中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2011年
12期
2105-2107
,共3页
朱春富%王仕忠%杨晓俊%徐皓%吴文溪
硃春富%王仕忠%楊曉俊%徐皓%吳文溪
주춘부%왕사충%양효준%서호%오문계
树突状细胞%MyD88%RNA干扰%Toll样受体
樹突狀細胞%MyD88%RNA榦擾%Toll樣受體
수돌상세포%MyD88%RNA간우%Toll양수체
Dendritic cells%MyD88%RNA interference%Toll-like receptor
目的 观察小于扰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-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iDC 48 h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(1 μg/500μl)和阳离子脂质体(3 μg/500μl)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吞噬能力无显著变化,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iDC MyD88基因沉默的一种有效、可行的方法.
目的 觀察小于擾RNA(siRNA)對大鼠骨髓來源不成熟樹突狀細胞(iDC) MyD88基因錶達的抑製作用.方法 採用粒細胞-巨噬細胞集落刺激因子體外培養大鼠骨髓來源iDC;設計併閤成針對MyD88基因的siRNA,暘離子脂質體作為轉染介質；siRNA轉染iDC 48 h後,分彆檢測iDC活力、轉染效率、錶麵分子錶達、吞噬能力、MyD88基因mRNA轉錄水平及蛋白錶達.結果 培養6d後收集iDC.採用適噹濃度siRNA(1 μg/500μl)和暘離子脂質體(3 μg/500μl)轉染48h,轉染iDC活力平均(98.50±1.04)％,轉染效率平均(85.60±3.50)％,轉染iDC錶麵分子與未轉染iDC比較無顯著差異,轉染前後iDC吞噬能力無顯著變化,MyD88基因mRNA轉錄水平和蛋白錶達分彆為內參的0.098±0.012和0.087±0.015.結論 化學閤成MyD88-siRNA是誘導iDC MyD88基因沉默的一種有效、可行的方法.
목적 관찰소우우RNA(siRNA)대대서골수래원불성숙수돌상세포(iDC) MyD88기인표체적억제작용.방법 채용립세포-거서세포집락자격인자체외배양대서골수래원iDC;설계병합성침대MyD88기인적siRNA,양리자지질체작위전염개질；siRNA전염iDC 48 h후,분별검측iDC활력、전염효솔、표면분자표체、탄서능력、MyD88기인mRNA전록수평급단백표체.결과 배양6d후수집iDC.채용괄당농도siRNA(1 μg/500μl)화양리자지질체(3 μg/500μl)전염48h,전염iDC활력평균(98.50±1.04)％,전염효솔평균(85.60±3.50)％,전염iDC표면분자여미전염iDC비교무현저차이,전염전후iDC탄서능력무현저변화,MyD88기인mRNA전록수평화단백표체분별위내삼적0.098±0.012화0.087±0.015.결론 화학합성MyD88-siRNA시유도iDC MyD88기인침묵적일충유효、가행적방법.
Objective To study the effects and efficiency of chemically synthesized small interfering RNA (siRNA) in knocking down the MyD88 gene expression of immature dendritic cells in rats.Methods Rat bone marrow-derived immature dendritic cells (iDCs) were generated by culturing bone marrow progenitor cells of rats with GM-CSF in vitro.Chemically synthesized MyD88-siRNA at the concentration of 1 μg/500 μl 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.
