【摘要】 目的 构建5个PAH基因无义突变位点慢病毒载体并转染细胞，比较3种不同类型药物PTC124、Amlexanox和G418对PAH基因无义突变的通读效率。方法 选择5个突变频率较高的人PAH基因无义突变位点(R111X、Y166X、R176X、W326X、Y356X)构建慢病毒载体，与野生型慢病毒载体(6个载体均带有Flag标签DDDDK)分别转染HEK293细胞。取野生型(WT)及5种突变型细胞，采用不加药物(0μmol/L)、PTC124(50、100μmol/L)、Amlexanox(250、500μmol/L)、G418(1、2、5 g/L)分别处理48 h,采用CCK-8实验检测72 h细胞增殖率，采用实时荧光定量PCR法检测DDDDK mRNA相对表达量，采用Western blot法检测PAH、DDDDK蛋白相对表达量。结果 (1)WT细胞经5 g/L G418处理72 h后细胞增殖率低于0μmol/L(P<0.05),其余药物及浓度处理后细胞增殖率均不受影响；5种突变型细胞经50、100μmol/L PTC124处理72 h后细胞增殖率均不受影响；W326X、Y356X突变型细胞经500μmol/L Amlexanox处理72 h后细胞增殖率低于0μmol/L(P<0.05),其余突变型细胞经250、500μmol/L Amlexanox处理后细胞增殖率不受影响；R111X、Y166X突变型细胞经5 g/L G418处理72 h后细胞增殖率低于0μmol/L(P<0.05),1、2 g/L G418处理后细胞增殖率不受影响，R176X、W326X和Y356X经1、2、5 g/L G418处理72 h后细胞增殖率均低于0μmol/L(P<0.05)。(2)WT细胞经50、100μmol/L PTC124和250、500μmol/L Amleanox处理后DDDDK mRNA及PAH、DDDDK蛋白相对表达量均高于0μmol/L(P<0.05),5 g/L G418处理后均低于0μmol/L(P<0.05),1、2 g/L G418处理后与0μmol/L比较差异均无统计学意义(P>0.05)。5种突变型细胞经250、500μmol/L Amleanox和5 g/L G418处理后DDDDK mRNA相对表达量均高于0μmol/L(P<0.05),经50、100μmol/L PTC124处理后DDDDK mRNA相对表达量与0μmol/L比较差异均无统计学意义(P>0.05);5种突变型细胞不加药物(0μmol/L)处理后DDDDK mRNA相对表达量均低于WT细胞(P<0.05)。5种突变型细胞经不加药物(0μmol/L)、50、100μmol/L PTC124和250、500μmol/L Amleanox处理后几乎不表达PAH、DDDDK蛋白；R111X、R176X、Y166X、Y356X突变型细胞经5 g/L G418处理后PAH、DDDDK蛋白相对表达量均高于0μmol/L(P<0.05),W326X突变型细胞经1、2、5 g/L G418处理后PAH、DDDDK蛋白相对表达量均高于0μmol/L(P<0.05),1、2 g/L G418均高于5 g/L G418(P<0.05),1 g/L G418与2 g/L G418比较差异均无统计学意义(P>0.05)。结论 Amlexanox和G418可提高PAH基因5种无义突变R111X、Y166X、R176X、W326X、Y356X转录水平，其中G418可使5种无义突变表达完整PAH蛋白；G418矫正W326X无义突变的有效浓度为1 g/L,而矫正其余4种无义突变的有效浓度为5 g/L。更多还原

【Abstract】 Objective To construct and transfect 5 lentiviral vectors carrying nonsense mutation sites of PAH gene, and to compare the read-through efficiencies of three different drugs as PTC124, Amlexanox and G418 On nonsense mutation of PAH gene.Methods Five high-frequency mutation sites of human PAH gene(R111X,Y166X,R176X,W326X,Y356X) were selected to construct lentiviral vectors. The above 5 vectors and wild-type lentiviral vector(all 6 vectors containing a Flag tag-DDDDK) were separately transfected into HEK293 cells.The wild-type and other 5 mutant cell lines were treated with no drug(0 μmol/L),PTC124(50,100 μmol/L),Amlexanox(250,500 μmol/L) and G418(1,2,5 g/L) for 48 h,The 72-h proliferation rate was detected by CCK-8 assay,the relative expression of DDDDK mRNA was detected by real-time fluorescence quantitative PCR,and the relative expressions of PAH and DDDDK proteins were detected by Western blot.Results(1) The proliferation rate of wild-type cells was lower after being treated with 5 g/L G418 for 72 h than that after being treated with 0 μmol/L(P<0.05).The proliferation rate was not affected by other drugs and concentrations.The proliferation rates of the 5 mutant cell lines were not affected after being treated with 50and 100 μmol/L PTC 124 for 72 h.After being treated with 500 μmol/L Amlexanox for 72 h,the proliferation rates of W326X and Y356X mutant cells were lower than those after being treated with 0 μmol/L(P<0.05),while the proliferation rates of the other mutant cell lines were not affected by 250 and 500 μmol/L Amlexanox.After being treated with 5 g/L G418 for 72 h, the proliferation rates of R111X and Y166X mutant cells were lower than those after being treated with 0 μmol/L(P<0.05).The proliferation rate was not affected by 1 and 2 g/L G418.The proliferation rates of R176X, W326X and Y356X mutant cells after being treated with 1,2 and 5 g/L G418 for 72 h were lower than those after being treated with 0 μmol/L(P<0.05).(2) The relative expressions of DDDDK mRNA and PAH and DDDDK proteins were higher after the wild-type cells were treated with 50 and 100 μmol/L PTC124 as well as 250 and500 μmol/L Amlexanox than those after being treated with 0 μmol/L(P<0.05),were lower after being treated with5 g/L G418 than those after being treated with 0 μmol/L(P<0.05),and showed no significant differences after being treated with 1 and 2 g/L G418 compared with those after being treated with 0 μmol/L(P>0.05). The relative expression of DDDDK mRNA was significantly higher after the 5 mutant cell lines were treated with 250 and 500 μmol/L Amleanox as well as 5 g/L G418 than that after being treated with 0 μmol/L(P<0.05), and showed no significant difference after being treated with 50 and 100 μmol/L PTC124 compared with that after being treated with 0 μmol/L(P>0.05). The relative expression of DDDDK mRNA in the 5 mutant cell lines after being treated with 0 μmol/L was lower than that in the wild-type cells(P<0.05).In the 5 mutant cell lines, PAH and DDDDK proteins were scarcely expressed after being treated with 0 μmol/L,50 and 100 μmol/L PTC124,and 250 and 500 μmol/L Amlexanox.The relative expressions of PAH and DDDDK proteins were higher in R111X,R176X,Y166X and Y356X mutant cell lines after being treated with 5 g/L G418 than those after being treated with 0 μmol/L(P<0.05),were higher after the W326X mutant cells were treated with 1,2 and 5 g/L G418 than those after being treated with 0 μmol/L, were higher after being treated with 1 and 2 g/L G418 than those after being with 5 g/L G418(P<0.05), and showed no significant differences between 1 and 2 g/L G418 treatments(P>0.05). Conclusions Amlexanox and G418 can increase the transcription levels of PAH gene in 5 nonsense mutant cells(R111X,Y166X,R176X,W326X,Y356X),in which G418enables the complete expression of PAH protein in these mutants.The effective concentraction of G418 is 1 g/L for correcting W326X nonsense mutation,and is 5 g/L for correcting the other 4 nonsense mutant cell lines.更多还原