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, 赵海霞,黄嘉璐,汪思嘉,陈康宁,周振华（400038重庆,第三军医大学西南医院神经内科） [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ZF-ATF,合成靶序列Oligo DNA, 退火形成双链DNA，与通过BamH Ⅰ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（human umbilical vein endothelial cell，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HUVEC后A20蛋白表达显著增加。ELISA法检测结果表明，在LPS刺激后，空载组及空白组细胞因子IL-8、IL-6、TNF-α表达均明显上调，而ZF-ATF转染组没有明显上调，与空载组及空白组比较，有统计学差异（P＜0.05，P＜0.01）；Western blot证实在LPS刺激后ZF-ATF转染组NF-κB p65的表达（19.8±4.5）较空载组（90.2±5.9）及空白组（96.3±3.3）显著下调（P＜0.01）。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 [基金项目] 国家自然科学基金 [通信作者] 陈康宁,电话:(023E-mail:ckn_640827@126.com 周振华,电话:(023E-mail:exploiter001@126.com 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 To 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．The HUVEC cells were stimulated with LPS and the NF-κBp65 was detected by Western blotting, The concentr