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目的研究热休克蛋白40(HSP40)和热休克蛋白70(HSP70)对N2a细胞内突变亨廷顿蛋白(htt)聚集物形成和毒性的影响。方法应用荧光显微镜术和免疫印迹技术检测单独或共同过表达HSP40和HSP70对N2a细胞内转染的突变htt(含有150个谷氨酰胺重复数,150Q)聚集物形成的影响;应用四甲基偶氮唑盐(MTT)法分析细胞活力和比色法检测细胞内活性氧(ROS)含量。结果单独过表达HSP40或HSP70,尤其是共同过表达HSP40和HSP70显著减少150Q htt在N2a细胞内的聚积,各组含有聚集物的细胞为(n=1 000):仅表达150Q htt组约50%,过表达HSP40组约12%,过表达HSP70组约15%,共同过表达HSP40和HSP70组约5%。MTT分析结果显示,单独尤其是共同过表达HSP40和HSP70能显著增加150Q细胞的活力(P<0.01,n=3),同时减少ROS产生(P<0.01,n=3)。结论HSP40和HSP70通过抑制细胞内突变htt聚积以及减少氧化应激而增加150Q N2a细胞活力。
Objective To investigate the effects of heat shock protein 40 (HSP40) and heat shock protein 70 (HSP70) on the formation and toxicity of mutated Huntington protein (htt) aggregates in N2a cells. Methods Fluorescence microscopy and Western blotting were used to detect the effect of HSP40 and HSP70 alone or co-overexpression on the formation of aggregates of mutant htt (containing 150 glutamine repeat, 150Q) transfection in N2a cells. Cell viability was assayed by MTT assay and intracellular reactive oxygen species (ROS) levels were measured by colorimetric assay. Results Overexpression of HSP40 or HSP70, especially co-overexpression of HSP40 and HSP70, significantly reduced 150Q htt accumulation in N2a cells (n = 1000): only about 50% , About 12% overexpression HSP40 group, about 15% overexpression HSP70 group, about 5% overexpression of HSP40 and HSP70 group. The results of MTT assay showed that overexpression of HSP40 alone and especially HSP70 could significantly increase the viability of 150Q cells (P <0.01, n = 3) and ROS production (P <0.01, n = 3). Conclusion HSP40 and HSP70 can increase the viability of 150Q N2a cells by inhibiting the accumulation of mutant intracellular htt and reducing oxidative stress.