小干扰RNA抑制大鼠不成熟树突状细胞MyD88基因的表达

Small interfering RNA-induced MyD88 gene silencing in bone marrow-derived immature dendritic cells of rats

目的观察小干扰RNA（siRNA）对大鼠骨髓来源不成熟树突状细胞（iDC）MyD88基因表达的抑制作用。方法采用粒细胞一巨噬细胞集落刺激因子体外培养大鼠骨髓来源iDC；设计并合成针对MyD88基因的siRNA，阳离子脂质体作为转染介质；siRNA转染iDC48h后，分别检测iDC活力、转染效率、表面分子表达、吞噬能力、MyD88基因mRNA转录水平及蛋白表达。结果培养6d后收集iDC。采用适当浓度siRNA（1ug／500ul）和阳离子脂质体（3ug/500ul）转染48h，转染iDC活力平均（98．50±1．04）％，转染效率平均（85．60±3．50）％，转染iDC表面分子与未转染iDC比较无显著差异，转染前后iDC吞噬能力无湿著变化，MyD88基因mRNA转录水平和蛋白表达分别为内参的0．098±0．012和0．087±0．015。结论化学合成MyD88-siRNA是诱导iDCMyD88基因沉默的一种有效、可行的方法。

Objective To study the effects and efficiency of chemically synthesized small interfering RNA （siRNA） in knocking down the MyD88 gene expression of immature dendritic ceils in rats. Methods Rat bone marrow-derived immature dendritic ceils （iDCs） were generated by culturing bone marrow progenitor ceils of rats with GM-CSF in vitro. Chemically synthesized MyD88-siRNA at the concentration of 1 ug/500 ul was transfected into iDCs by lipofection. The viability of transfected iDCs was determined by Trypan blue dye exclusion assay. The efficiency of transfection and the expression of MHC II and costimulatory molecules of MyD88-silenced iDCs were analyzed by using flow cytometry. Reverse transcription-polymerase chain reaction （RT-PCR） assay was used to detect MyD88 mRNA transcription, and Western blotting to detect the expression of MyD88 protein. The function of endocytosis was evaluated by uptaking of FITC-dextran by iDCs by using flow cytometry. Results MyD88-siRNA was transfected into iDCs with high transfection efficiency. The transfection procedures affected neither the immature phenotype property nor the viability of iDCs. RT-PCR and Western blotting indicated that MyD88 mRNA transcription and the expression of MyD88 protein in MyD88-silenced iDCs were at a very low level. Analysis of endocytosis showed similar abilities to uptake FITC-dextran by various groups of iDCs. Conclusion Transfection of MyD88-siRNA by lipofectamine is a practical and effective way to induce MyD88 gene silencing in iDCs of rats.