ALDH2 shRNA质粒构建及其对酒精致HepG2细胞毒性的影响  

Effect of ALDH2 shRNA on the survival of HepG2 cell treated with ethanol

作　　者：郭涛[1] 王婷[1] 王飞[1] 曾妮[1] 李若碧[1] 江红梅[1] 王庆[1] 

机构地区：[1]中山大学公共卫生学院预防医学系,广东广州510080

出　　处：《热带医学杂志》2017年第4期416-420,F0004,共6页Journal of Tropical Medicine

基　　金：国家自然科学基金(81673140);广州市科技计划项目(201605122117078);中山大学青年教师培育项目(15ykpy06)

摘　　要：目的构建抑制人乙醛脱氢酶2(ALDH2)基因表达的shALDH2-PLKO.1重组慢病毒质粒,并将重组质粒转染HepG2细胞株,为构建ALDH2基因沉默细胞株及研究ALDH2在酒精致HepG2细胞毒性中的作用奠定基础。方法设计3对ALDH2 sh RNA序列并插入到PLKO.1-GFP载体中,通过测序鉴定。将ALDH2 sh RNA和包装质粒共转染293FT细胞,收集24、48和72 h病毒液进行浓缩,通过梯度稀释法测定病毒液滴度。Western blot和qRTPCR检测转染sh RNA的HepG2细胞ALDH2蛋白和mRNA表达量。MTS比色法检测转染sh RNA的HepG2细胞在酒精染毒后的增殖能力。结果测序结果显示shALDH2-PLKO.1质粒构建成功;滴度测定结果显示包装系统成功包装出病毒颗粒,病毒滴度为:4×107TU/ml;Western blot及qRT-PCR结果显示,与对照组比较,转染了shALDH2-1和shALDH2-3的HepG2细胞蛋白和mRNA表达量明显下调,其中shALDH2-1组抑制率为51%(P=0.046),shALDH2-3组抑制率为72%(P=0.008),差异有统计学意义;MTS结果显示转染sh RNA的HepG2细胞株酒精染毒后细胞增殖能力明显下降。结论成功构建了靶向ALDH2基因的重组慢病毒表达质粒,而且shALDH2-PLKO.1可以有效抑制He PG2细胞中ALDH2基因的表达,为研究ALDH2基因在酒精性肝病和肝癌中的作用机制奠定了基础。Objective To study of effect of ALDH2 sh RNA on the survival of HepG2 cell treated with ethanol.Methods Three pairs of ALDH2 sh RNA were designed and inserted into PLKO.1 vector. The recombinant shALDH2-PLKO.1 vectors were transfected into 293 FT cell to produce recombinant viruses. Recombinant viruses were harvested at24,48 and 72 h and the virus titer was determined through serial dilution. Protein and mRNA of ALDH2 were detected by Western blot and qRT-PCR,respectively. MTS assay was used to determine HepG2 cell viability. Results shALDH2-PLKO.1 vectors were constructed successfully demonstrated by DNA sequencing. The lentivirus packaging system was effective to produce virus particles,and the virus titer was 4×107TU/ml. Western blot and qRT-PCR showed that shALDH2-1and shALDH2-3 could knock down the expression of ALDH2 effectively;the inhibition rate of shALDH2-1 was 51%（P=0.046）,and the inhibition rate of shALDH2-3 was 72%（P=0.008）. MTS assay indicated that the viability of ethanol-treated HepG2 cell which transfected with sh RNA decreased dramatically. Conclusions The shALDH2-PLKO.1recombinant plasmids were constructed successfully. Knock down of the ALDH2 could cause cell death of the HepG2 cells when treated with ethanol,and may be a target treatment for liver carcinoma.

关 键 词：ALDH2 慢病毒质粒 RNA干扰 酒精性肝脏疾病 

分 类 号：R575.5[医药卫生—消化系统]