CAJ | 학술논문

目的设计并制备有效干扰细胞内锌指蛋白A20(简称A20)表达的基因沉默载体,初步用于观察A20基因沉默对细胞炎症应答的影响. 方法人工设计并合成A20特异性RNA干扰寡核苷酸片段(ASRF),构建A20基因沉默载体pSUPER-EGFP-A20 siRNA.采用基因转染技术使人单核细胞株THP1感染pSUPER-EGFP-A20 siRNA,用适时荧光定量PCR技术鉴定转染细胞A20基因的沉默率;用ELISA测定细胞核内NF-κB活性及培养上清液中的TNF-α水平.结果在设计的2条ASRF中,以M59465-385R/F对细胞A20表达的抑制效果最佳,基因沉默率达83.86%.初步应用表明,A20基因沉默后THP1内NF-κB活性水平增加了78.13%,释放的TNF-α增加了49.30%. 结论成功得到了高效A20基因沉默载体,初步应用研究提示A20表达的意义在于下调细胞炎症应答程度.
목적 설계병제비유효간우세포내자지단백A20(간칭A20)표체적기인침묵재체,초보용우관찰A20기인침묵대세포염증응답적영향. 방법 인공설계병합성A20특이성RNA간우과핵감산편단(ASRF),구건A20기인침묵재체pSUPER-EGFP-A20 siRNA.채용기인전염기술사인단핵세포주THP1감염pSUPER-EGFP-A20 siRNA,용괄시형광정량PCR기술감정전염세포A20기인적침묵솔;용ELISA측정세포핵내NF-κB활성급배양상청액중적TNF-α수평.결과 재설계적2조ASRF중,이M59465-385R/F대세포A20표체적억제효과최가,기인침묵솔체83.86%.초보응용표명,A20기인침묵후THP1내NF-κB활성수평증가료78.13%,석방적TNF-α증가료49.30%. 결론 성공득도료고효A20기인침묵재체,초보응용연구제시A20표체적의의재우하조세포염증응답정도.
Objective To design and prepare an RNA interfering vector for effectively inhibiting the cellular expression of zinc finger protein A20 and observe the effect of A20 gene silence on cellular inflammatory response. Methods Specific RNA interfering oligonucleotide fragments (ASRF) were designed and synthesized artificially and the A20 RNA interfering vector pSUPER-EGFP-A20 siRNA constructed. Human monocyte cell line THP1 was used to infect the pSUPER-EGFP-A20 siRNA by means of genetic transfection technique; then, silence rate of cellular A20 was analyzed by real-time polymerase chain reaction (PCR). In the meantime, the activity of nuclear transcription factor nuclear factor-κB (NF-κB) and the level of tumor necrosis factor-α (TNF-α) in culture supernatant were measured by ELISA. Results Of two specific inhibitory oligonueleotide fragments of A20, the fragment M59465-385R/F had a higher inhibition to A20 expression, with rate of A20 gene silence of 83.86%. Preliminary application showed that after A20 gene silence, the activity of NF-κB was increased by 78.13% and the level of TNF-α in cell culture supernatant was increased by 49.30%. Conclusions Vector of A20gene silence with a high efficiency is obtained successfully. Preliminary application indicates that the expression of A20 can down-regulate the degree of cellular inflammatory responses.