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目的探讨轴突型腓骨肌萎缩症2L型(axonal Charcot-Marie-Tooth disease type 2L,CMT2L)致病基因小分子热休克蛋白HSPB8(smallheatshockproteinHSPB8,HSPB8)的K141N突变导致细胞内聚集物形成的可能机理。方法建立pEGFPN1-HSPB8、pEGFPN1-K141NHSPB8瞬时表达细胞模型,并进行EGFP-K141NHSPB8与小分子热休克蛋白HSPB1(smallheatshockproteinHSPB1,HSPB1)、神经丝轻链(neurofilamentlightchain,NEFL)的免疫荧光共定位分析,观察EGFP-K141NHSPB8在不同内源性表达细胞系的聚集物形成情况,采用t检验和单因素方差分析的统计学方法分析聚集物形成的可能机理。结果EGFP-K141NHSPB8形成以核周分布为主的聚集物,EGFP-K141NHSPB8与HSPB1、NEFL均存在免疫荧光共定位。EGFP-K141NHSPB8在不同内源性表达细胞系的聚集物形成百分率的差异有统计学意义。结论突变型HSPB8(K141N)形成以核周分布为主的胞内聚集物,聚集物中K141NHSPB8与HSPB1、NEFL均存在共定位。聚集物形成的可能机理包括K141NHSPB8多肽链构象发生改变后不能维持稳态而出现自身异常聚集;与家族内其他成员特别是HSPB1结合成异常的异源多聚体,在胞内形成不可溶性大分子后产生聚集。
Objective To investigate the possible mechanism of the K141N mutation in the small heat shock protein HSPB8 (HSPH8) of axonal Charcot-Marie-Tooth disease type 2L (CMT2L) . Methods The transient expression cell models of pEGFPN1-HSPB8 and pEGFPN1-K141NHSPB8 were established. Immunofluorescent co-localization analysis of EGFP-K141NHSPB8 and small heat shock protein HSPB1 (HSPB1) and neurofilament light chain (NEFL) -K141NHSPB8 aggregates formation in different endogenously expressed cell lines, the possible mechanism of aggregates formation was analyzed using t-test and one-way analysis of variance using statistical methods. Results EGFP-K141NHSPB8 formed perinuclear distribution of aggregates, EGFP-K141NHSPB8 and HSPB1, NEFL exist immunofluorescence co-localization. The difference in the percentage of EGFP-K141NHSPB8 aggregates formation between different endogenously expressed cell lines was statistically significant. Conclusions Mutant HSPB8 (K141N) forms intracellular aggregates with perinuclear distribution. Co-localization of K141NHSPB8 with HSPB1 and NEFL exists in the aggregates. Possible mechanisms for the formation of aggregates include that the K141NHSPB8 polypeptide chain can not maintain its homeostasis after being altered in its conformation; heterogeneous multimers that bind abnormally with other members of the family, especially HSPB1, form insoluble intracellular macromolecules After the accumulation.