Uhrf1基因重组腺病毒载体构建及其在小鼠心肌细胞DNA损伤修复中的作用研究

Construction of recombinant adenovirus vector of Uhrf1 gene and its role in DNA damage repair of cardiomyocytes

目的:构建携带小鼠泛素样同源域和环指结构域1(Uhrf1)基因的重组腺病毒载体,验证Uhrf1基因在原代乳鼠心肌细胞中的表达情况,并探究其在过氧化氢(H_(2)O2)诱导的心肌细胞DNA损伤中的作用。方法:利用PCR扩增小鼠Uhrf1基因的编码序列,将其酶切后插入pADM-CMV-C-FH载体,获得重组腺病毒质粒ADM-Uhrf1。将该质粒转染至HEK293T细胞包装成重组腺病毒颗粒,数代扩增后进行腺病毒的纯化及滴度检测。分离25只1日龄ICR小鼠原代心肌细胞,分为两组,以感染复数(MOI)为50的比例分别感染ADM-Uhrf1及ADM-control(ADMCtrl),通过Western blot及免疫荧光染色验证重组腺病毒介导的UHRF1蛋白的表达,并利用H_(2)O2诱导心肌细胞DNA损伤,进而探究Uhrf1在DNA损伤修复过程中的作用。结果:通过壳蛋白免疫法检测得到的ADM-Uhrf1病毒滴度为1.8×10^(13) pfu/L。Western blot验证显示UHRF1蛋白表达水平显著升高(P<0.05),免疫荧光染色显示UHRF1主要表达在细胞核内,且Uhrf1的过表达能够显著抑制DNA损伤标志物磷酸化组蛋白H_(2)A变异体(γH_(2)AX)蛋白的表达(P<0.01)。结论:成功构建了携带小鼠Uhrf1基因的重组过表达腺病毒载体,并通过腺病毒递送系统在心肌细胞中实现了Uhrf1的过表达,且Uhrf1的过表达有效减轻了H_(2)O2诱导的心肌细胞DNA损伤。

AIM:To construct a recombinant adenovirus vector carrying the mouse ubiquitin-like with plant homeodomain and RING finger domains 1(Uhrf1)gene,validate the expression of Uhrf1 in neonatal mouse cardiomyocytes and explored its role in hydrogen peroxide(H_(2)O2)-induced DNA damage.METHODS:The mouse Uhrf1 gene coding sequence was amplified by polymerase chain reaction(PCR),digested,and inserted into the pADM-CMV-C-FH vector to create the recombinant adenoviral plasmid ADM-Uhrf1.Following transfection into HEK293T cells,we generated recombinant adenoviral particles,amplified,purified,and determined the titer.Neonatal mouse cardiomyocytes were infected at an multiplicity of infection(MOI)of 50,UHRF1 protein expression was validated via Western blot and immunofluorescence staining.H_(2)O2-induced DNA damage was explored along with adenovirus-mediated Uhrf1 overexpression to investigate its role in DNA damage repair.RESULTS:ADM-Uhrf1 virus titer,determined by capsid immunofluorescence assay,was 1.8×10^(13 )pfu/L.Western blot confirmed a significant increase in UHRF1 protein expression(P<0.05),with immunofluorescence indicating predominant nuclear localization.Uhrf1 overexpression effectively inhibited the expression of the DNA damage marker,phosphorylated H_(2)AX protein(γH_(2)AX)(P<0.01).CONCLUSION:We successfully constructed a recombinant adenoviral vector carrying the mouse Uhrf1 gene,facilitating Uhrf1 overexpression in neonatal mouse cardiomyocytes.Furthermore,this overexpression effectively alleviated DNA damage in cardiomyocytes.