人工转录因子抑制脂多糖诱导的内皮细胞凋亡及其-第三军医大学学报.DOC

A20人工锌指转录因子真核载体的构建及其对内皮细胞炎症反应的影响 孟召友 黄嘉璐 赵海霞 汪思嘉 陈康宁 周振华 （第三军医大学西南医院神经内科， 重庆400038） 摘要：目的 观察A20基因的人工锌指转录因子（ZF-ATF）对内源性A20基因及内皮细胞炎症反应的影响。方法 根据人A20基因的序列，设计出其人工锌指转录因子（ZF-ATF）, 合成靶序列Oligo DNA, 退火形成双链DNA，与通过BamHI和KpnI酶切后的载体连接产生ZF-ATF真核载体, 质粒转化感受态细菌DH5α，筛选阳性克隆，提取质粒，限制性内切酶酶切鉴定，鉴定阳性克隆进行测序。质粒DNA转染人脐静脉内皮细胞（HUVEC），通过Western blot检测A20蛋白的表达，用脂多糖（LPS）诱导 HUVEC细胞产生炎症反应，利用Western blot检测NF-κBp65的表达情况，利用ELISA检测TNF-α、IL-6、IL-8等，进一步探讨其机制。结果 限制性内切酶酶切鉴定与DNA测序证实合成的含ATF的真核载体寡核苷酸链插入正确，Western-blot证实转染内皮细胞后A20蛋白表达显著增加，Western-blot、ELSA证明LPS诱导HUVEC细胞后实验组NF-κB p65、TNF-α（P＜0.05）、IL-6(P＜0.01)、IL-8（P＜0.01）表达明显下调。结论 成功构建人工锌指转录因子真核表达载体，且能够启动内源性A20的稳定表达，并发挥其抗炎等作用。 关键词：A20； 人工转录因子； 抗炎作用 [中图法分类号] R34；R392.12；R743.33 [文献标志码] A Construction of the recombinant plasmid ZF-ATF and evaluation of the roles in anti-inflammation of HUVEC cell line Meng Zhaoyou Huang Jialu Zhao Haixia Wang Sijia Chen Kangning Zhou Zhenhua Department of Neurology, Southwest Hospital, Third Military Medical University, Chongqing, 400038，China [Abstract]：Objective o construct eukaryotic expression vector of A20 artificial zinc finger transcription factor (ZF-ATF), make it express A20 stably and evaluate the effects of anti-inflammation in HUVEC cells. Methods A20 artificial zinc finger transcription factor (ZF -ATF) was designed based on the gene sequence of A20, Then ZF -ATF was subcloned into the eukaryotic expression vector PCDNA-NLS-P65-Flag, which was identified and confirmed by restricting enzyme digestion and DNA sequencing. Subsequently, the PCDNA-NLS-ZF-ATF-P65-Flag plasmid was transfected in HUVEC cell and the expression of protein A20 was detected by Western blotting．HUVEC cells were stimulated with LPS and the NF-κBp65 was detected by Western blotting, The concentration of the TNF-α,IL-6 and IL-8 in the supernatants were determinated by cytokine ELISA kits. Results In 24 hours after transfection with recombinant vector, the expression of protein A20 was upregulation . After stimulation with LPS