CRX基因突变影响视紫红质转录表达和NRL蛋白稳定性的细胞学研究

摘要

目的 在细胞水平上,探讨存在和缺乏神经视网膜亮氨酸拉链(NRL)的条件下,视锥-视杆细胞同源异形框基因(CRX)突变型对调控视紫红质基因转录表达的影响,并分析CRX突变型对CRX和NRL蛋白稳定性的影响.方法 实验研究.分别构建携带野生型和c.C766T (p.Gln256X)无义突变型的CRX基因,以及共表达NRL基因和牛视紫红质基因启动子萤光素酶(pBR130-1uc)的表达载体,分别转染体外培养的293T细胞和ARPE-19细胞,再行双荧光素酶检测分析和Western blotting实验.以持家蛋白GAPDH作为内参照.结果 与野生型CRX蛋白的表达导致牛视紫红质启动子表达5倍的激活率相比,CRX/c.C766T(p.Q256X)突变所造成的相应激活率降至1/4.共表达野生型CRX和NRL基因后,激活率增高至30倍,而共转染CRX/c.C766T和NRL后,牛视紫红质蛋白的活性降至1/13.以持家蛋白GAPDH作为内参,可见存在CRX/c.C766T突变时,CRX蛋白的稳态水平大幅下降,且CRX/c.C766T突变型对NRL蛋白的稳态有一定的影响,出现NRL增高的现象.结论 在细胞水平上,CRX基因的c.C766T(p.Q256X)突变可降低所调控的视紫红质蛋白的表达,并对CRX蛋白和NRL蛋白的稳态均造成一定的影响.%Objective To investigate the effect of cone-rod homeobox containing gene (CRX) mutation on the transcriptional expression of the rhodopsin gene and the stability of the neural retina leucine zipper (NRL) protein,both in the presence or absence of the NRL gene.Methods In this experimental study,different expression vectors carrying the wild-type (WT) or the c.C766T (p.Q256X)nonsense mutant CRX were constructed with the NRL and the bovine rhodopsin gene promoter sub-luciferase (pBR130-1uc).The vectors were then transfected into 293T and ARPE-19 cell lines,respectively.Then,dual-luciferase assays and Western blotting analyses were performed The housekeeping protein glyceraldehyde-3-phosphate dehydrogenase was used as an internal reference marker.Results The expression vector of CRX/WT caused a 5-fold increased activation of bovine rhodopsin promoter compared to a 25％ decrease by CRX/c.C766T.Co-expression of CRX/WT and NRL increased activation by 30-fold compared to a 7.7％ decrease by the co-expression of CRX/c.C766T and NRL.Also,expression of NRL protein increased with the co-expression of CRX/c.C766T and NRL.Conclusion The in vitro experiments suggest that the c.C766T (p.Q256X) mutant of the CRX gene down-regulated the transcriptional expression of rhodopsin and interfered with the homeostasis of CRX and NRL proteins.