaction of nuclefection to DC,including viability,expression of co-stimulatory molecules and the level of cytokines secretion by DC.Methods：p/IRES-EGFP was nuclefected into DC which was differentiated from PBMC in vitro.The un-nuclefected DC was used as control.typan blue exclusion assay was used to detect the viability of nuclefected DC.Flow cytometry was used to analyze the variation of CD80,CD83,CD86,HLA-DR and CD11c.The level of TNF-α and IL-12 produced by DC were measured by ELISA.Results：The viability was progressively decreased after nuclefection.EGFP could be detected in nuclefected-DC in 4 h after nulcefection,and the nuclefection efficiency was increasing gradually.The nuclefection efficiency was 56%±12% and 45±15% in 24 h and 48 h,respectively.The level of CD80,CD83 and CD86 decreased significantly in 48 h,but the level of HLA-DR and CD11c did not change.IL-12 and TNF-α produced by nuclefected DC were dramatically higher than that in control group（P0.05）.Conclusion：Nuclefection is an effective method for DC transfection,and even can induce the secretion of proinflammatory cytokines.