桥粒芯糖蛋白基因沉默对HL-1细胞的影响  

RNA interference of desmoglein-2 gene and its effect on HL-1 cells

作　　者：丰尚鹏[1] 杨兵[1] 陈明龙[1] 周秀娟[1] 王静[1] 王本琪[1] 王薇[1] 李丹丹[1] 宋华连[1] 周本军[1] 陈红武[1] 张凤祥[1] 居维竹[1] 郦明芳[1] 曹克将[1] 

机构地区：[1]南京医科大学第一附属医院心脏科,210029

出　　处：《中华心律失常学杂志》2012年第3期219-223,共5页Chinese Journal of Cardiac Arrhythmias

基　　金：国家自然科学基金

摘　　要：目的探讨桥粒芯糖蛋白（DSG2）基因沉默对HL-1细胞超微结构、细胞凋亡以及纤维化、脂肪化相关基因表达的影响。方法设计并合成针对DSG2基因编码区的干扰序列，构建真核细胞表达质粒并转染HL-1细胞。荧光定量PCR（RT—PCR）和蛋白免疫印迹（Westernblot）技术检测DSG2在mRNA和蛋白水平表达变化，筛选出沉默效率最佳的细胞株。观察其超微结构、细胞凋亡和纤维化与脂肪化相关基因mRNA水平表达的改变。结果成功构建了4种ShDSG2重组质粒，转染HL-1细胞并获得稳定转染细胞株，筛选出对DSG2基因mRNA水平（71．67％对0，P〈0．01）和蛋白水平表达（51．72％对0，P〈0．01）的抑制效率最显著的ShDSG2—273组。电镜扫描显示后者细胞空泡样变性、线粒体肿胀和嵴消失，流式细胞仪检查提示细胞凋亡显苫增加，RT-PCR示纤维化相关基因（Collal、Colla2、C013a1）与脂肪化相关基因（Adiponectin、PPAR-γ和C／EBP-α）的mRNA表达显著升高。结论成功建立能有效抑制DSG2表达的HL-1细胞株，表现出符合致心律失常型右心室心肌病（ARVC）患者病理和分子生物学特点的表型特征，提示其可作为深入研究ARVC致病机制的工具细胞。Objective To investigate the effects of down-regulation of desmoglein-2 （DSG2）on ultra- strueture,apoptosis,adipogenesis and fibrogenesis in HL-1 cells. Methods A set of DSG2-specific siRNAs and control siRNA （siNC）were synthesized and cloned into pGPU6/GFP/Neo vector. The recombinant plasmids （ShDSG2 and ShNC）were transfected into HL-1 cells, and the positive clones were selected with C,418. The effi- ciency of DSG2 knock down on the levels of mRNA and protein was determined by real time-PCR（RT-PCR） and Western blot in order to pick the optimal cell line. The changes of cell apoptosis and ultrastrncture were evaluated by flow cytometry and transmission electron microscope（ TEM）, respectively. RT-PCR was used to measure the ex- pression of Collal ,Colla2,Col3al ,Adiponectin,PPAR-γ,and C/EBP-α on mRNA level. Results Four recom- binant ShDSG2 plasmids were constructed successfully and transtected into HL-1 cells,and stable cell lines were established by RNA interference. Compared with the control group ,the levels of DSG2 knock down on mRNA and protein in ShDSG2-273 group were 71.67% vs 0 （ P 〈 0. 01 ） and 51.72% vs 0 （ P 〈 0. 01 ）, respectively. In ShDSG2-273 group,TEM showed that cellular vacuoles, swollen mho ehondria and disappeared cristae and Flow Cytometry further demonstrated that the apoptosis was increased in the stable cell lines. A marked increase in ex- pression levels of two major regulators of adipogenesis, namely PPAR-γ, and C/EBP-α, and their target gene adi- poneetin, were also observed. The expression levels of procollagengenes Co]lal, Colla2 and Col3al were increases remarkably. Conclusions We established DSG2-defieient HL-1 cell line,which exhibited increased apoptosis, adipo genesis and fibrogenesis, thus recapitulating the phenotype of human arrhythmogenic right ventricular cardiomy- opathy（ARVC）. This cellular tool could provide the opportunity to explore the pathogenesis and novel therapeu- tic targets of ARVC in vitro.

关 键 词：桥粒芯糖蛋白 RNA干扰 HL-1细胞 致心律失常型右心室心肌病 细胞凋亡 

分 类 号：R542.2[医药卫生—心血管疾病]