| Phenylketonuria?PKU?is an autosomal recessive disorder caused by abnormal metabolism of phenylalanine?Phe?.PKU is widely distributed in the world and has a high incidence,which will seriously affect patients'cognition and psychology.At present,PKU patients are mainly controlled by diet therapy,which not only reduces the quality of life of patients,but also brings great economic burden to society.Therefore,new therapies are needed clinically.The cause of some PKU patients is a nonsense mutation of phenylalanine hydroxylase?PAH?,which can be corrected by a new compound PTC124 in some experiments.In this study,HEK293 cells transfected with nonsense mutation PAH gene(Y356XPAH and W326XPAH)vectors were treated by PTC124,and the changes of PAH expression at transcriptional and translation levels were detected.The corrective effects of PTC124 on the expression of Y356XPAH and W326XPAH were explored in order to provide a new strategy for the treatment of PKU.1.Constructing related vectorsIn this study,eukaryotic expression vectors were successfully constructed by genetic engineering technology.One type was with red fluorescent markers:pCMV-DsRed-PAH,pCMV-DsRed-Y356XPAH and pCMV-DsRed-W326XPAH,the other was with G418 markers:pcDNA3.1-PAH,pcDNA3.1-Y356XPAH and pcDNA3.1-W326XPAH.2.Identification of expression system in vitroHEK293 cells were selected to express vector genes through literature review.After purchasing cell lines,HEK293 cells were expanded by conventional cell culture technology,and the expression of PAH gene was preliminarily analyzed.No PAH gene expression was detected,which proved that HEK293 cells met the experimental requirements.3.Vector transfection and cell screeningIn this study,the vector gene was transfected into HEK293 cells by liposome transfection.ThecellstransfectedwithpCMV-DsRed-PAH,pCMV-DsRed-Y356XPAH and pCMV-DsRed-W326XPAH were detected by flow cytometry.The transfection efficiency reached 72%.The cells transfected with pcDNA3.1-PAH,pcDNA3.1-Y356XPAH and pcDNA3.1-W326XPAH were screened by G418.The surviving cells were positive cells.4.Determination of PTC124 concentrationHEK293 cells were treated with PTC124 with concentrations of 0?M,50?M,100?M and 200?M respectively.Cell viability,cell cycle,cell apoptosis and cell proliferation were analyzed.It was found that PTC124 with concentration of 50?M and 100?M was relatively safe for cells.PTC124 with concentration of 200?M had negative effects on cell viability and proliferation,blocked cell in S phase and promoted cell apoptosis.Finally,100?M was chosen as the treatment concentration of PTC124 in the follow-up experiment.5.Detection of each experimental groupHEK293 cells were respectively transfected with pCMV-DsRed-PAH,pCMV-DsRed-Y356XPAH,pCMV-DsRed-W326XPAH,pcDNA3.1-PAH,pcDNA3.1-Y356XPAH and pcDNA3.1-W326XPAH,and then divided into two parts.The control group was transfected with PAH gene and cultured with normal mathod.The experimental group was transfected with nonsense mutation PAH gene,and the other part was treated with PTC124 with a concentration of 100?M was used for treatment.After 48 hours,qPCR and Western Blot were detected respectively.The results of qPCR showed that PAH was expressed in all experimental groups.The expression of nonsense mutation PAH gene in corrected culture was significantly higher than that in normal culture,but still lower than that in transfected PAH gene.Western Blot experiment showed that PAH protein was successfully expressed in all experimental groups.After corrected culture,PAH increased in nonsense mutation group.The activity of wild type and nonsense mutant PAH was detected by enzyme activityassay.ThecellsweretransfectedwithpcDNA3.1-PAH,pcDNA3.1-Y356XPAH and pcDNA3.1-W326XPAH vectors respectively,and the cells transfected with PAH gene were cultured in normal culture as positive reference.The cells transfected with nonsense mutant PAH gene were cultured in normal culture and corrected by 100?M PTC124 respectively.It was found that the cells transfected with wild-type PAH gene synthesized active PAH.In the cells transfected with pcDNA3.1-Y356XPAH and pcDNA3.1-W326XPAH,the cells were cultured without detection of active PAH and corrected with PTC124 to synthesize active PAH.Compared with the cells transfected with wild-type PAH gene,the activity of transfected vector pcDNA3.1-Y356XPAH reached 19%.The activity of pcDNA3.1-W326XPAH reached 13%.In summary,PTC124 can play a corrective role in the expression of nonsense mutation PAH genes Y356XPAH and W326XPAH,so that it can synthesize active PAH proteins. |
PDF Full Text Request