内皮型一氧化氮合酶重组腺病毒表达载体的构建及其在食管平滑肌细胞中的表达和调控  

Construction of Recombinant Adenovirus Carrying Endothelial Nitric Oxide Synthase Gene and its Expression and Control in Esophageal Smooth Muscle Cells

作　　者：宁守斌[1] 张忠兵[1] 谢渭芬[1] 杨秀疆[1] 邱镇[1] 

机构地区：[1]第二军医大学长征医院消化内科,200003

出　　处：《胃肠病学》2003年第1期29-32,60,共5页Chinese Journal of Gastroenterology

摘　　要：背景:一氧化氮(NO)是一种重要的抑制性神经递质,其合成障碍与多种胃肠道动力障碍性疾病的发生密切相关。目的:构建四环素调控的内皮型一氧化氮合酶(eNOS)重组腺病毒表达载体,实现eNOS在食管平滑肌细胞(ESMC)中表达的精确调控。方法:可调控重组腺病毒表达载体的构建分为3个步骤:首先,将编码目的基因eNOS的全长cDNA定向克隆于穿梭质粒pTRE-Shuttle中,获得一个能被四环素或其类似物强力霉素调控的表达盒(Tet-responsive expression cassette):然后,将表达盒与腺病毒DNA(Adeno.XTM viral DNA)体外连接,形成重组腺病毒质粒Ad-eNOS;最后,Ad-eNOS转染人胚肾293细胞后被包装成有感染能力的重组腺病毒颗粒Adeno-X-TRE-eNOS。用Adeno-X-TRE-eNOS和调控病毒Adeno-X.Tet-off共感染体外培养的ESMC,采用逆转录聚合酶链反应(RT-PCR)和Western blot评价eNOS在靶细胞中的表达以及强力霉素对表达强度的调控情况。结果:体外连接法成功构建了可调控重组腺病毒表达载体Adeno-X-TRE-eNOS,其与调控病毒共感染ESMC后,RT-PCR和Western blot出现阳性结果。加入不同浓度的强力霉素可以抑制eNOS基因的转录强度,其抑制作用呈剂量-效应关系,强力霉素浓度达到0.01μg/ml时可以完全关闭目的基因的表达。结论:采用体外连接法成功构建了可?Background: Nitric oxide (NO) is a major inhibitory neurotransmitter, and its deficiency plays an important role in the pathogenesis of motility disorders of gastrointestinal tract. Aims: To generate a recombinant adenovirus containing tetracycline-regulated endothelial nitric oxide synthase (eNOS) gene, and to detect the expression and control of eNOS gene in esophageal smooth muscle cells (ESMC). Methods: The construction of controllable recombinant adenovirus was completed in three phases: First, a Tet-responsive expression cassette which could be regulated by tetracycline or its analogue, doxycycline, was made by cloning the full-length cDNA encoding eNOS into pTRE-Shuttle vector. Next, the expression cassette was transferred to Adeno-X?viral DNA to form a recombinant adenoviral plasmid Ad-eNOS by means of an in vitro ligation reaction. Finally, the Ad-eNOS was packaged into infectious recombinant adenoviral particles Adeno-X-TRE-eNOS by transfecting human embryonic kidney 293 cells. ESMC cultured in vitro was coinfected by Adeno-X-TRE-eNOS and regulation virus Adeno-X-Tet-off, and the doxycycline-regulated eNOS expression was detected by reverse trans-criptase polymerase chain reaction (RT-PCR) and Western blot. Results: The controllable recombinant adenovirus Adeno-X-TRE-eNOS was generated successfully by in vitro ligation reaction. The expression of eNOS gene in the coinfected ESMC was confirmed by the results of RT-PCR and Western blot. Furthermore,the transcription could be precisely regulated in a dose-dependent manner in a series of concentrations of doxycycline, and it was completely turned off when the concentration reached 0.01 Hg/ml. Conclusions: A controllable recombinant adenovirus carrying eNOS can be generated successfully by in vitro ligation reaction, and its controllable expression in ESMC cultured in vitro can be realized, which may provide the basis for using eNOS in gene therapy.

关 键 词：内皮型一氧化氮合酶 腺病毒 抑制性神经递质 胃动力障碍性疾病 肠道动力障碍性疾病 

分 类 号：R571[医药卫生—消化系统]