n-6脂肪酸去饱和酶fat-1基因对神经元细胞凋亡的抑制作用

Expression and Effect of fat-1 Gene Encoding n-6 Fatty Acid Desaturase in Rat Cortical Neurons

将C.elegans n-6脂肪酸去饱和酶基因fat-1的cDNA插入到腺病毒的穿梭载体pAd-CMV中,并与骨架载体同源重组,构建腺病毒重组载体(Ad.GFP.fat1),通过包装细胞系(293)产生重组腺病毒,感染原代培养的大鼠皮层细胞.在显微镜下观察、细胞增殖试剂盒(MTT)和凋亡染色试剂盒分析fat-1基因对大鼠皮层细胞凋亡的影响,核糖核酸酶保护性分析,检测fat-1基因在大鼠皮层细胞内的表达,酶联免疫分析花生四烯酸类(Eicosanoids)前列腺素(Prostaglandin E2)的含量.结果表明,通过基因重组技术,得到预期的重组病毒;fat-1基因在原代培养的大鼠皮层细胞中能有效异源表达,2 d后,可检测到fat-1 mRNA的条带.与对照Ad.GFP细胞相比,fat-1基因明显抑制了大鼠皮层细胞因诱导产生的凋亡(35%),受保护细胞的前列腺素含量也明显地减少(30%).

The fat-1 gene encoding n-6 fatty acid desaturase eDNA was cloned into adenovirus shuttle vector pAd-CMV and then homologously recombinated with backbone vector to construct a recombinant adenoviral vector pAd.GFP, fat1 ;The vector was transfected into 293 cell to get the recombinant virus,infected primary cultural rat cortical neuron cell;The infected cell was observed under microscope,and was analyzed the effect of fat-1 gene on proliferation and apoptosis of the cell using MTT kit and apoptosis kit, expression of fat-1 using Ribonuclease Protection Assay kit, Eicosanoids （Prostaglandin E2） was measured by enzyme immunoassay kit. The expectant recombinant virus was obtained; fat-1 gene could express at high level in rat conical neuron cell, fat-1 mRNA band appeared two days after infection. Compared with control Ad. GFP, fat-1 gene markedly inhibited apoptosis of rat conical neuron cell caused by induction（35%）, amount of eieosanoids（prostaglandin E2） was visibly reduced （30%）.