中华医学遗传学杂志
中華醫學遺傳學雜誌
중화의학유전학잡지
CHINESE JOURNAL OF MEDICAL GENETICS
2011年
5期
496-500
,共5页
张如旭%杨茜%资晓宏%李小波%刘婷%刘三妹%李琳%占雅静%厉兰%夏昆%潘乾%唐北沙
張如旭%楊茜%資曉宏%李小波%劉婷%劉三妹%李琳%佔雅靜%厲蘭%夏昆%潘乾%唐北沙
장여욱%양천%자효굉%리소파%류정%류삼매%리림%점아정%려란%하곤%반건%당북사
腓骨肌萎缩症%热休克蛋白B1%神经丝轻链蛋白
腓骨肌萎縮癥%熱休剋蛋白B1%神經絲輕鏈蛋白
비골기위축증%열휴극단백B1%신경사경련단백
Charcot-Marie-Tooth disease%heat shock protein B1%neurofilament light chain protein
目的 观察HSPB1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-R127W HSPB1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-wt HSPB1 和EGFP-R127W HSPB1的胞内分布情况.pCL-NFL与pEGFPN1-wt HSPB1、pEGFPN1-R127W HSPB1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R127W HSPB1与NFL是否存在共定位.结果 EGFP-R127W HSPB1在胞浆内形成以核周分布为主的聚集物,EGFP-wt HSPB1在胞浆和胞核中均匀分布；NFL与wt HSPB1共表达时在胞浆形成均一结构,与R127W HSPB共表达时形成胞内聚合物,聚合物中NFL与R127W HSPB1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R127W HSPB在腓骨肌萎缩症的主要致病机理.
目的 觀察HSPB1的R127W突變蛋白在細胞內的分佈,對神經絲輕鏈(neurofilament light chain,NFL)蛋白自身組裝的影響和二者的共定位情況.方法 構建pEGFPN1-wt HSPB1和pEGFPN1-R127W HSPB1真覈錶達載體,瞬間轉染Hela細胞後在激光共聚焦顯微鏡下觀察EGFP-wt HSPB1 和EGFP-R127W HSPB1的胞內分佈情況.pCL-NFL與pEGFPN1-wt HSPB1、pEGFPN1-R127W HSPB1共轉染Hela細胞,通過間接免疫熒光技術在激光共聚焦顯微鏡下觀察NFL胞內錶達情況及R127W HSPB1與NFL是否存在共定位.結果 EGFP-R127W HSPB1在胞漿內形成以覈週分佈為主的聚集物,EGFP-wt HSPB1在胞漿和胞覈中均勻分佈；NFL與wt HSPB1共錶達時在胞漿形成均一結構,與R127W HSPB共錶達時形成胞內聚閤物,聚閤物中NFL與R127W HSPB1存在共定位.結論 HSPB1的R127W突變可能導緻蛋白異常聚集、神經元細胞骨架不能維持穩定和正確組裝.軸索細胞骨架的不穩定和物質轉運障礙可能是R127W HSPB在腓骨肌萎縮癥的主要緻病機理.
목적 관찰HSPB1적R127W돌변단백재세포내적분포,대신경사경련(neurofilament light chain,NFL)단백자신조장적영향화이자적공정위정황.방법 구건pEGFPN1-wt HSPB1화pEGFPN1-R127W HSPB1진핵표체재체,순간전염Hela세포후재격광공취초현미경하관찰EGFP-wt HSPB1 화EGFP-R127W HSPB1적포내분포정황.pCL-NFL여pEGFPN1-wt HSPB1、pEGFPN1-R127W HSPB1공전염Hela세포,통과간접면역형광기술재격광공취초현미경하관찰NFL포내표체정황급R127W HSPB1여NFL시부존재공정위.결과 EGFP-R127W HSPB1재포장내형성이핵주분포위주적취집물,EGFP-wt HSPB1재포장화포핵중균균분포；NFL여wt HSPB1공표체시재포장형성균일결구,여R127W HSPB공표체시형성포내취합물,취합물중NFL여R127W HSPB1존재공정위.결론 HSPB1적R127W돌변가능도치단백이상취집、신경원세포골가불능유지은정화정학조장.축색세포골가적불은정화물질전운장애가능시R127W HSPB재비골기위축증적주요치병궤리.
Objective To observe the cellular expression of R127W HSPB1 and its influence on neurofilament light chain (NFL) self-assembly 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-wt HSPB1,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 R127W HSPB1 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 R127W mutation of HSPB1 in the pathogenesis of Charcot-Marie-Tooth disease.
