双链寡聚脱氧核苷酸/载体复合物在急性肺损伤肺泡巨噬细胞中的体外转染效率研究  

A study of transfection efficiency by NF-кB decoy dsODNs /carrier in the alveolar macrophages in vito during acute lung injury

作　　者：尹文[1] 王倩梅[1] 冯洋[1] 朱金文[1] 李明月[2] 高路[3] 

机构地区：[1]第四军医大学西京医院急诊科,陕西西安710032 [2]西安市儿童医院,陕西710003 [3]西安交通大学医学院第一附属医院,陕西710061

出　　处：《创伤外科杂志》2014年第6期538-542,共5页Journal of Traumatic Surgery

基　　金：国家自然科学基金项目(81070063);陕西省自然科学基金重点项目(2014JM4186)

摘　　要：目的利用圈套dsODNs/载体基因技术,通过体外实验研究NF-кB decoy dsODNs/载体复合物在急性肺损伤肺泡巨噬细胞中的体外转染效率,为进一步的体内试验提供依据,从而在细胞分子水平为临床防治全血性炎症反应综合征/急性肺损伤(SIRS/ALI)筛选精确药物靶点和寻求高效合理的治疗方案提供前瞻性的实验依据。方法制备失血性休克致ALI的实验动物模型,以Lipofectamine2000为载体,对巨噬细胞分组进行dsODNs干预:对照组、脂多糖(lipopolysaccharide,LPS)组、Lipofectamine2000组(Lipo2000组)、NF-κB dsODNs/Lipofectamine2000组(dsODNs组)。乳酸脱氢酶(LDH)试剂盒检测巨噬细胞(AM)毒性;观察并测定巨噬细胞的转染活性、最佳转染浓度和转染效率。结果 dsODNs/Lipofectamine2000浓度比率为1∶5,体外转染6h时,转染效率、荧光强度和细胞状态最佳,细胞毒性适中。结论 NF-кB圈套dsODNs与Lipofectamine2000在一定浓度范围内能有效提高圈套dsODNs的转染效率,并且最大限度降低各种刺激对细胞造成的损伤,为SIRS/ALI的临床防治提供了新的思路和途径,为临床治疗ALI筛选特异性的炎症治疗药物和开发临床药物确定了精确靶点。Objective To study the transfection efficiency by NF -кB decoy dsODNs/carrier in the alveo-lar macrophages in vito during acute lung injury by NF -кB decoy dsODNs/liposome technology .Intended results will provide a preliminary experimental basis in vitro .It could provide the feasibe experiment basis for screening drug targets and seek dfficient and reasonable treatment to prevent and cure SIRS /ALI.Methods Animal model of hemorrhagic shock caused acute lung injury was established .Different stimulation and complex processing were giv-en to alveolar macrophages in vitro using Lipofectamine 2000 as carrier.The transfection efficiency of each phase and the best time to determine dfficient transfection was measured .The LDH level trasformation by different pre-treatment were detected in different time points .Results When NF-кB decoy dsODNs/Lipofectamine2000 rate was 1∶5 and transfected time was six hours ,the transfection efficiency was highest ,and the cytotoxicity was moder-ate.Conclusion NF-кB decoy dsODNs could significantly improve the transfection efficiency within a proper dsODNs/Lipofectamine2000 rate and reduce injury of alveolar macrophages by different stimulations as much as pos -sible.Decoy dsODNs strategy provides new ideas and approaches for preventing and treating for SIRS /ALI, deter-mines the precise target and screening specific inflammatory therapy for the clinical treatment , and determines the precise target of the clinical drug development .

关 键 词：肺损伤 失血性休克 基因 细胞 

分 类 号：R331.4[医药卫生—人体生理学]