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�
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(0μmol/L),PTC124(50,100μmol/L),Amlexanox(250,500μmol/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 0μmol/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 100μmol/L PTC 124 for 72 h.After being treated with 500μmol/L Amlexanox for 72 h,the proliferation rates of W326X and Y356X mutant cells were lower than those after being treated with 0μmol/L(P<0.05),while the proliferation rates of the other mutant cell lines were not affected by 250 and 500μmol/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 0μmol/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 0μmol/L(P<0.05).(2)The relative expressions of DDDDK mRNA and PAH and DDDDK proteins were higher after the wild
作者
侯丽青
侯东霞
冀云鹏
朱博
周燕
董弘
刘小霞
王晓华
HOU Liqing;HOU Dongxia;JI Yunpeng;ZHU Bo;ZHOU Yan;DONG Hong;LIU Xiaoxia;WANG Xiaohua(Department of Genetics and Eugenics,Inner Mongolia Maternal and Child Health Care Hospital,Hohhot,Inner Mongolia Autonomous Region O10021,China;School of Public Health,Baotou Medical College of Inner Mongolia University of Science&Technology,Baotou,Inner Mongolia Autonomous Region 014000,China)
出处
《中华实用诊断与治疗杂志》
2023年第8期802-808,共7页
Journal of Chinese Practical Diagnosis and Therapy
基金
国家自然科学基金(81860168)
内蒙古自治区自然科学基金(2016MS0858)。
