大鼠过氧化物酶体增生因子活化受体α基因重组腺病毒载体及在乳鼠心肌细胞中的表达

Construction of a recombinant adenovirus vector of peroxisome proliferators-activated receptor alpha gene and its expression in primary neonatal rat cardiomyocytes

背景:过氧化物酶体增生因子活化受体α是调节心脏脂肪酸氧化的重要核转录因子,利用基因工程技术构建其重组腺病毒载体对研究心脏能量代谢障碍机制具有重要意义。目的:构建大鼠过氧化物酶体增生因子活化受体α基因重组腺病毒载体,评价其在原代培养的乳鼠心肌细胞中的表达效率。方法:采用RT-PCR法自SD大鼠肝脏扩增过氧化物酶体增生因子活化受体α基因,将其克隆至带有增强型绿色荧光蛋白的穿梭质粒载体pAd-Track-CMV。线性化重组穿梭质粒载体并转化入含有腺病毒骨架质粒pAd-Easy-1的感受态大肠杆菌BJ5183构建重组腺病毒载体质粒。用脂质体将线性化的重组腺病毒载体质粒转染于人胚肾293细胞以包装、扩增,纯化病毒后计算滴度。用纯化的重组腺病毒转染心肌细胞,荧光显微镜观察转染效率;荧光定量PCR、免疫印迹法检测细胞过氧化物酶体增生因子活化受体αmRNA、蛋白表达。结果与结论:成功构建携带大鼠过氧化物酶体增生因子活化受体α基因的重组腺病毒载体,病毒滴度为3.5×1011pfu/L。重组腺病毒转染心肌细胞的效率达90%以上,被转染细胞目的基因mRNA、蛋白表达显著增高。该载体的成功构建为研究过氧化物酶体增生因子活化受体α基因在心肌能量代谢障碍中的作用奠定基础。

BACKGROUND： Peroxisome proliferators-activated receptor a （PPARa） has been thought as a key nuclear transcription factor in the regulation of cardiac free fatty acid metabolism, which may be involved in the determination of substrate utilization during the development of cardiac dysfunction. Sustained-overexpression of PPARe mediated by adenovirus vector can contribute to clarify the mechanisms of cardiac metabolism dysfunction. OBJECTIVE： To construct an adenovirus vector carrying rat PPARa gene and to observe its expression in primary neonatal cardiomyocytes. METHODS： The full-length of PPARe gene cDNA was acquired by reverse transcription-PCR from the rat liver and cloned into shuttle plasmid pAd-Track-CMV. After linearization with pme 1, the recombinant shuttle plasmid （pAd-Track-CMV-PPARe） was transformed into E.coli BJ5183 by electroporation to construct the recombinant adenovirus plasmid AdEasy-track-CMV-PPARa. The recombinant adenovirus plasmid was linearized and transfected into HEK293 cells using Lipofectamine 2000 for packaging and amplification. The adenovirus particles were further purified, quantified, and sequentially transfected to neonatal rat cardiomyocytes. The transfection efficiency of recombinant adenovirus was observed by fluorescent microscope. The expression of PPARa was detected by quantified PCR and western-blot method at 48 hours after deliver＇）,. RESULTS AND CONCLUSION： The recombinant adenovirus vector of PPARa was successfully constructed with a high yield of 3.5x1011 pfu/L. It was successfully transfected into cardiomyocytes （over 90%） observed by fluorescent microscope. The mRNA and protein expression of PPARa were markedly increased in the transfected cardiomyocytes. The＇construction of recombinant adenovirus vector could facilitate further investigation on the role of PPARa gene in the progression of cardiac metabolism dysfunction.