锌指蛋白A20基因沉默载体制备及初步应用

Preparation of vector of zinc finger protein A20 gene silence and its preliminary application

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。

Objective To design and prepare an RNA interfering vector for effectively inhibiting the cellular expression of zinc finger protein 3220 and observe the effect of 3220 gene silence on cellular inflammatory response. Methods Specific RNA interfering oligonucleotide fragments （ASRF） were designed and synthesized artificially and the 3220 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 3220 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 oligonucleotide fragments of 3220, the fragment M59465385R/F had a higher inhibition to 3220 expression, with rate of 3220 gene silence of 83.86%. Preliminary application showed that after 3220 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 3220 gene silence with a high efficiency is obtained successfully. Preliminary application indicates that the exrpession of 3220 can down-regulate the degree of cellular inflammatory responses.