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作 者:赵娜[1] 吕雅素[2] 陆勤康[1] 王红婷[1] 王惠云[1] 童奇湖[1] 张咸宁[2]
机构地区:[1]宁波市鄞州人民医院眼科中心宁波大学医学院附属鄞州医院眼科中心,315040 [2]浙江大学医学部细胞生物学研究所,310058
出 处:《中华眼视光学与视觉科学杂志》2017年第1期47-52,共6页Chinese Journal Of Optometry Ophthalmology And Visual Science
基 金:浙江省自然科学基金(LYl2H12001);宁波市社会民生科技重大项目(2014C50091);浙江省医药卫生科技项目(2017KY616);宁波市鄞州区科技项目(鄞科2013-90)
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-luc)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-luc). 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.
关 键 词:视锥-视杆细胞同源异形框基因 神经视网膜亮氨酸拉链 2型视锥-视杆细胞营养不良 遗传性视网膜变性 双荧光素酶实验 蛋白质印迹法
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