整合稳定靶向高迁移率族蛋白B1基因shRNA载体的人脐静脉内皮细胞株建立  

Construction of stable human umbilical vein endothelial cells line expressing short hairpin RNA （shRNA） targeting high mobility group box-1 gene

作　　者：张晓娟[1] 栾正刚[1] 马晓春[1] 

机构地区：[1]中国医科大学附属第一医院重症医学科,辽宁沈阳110001

出　　处：《中国危重病急救医学》2012年第9期522-525,共4页Chinese Critical Care Medicine

基　　金：国家自然科学基金项目（30901438）

摘　　要：目的构建针对高迁移率族蛋白B1（HMGBl）基因的特异性RNA干扰载体，建立稳定转染该干扰载体的人脐静脉内皮细胞株（HUVEC）。方法根据HMGBl基因的编码序列及短发夹RNA（shRNA）设计原则，设计并合成针对HMGB1基因的特异性shRNA，将其定向克隆到pRNA—u6．1／Neo载体中，同时设立阴性对照，重组载体经脂质体转染HUVEC；转染细胞经类氨基糖抗生素样物质G418筛选，采用实时荧光逆转录-聚合酶链反应检测HMGB1mRNA表达，蛋白质免疫印迹法检测HMGB1蛋白表达。结果酶切鉴定和测序证实，重组质粒shRNA序列与设计的片段完全一致。HMGB1基因特异性RNA干扰载体能够显著抑制HMGBl在HUVEC细胞中的表达（mRNA：0．4635±0．0342比1．0340±0．0352，蛋白：0．4510±0．0200比1．0210±0．0110，均P〈0．05）。结论成功构建了靶向HMGB1基因的shRNA真核表达载体pRNA—HMGB1，并获得了稳定表达靶向HMGB1基因的shRNA的HUVEC细胞株，为进一步研究HMGB1在HUVEC细胞株中的生物学功能和作用机制奠定了基础。Objective To construct the short hairpin RNA （shRNA） targeting high mobility group box-1 （HMGB1） and culture the stable human umbilical vein endothelial cell （HUVEC） line expressing this shRNA. Methods Based on the HMGB1 gene sequence, shRNA was designed, synthesizedandsubclonedintothepRNA-u6.1/ Neo vector, while negative controls were also established. Then the recombinant vector was transfeeted into HUVEC cell line and the cell was screened with G418 and assayed by using real time reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting. Results Restriction endonuclease digestion test and sequencing verification showed that the recombinant pRNA-u6.1/Neo vector expressing shRNA targeting HMGB 1 was successfully constructed and the stable HUVEC cell line expressing this shRNA was developed. The real time RT-PCR and Western blotting was used to detect that recombinant plasmid in HUVEC cell effect on expression of HMGB1 was reduced （mRNA： 0.4635 ± 0.0342 vs. 1.0340 ± 0.0352, protein： 0.4510 ± 0.0200 vs. 1.0210 ± 0.0110, both P〈0.05）. Conclusion The recombinant pRNA-u6.1/Neo vector expressing shRNA targeting HMGB1 was successfully constructed and the stable HUVEC cell line expressing this shRNA was developed, and therefore allowed further investigation regarding the function of HMGB1 gene in the HUVEC cell line.

关 键 词：高迁移率族蛋白B1 人脐静脉内皮细胞 转染 脓毒症 短发夹RNA 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]