转基因小鼠模型的载体构建和在人肝癌HepG2细胞中的表达

Construction of Vector of Transgenic Mice Model and Expression of Vector in Human Liver Cancer HepG2 Cells

目的:构建转基因小鼠模型的载体并检测在人肝癌HepG2中的表达效果。方法:将n-3多不饱和脂肪酸脱氢酶基因fat-1插入到真核表达载体(pcDNA3.1(+)myc-His A)中,构建重组表达载体pcDNA3.1(+)myc-His A-fat-1,用脂质体介导的方法转染到人肝癌HepG2细胞中,RT-PCR检测fat-1基因的表达,MTT法分析fat-1基因对HepG2细胞增殖的影响,气相色谱分析检测fat-1基因对HepG2细胞n-6/n-3多聚不饱和脂肪酸(PUFAs)比例的影响。结果:成功地构建了真核表达载体pcDNA3.1(+)myc-HisA-fat-1,并能在HepG2细胞内有效异源表达,48h后可检测到fat-1mRMA的条带。与对照细胞相比,fat-1基因有效地抑制了人肝癌细胞HepG2细胞的增殖(70%,p<0.01),降低了n-6/n-3PUFAs比例。结论:pcDNA3.1(+)myc-His A-fat-1重组载体构建成功并能在肝癌细胞中有效的表达,可以作为下一步转基因小鼠的合适载体。

Objective： To construct the vector of transgenic mice model and detect its expression in human liver cancer HepG2 cells. Method： Polyunsaturated fatty acids dehydrogenase fat-1 cDNA was inserted into eukaryotic expression vector pcDNA3.1 （＋） myc-His A to form a recombinant vector and the recombinant vector was transfected into HepG2 cell by liposomal reagent. The expression of fat-1 gene was detected by RT-PCR. MTT and Gas chromatography were used to examine the proliferation of cells and the change of n-6/n-3 PUFAs ratio of HepG2 cell membrane. Results： The proposed recombinant plasmid was got through DNA recombinant technique; fat-lgene expressed effectively in human liver cancer HepG2 cells; fat-1 mRNA band appeared 48h after transfection ofpcD- NA3.1 （＋）myc-His A-fat-1. Compared with the control cell （pcDNA3.1 （＋）myc-His A-control）, proliferation of HepG2 cells was markedly inhibited（70 %,p〈0.01）. Moreover, fat-lgene significantly decreased the ratio of n-6/n-3 PUFAs of HepG2 cells membrane. Consequent ly, fat-1 gene could heterologously express in human liver cancer cells HepG2 and inhibit proliferation of cells by decreasing the ratio of n-6/n-3 PUFAs of cell membrane. Conclusion： pcDNA3.1 （＋）myc-His A-fat-1 being successfully constructed and effectively expressed in animal cell can provide a suitable vector for transgenic mice model.