摘要
目的观察HSPBI的R127W突变蛋白在细胞内的分布，对神经丝轻链（neurofilament light chain，NFL）蛋白自身组装的影响和二者的共定位情况。方法构建pEGFPN1-wt HSPB1和pEGFPN1-R127WHSPB1真核表达载体，瞬间转染Hela细胞后在激光共聚焦显微镜下观察EGFP-wtHSPB1和EGFP-R127WHSPBl的胞内分布情况。pCL—NFL与pEGFPN1wtHSPB1、pEGFPN1-R127WHSPB1共转染Hela细胞，通过间接免疫荧光技术在激光共聚焦显微镜下观察NFL胞内表达情况及R127WHSPB1与NFL是否存在共定位。结果EGFP-R127WHSPB1在胞浆内形成以核周分布为主的聚集物，EGFP-wtHSPB1在胞浆和胞核中均匀分布；NFL与wtHSPB1共表达时在胞浆形成均一结构，与R127WHSPB共表达时形成胞内聚合物，聚合物中NFL与R127WHSPB1存在共定位。结论HSPB1的R127W突变可能导致蛋白异常聚集、神经元细胞骨架不能维持稳定和正确组装。轴索细胞骨架的不稳定和物质转运障碍可能是R127WHSPB在腓骨肌萎缩症的主要致病机理。
Objective To observe the cellular expression of R127WHSPB1 and its influence on neurofilament light chain （NFL） self-assemhly and co localization with NFL. Methods Eukaryotic expression vectors pEGFPN1-wt HSPB1 and pEGFPN1-R127W HSPB1 were constructed. Hela cells were transiently transfected with pEGFPN1-wt HSPB1 or pEGFPN1-R127W HSPB1 and observed under a confocal microscope. Hela cells were also transiently co-transfected with pCL-NFL and pEGFPN1-wt HSPB1, or pCL-NFL and pEGFPN1 R127W HSPB1. The self-assembly of NFL was observed and the co-localization study of HSPB1/R127W HSPB1 with NFL was carried out in these two cell models by immunofluorescence technique. Results The aggregates formed by EGFP -R127W HSPB1 predominantly located around the nucleus, and EGFP-wt, HSPB1 showed diffusion pattern in Hela cells. When co-expressed with EGFP- wtHSPB1, NFL formed homogeneous structure in cytosol. When co-expressed with EGFP-R127W HSPB1, however, NFL had amorphous staining pattern predominantly consisting of NFL aggregates, and NFL colocalized with R127WHSPB1 in these aggregates. Conclusion The R127W mutant of HSPB1 may have reduced capacity to serve as a chaperone to prevent aggregate formation, and fail to correctly organize the neurofilament network. Dysfunction of the axon cytoskeleton and axon transport may be the primary mechanism of R127TW mutation of HSPB1 in the pathogenesis of Charcot-Marie-Tooth disease.
出处
《中华医学遗传学杂志》
CAS
CSCD
北大核心
2011年第5期496-500,共5页
Chinese Journal of Medical Genetics
基金
基金项目：国家自然科学基金（81071001、30600200）
湖南省自然科学基金（2006JJ30009）
关键词
腓骨肌萎缩症
热休克蛋白B1
神经丝轻链蛋白
Charcot-Marie-Tooth disease
heat shock protein B1
neurofilament light chainprotein
