【摘要】 目的探讨PAI的发卡样siRNA真核表达载体PAI siRNA对人肝癌HpG2细胞VEGF表达的影响。方法设计短发夹状PAI siRNA的寡核苷酸链,将其插入到真核细胞表达载体pSilencerTM 2.1-U6 neo载体中,构建的重组载体后;采用脂质体转染方法将构建的重组载体导入人肝癌细胞HpG2中,采用逆转录聚合酶链反应(RT-PCR)、westernblot法分别检测转染细胞PAI mRNA和蛋白的表达变化以及对VEGF表达的影响。结果经测序证明PAI siRNA序列正确;转染PAI siRNA载体后,HpG2细胞PAI mRNA和蛋白表达均较对照组明显下降(P<0.01)。转染后,细胞中PAI含量降低,V EGF的表达也减少。结论测序结果表明发卡样的PAI siRNA真核表达载体构建成功,转染HpG2细胞后获得稳定表达,并可特异性封闭PAI的表达,PAI siRNA能有效阻断PAI的蛋白合成,同时抑制细胞中V EGF的表达。更多还原

【Abstract】 Objective To investigate the alternative expression level of VEGF by PAI short interfering RNA(siRNA) into HpG2 cells.Methods A PAI siRNA targeting human mRNA common sequence was synthesized and was inserted into BamHI /HindIIIl in pSilencerTM neo U6 2.1 vector.The sequence of PAI siRNA plasmid was analyzed by DNA sequencer.The recombinant plasmid was transfected into HpG2(human liver cancer cell line) by lipofectamine 2 000.Results Results It was verified that the sequence of constructed recombinant plasmid was corrected by DNA sequencing.The expressions of PAI mRNA and protein in HpG2 cells of experimental groups were significantly decreased,compared to that of controls(P<0.01),the expression level of VEGF were also decreased.Conclusion It might playa specific inhibitory role in the tumor cell lines.Down regulation of PAI by siRNA may alter the expression level of VEGF.This study laid experimental foundation for further research of the therapy of PAI siRNA vector.更多还原