大鼠乙醛脱氢酶2基因调控腺病毒载体构建方法及意义  

Methodology on construction of rat aldehyde dehydrogenase 2 gene regulation recombinant adenovirus vectors

作　　者：李鸿博[1] 郎小娥[2] 

机构地区：[1]北京协和医学院临床医学系,北京100005 [2]山西医科大学第一医院精神卫生科,太原030001

出　　处：《中国临床药理学杂志》2014年第10期922-925,共4页The Chinese Journal of Clinical Pharmacology

摘　　要：目的观察持续活化突变体腺病毒转染大鼠心肌细胞的转染效果及对乙醛脱氢酶2(ALDH2)表达的影响。方法分别将扩增得到的ALDH2持续活化突变体基因及合成ALDH2-siRNA序列颈环状DNA,连接相应载体后,得到重组穿梭质粒;对2种穿梭质粒分别进行扩增和酶切鉴定,并导入pAdeno腺病毒载体,转染293细胞进行扩增与纯化。将1日龄雄性SD大鼠心肌细胞进行培养,将2种重组腺病毒及对照空载体分别感染细胞,随后检测ALDH2表达量。结果 2种载体构建正确,纯化后两者滴度分别为2×1010,1.6×1010PFU·mL-1。实验组ALDH2表达量与对照组相比差异具有统计学意义(P<0.01)。结论成功构建大鼠ALDH2基因双向调控腺病毒载体,可以有效调控离体大鼠心肌细胞ALDH2表达。Objective To construct adenovirus specific for rat aldehyde dehydrogenase 2 （ALDH2） gene interference and consistent activation and transfect the viruses into rat cardiomyocytes to observe transfection effect and its influence on ALDH2 expression.Methods Consistently active ALDH2 （CA -ALDH2） mutant gene was amplified and linked to shuttle vector, thus recombinant shuttle plasmid was subsequently con -structed.Stem -loop DNA for ALDH2 silencing RNA （ ALDH2 -siRNA） sequence was synthesized and loaded it into vector thus recombi -nant shuttle plasmid was constructed .Both kinds of plasmid were imple -mented amplification and enzyme identification.Verified plasmids were loaded into pAdeno adenovirus vectors.The viruses were then transfected into 293 cell linage to replicate and be purified.Treat cultured cardio-myocytes from 1 -day -old neonatal male Sprague Dawley （SD） rat with empty adenovirus vector control and both kinds of recombinant adenovirus vector, and perform subsequent assay for ALDH2 expression.Results Both vectors are identified by endonuclease with titre of 2 ×10^10 , 1.6 ×10^10 PFU · mL-1 respectively after purification.The change in ALDH2 expression after infection are both observable （ P 〈0.01 ） . Conclusion Both of the double -way regulation recombinant vectors for rat ALDH2 gene are successfully constructed , which are capable of effec-tive regulation of the gene expression in rat cardiomyocytes .

关 键 词：乙醛脱氢酶2 腺病毒载体 缺血-再灌注 心肌保护 

分 类 号：R965.2[医药卫生—药理学] R977.3[医药卫生—药学]