Nrdp1 siRNA重组腺病毒载体的构建及其对心肌肥大的影响  

Construction of Recombinant Adenovirus Vector of Nrdp1 siRNA and Its Effect of Promoting Cardiomyocyte Hypertrophy

作　　者：张圆[1] 韩丽莎[1] 胡海[1] 张坤[1] 王艳国[1] 刘佳[1] 牛常存[2] 

机构地区：[1]包头医学院病理生理学教研室,内蒙古包头市014060 [2]包头医学院第一附属医院神经外科,内蒙古包头市014010

出　　处：《中国动脉硬化杂志》2015年第4期352-356,共5页Chinese Journal of Arteriosclerosis

基　　金：国家自然科学基金资助(81200124);内蒙古自治区自然科学基金资助(2012MS1107);内蒙古自治区高等学校青年科技英才支持计划资助(NJYT-13-B19)

摘　　要：目的构建及鉴定神经调节素受体蛋白1(Nrdp1)基因siRNA重组腺病毒载体,初步研究Nrdp1基因对心肌细胞肥大的影响。方法设计并合成Nrdp1的siRNA靶向DNA序列,克隆至穿梭载体GV119中,并与腺病毒骨架质粒Ad Max在BJ5183细菌中进行同源重组,转染HEK293细胞,包装得到含si Nrdp1的重组腺病毒,实时定量PCR及Western blot检测重组腺病毒对新生大鼠原代心肌细胞Nrdp1表达的影响。然后利用血管紧张素Ⅱ和沉默Nrdp1的腺病毒干预体外培养的新生大鼠原代心肌细胞,实时定量PCR检测心肌肥大标志基因(ANF、β-MHC、Skeletal-α-actin)的表达。结果酶切后PCR分析、测序鉴定表明,干扰Nrdp1腺病毒构建成功,病毒纯化后滴度为1.5E+9 PFU/m L;重组干扰Nrdp1腺病毒可在蛋白水平和mRNA水平明显抑制Nrdp1的表达(P<0.001);沉默Nrdp1基因后,可明显上调血管紧张素Ⅱ诱导的心肌细胞肥大标志基因ANF、β-MHC和Skeletal-α-actin的表达(P<0.01)。结论构建的Nrdp1 siRNA重组腺病毒能有效地抑制新生大鼠原代心肌细胞中Nrdp1表达,且Nrdp1基因沉默后可加重AngⅡ诱导的心肌细胞肥大。Aim To construct and identificate recombinant adenovirus with si Nrdp1 gene using Ad Max system,and investigate the effect of Nrdp1 gene on cardiomyocyte hypertrophy. Methods Designing and synthesizing siRNA sequences targeting of Nrdp1 DNA,then cloned into the shuttle vector GV119 and homologous recombinated with adenovirus backbone plasmid Ad Max in BJ5183 bacteria transfected HEK293 cells,and got adenovirus containing Nrdp1-siRNA gene through packaging. Real-time quantitative PCR and Western blot were used to detect Nrdp1 expression in primary rat neonatal cardiomyocytes. After adenoviral containing si Nrdp1 transfection and angiotensin Ⅱ( AngⅡ) stimulation,realtime quantitative PCR was used to detect the expression of myocardial hypertrophy marker gene( ANF,β-MHC and Skeletal-α-actin) of rat neonatal cardiomyocytes. Results Digested PCR analysis and sequencing showed that interference Nrdp1 adenovirus was successfully constructed,and the titer of virus was 1. 5E + 9 PFU / m L. Real-time PCR and Western blot indicated that the expressions of Nrdp1 mRNA and protein were greatly inhibited after infection in rat primary cardiomyocytes with recombinant adenovirus particles( P < 0. 001). Nrdp1 gene silencing cloud significantly increase expression of AngⅡ induced cardiomyocyte hypertrophy marker genes including ANF,β-MHC and Skeletal-α-actin( P <0. 01). Conclusion The recombinant adenovirus vector containing the Nrdp1-siRNA gene was successfully constructed,which can effectively silence Nrdp1 gene and enhance AngⅡ induced cardiomyocytes hypertrophy in vitro.

关 键 词：Nrdp1基因 小干扰RNA 腺病毒载体 心肌肥大 

分 类 号：R542.2[医药卫生—心血管疾病]