CAJ | 학술논문

目的利用RelB短发卡RNA(shRNA)慢病毒转染树突状细胞(DC)诱导耐受性DC的产生.方法将Lenti-RelB shRNA转染未成熟DC后,应用逆转录-聚合酶链反应(RT-PCR)和Western blot检测DC中RelB的表达；流式细胞仪检测DC凋亡以及表型分子人类主要组织相容性复合体(MHC)-Ⅱ、CD80、CD86、CD83的表达；酶联免疫吸附试验(ELISA)检测分泌的细胞因子白细胞介素(IL)-10、IL-12、转化生长因子-β1(TGF-β1)以及核因子(NF)-κB各亚基的DNA结合活性.结果比较不成熟组、成熟组、空载体对照组DC 、Lenti-RelB shRNA-DC下调RelB的表达；而凋亡率同其余3组比较差异无统计学意义(P＞0.05)；同时下调DC表型分子MHC-Ⅱ(0.34±0.04)、CD80(0.38±0.03)、CD86(0.17 ±0.02)、CD83(0.21 ±0.02)的表达；转染后的DC低表达IL-12[(96.3±32.8)ng/L],高表达IL-10[(218.2 ±43.3) ng/L]；而RelB的DNA结合活性(0.28±0.06)较其他组显著下降,但其他亚基的DNA结合活性各组差异无统计学意义(P＞0.05).结论利用LentiRelB shRNA沉默RelB表达可体外诱导耐受性DC的产生.
목적 이용RelB단발잡RNA(shRNA)만병독전염수돌상세포(DC)유도내수성DC적산생.방법 장Lenti-RelB shRNA전염미성숙DC후,응용역전록-취합매련반응(RT-PCR)화Western blot검측DC중RelB적표체；류식세포의검측DC조망이급표형분자인류주요조직상용성복합체(MHC)-Ⅱ、CD80、CD86、CD83적표체；매련면역흡부시험(ELISA)검측분비적세포인자백세포개소(IL)-10、IL-12、전화생장인자-β1(TGF-β1)이급핵인자(NF)-κB각아기적DNA결합활성.결과 비교불성숙조、성숙조、공재체대조조DC 、Lenti-RelB shRNA-DC하조RelB적표체；이조망솔동기여3조비교차이무통계학의의(P＞0.05)；동시하조DC표형분자MHC-Ⅱ(0.34±0.04)、CD80(0.38±0.03)、CD86(0.17 ±0.02)、CD83(0.21 ±0.02)적표체；전염후적DC저표체IL-12[(96.3±32.8)ng/L],고표체IL-10[(218.2 ±43.3) ng/L]；이RelB적DNA결합활성(0.28±0.06)교기타조현저하강,단기타아기적DNA결합활성각조차이무통계학의의(P＞0.05).결론 이용LentiRelB shRNA침묵RelB표체가체외유도내수성DC적산생.
Objective Lentiviral-mediated short hairpin RNA (shRNA) against RelB was used to produce tolerogenic dendritic cells from murine bone marrow derived dendritic cells (BMDCs).Methods RelB expression in the BMDCs was silenced by Lentivirus carrying RelB shRNA.The RelB expression was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting.The apoptosis rate and surface markers of dendritic cells (DCs) were assessed by flow cytometry.Interleukin (IL)-12,IL-10,transforming growth factor-β1 (TGF-β1) secreted by DCs and DNA binding capacity of nuclear factor-κB (NF-κB) subunits in the nucleus were measured by enzyme linked immunosorbent assay (ELISA),independently.Results RelB expression was significantly inhibited in DCs following lentiviral mediated delivery of RelB specific shRNA.The RelB shRNA-DC produced lower IL-12 [(96.3 ± 32.8) ng/L] and higher IL-10 [(218.2 ± 43.3) ng/L] than mature dendritic cells (mDCs) and silencing control DCs.There were no difference in the apoptosis rate between shRNA ReIB-DCs and mDCs.The expression levels of co-stimulatory molecules MHC-Ⅱ (0.34 ± 0.04),CD80 (0.38 ± 0.03),CD86 (0.17 ± 0.02),CD83 (0.21 ± 0.02)were lower in the RelB shRNA-DCs than in the mDCs and silencing control DCs.In addition,RelB shRNA also inhibited the RelB DNA binding capacity (0.28 ±0.06) but had no effect on otherNF-κB subunits.Conclusion RelB shRNA transfection of DCs can induce the immature status,and produce tolerogenic DCs.