热休克蛋白40和热休克蛋白70抑制N2a细胞内突变亨廷顿蛋白聚积和毒性

HSP40 and HSP70 inhibit the accumulation and toxicity of mutant huntingtin in N2a cells

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＆lt;0.01,n=3）,同时减少ROS产生（P＆lt;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 study the effect of heat shock protein 40（HSP40） and heat shock protein 70（HSP70） on the aggregate formation of mutant huntingtin （htt） and its toxicity in N2a cells. Methods The effect of the over-expression of HSP40 and HSP70 alone or co-over-expression of them on aggregate formation of transfected mutant htt （containing 150 glutamine repeats, 150Q） in N2a cells was detected by fluorescence microcopy and Western blotting. Cell viabilities were detected by MTT assay. Reactive oxygen species （ROS） production was detected by colorimetric method. Results The over-expression of HSP40 and HSP70 alone, especially the co-over-expression of them, dramatically decreases the formation of 150Q htt aggregates in N2a cells. The numbers of aggregates in each group are as follows （ n = 1 000） ： about 50% in the group of 150Q htt-expressing alone, about 12% in the group of over-expression of HSP40, about 15% in the group of over-expression of HSP70, about 5% in the group of co-over-expression of HSP40 and HSP70. The result of MTr assay shows that the over-expression of HSP40 and HSP70 alone, especially the co-over-expression of them, dramatically increases cell viabilities （P 〈 0.01, n = 3） and reduces the production of ROS （P 〈 0.01, n = 3）. Conclusion HSP40 and HSP70 can increase cell viabilities of 150Q N2a cells via inhibiting the aggregates formation of mutant huntingtin and reducing oxidative stress.