【摘要】 目的探索在鱼藤酮(Rotenone)染毒的SH-SY5Y细胞中,核仁素(Nucleolin,NCL)与两种已知与帕金森病(Parkinson Disease,PD)相关的基因α-Synuclein和PARK7(Parkinson disease protein 7,又称DJ-1)编码的蛋白α-突触核蛋白(α-Synuclein,α-Syn)和DJ-1的相互作用。方法采用MTT法检测鱼藤酮(20、100和500 nmol/L)染毒SH-SY5Y细胞24 h和3 d后对细胞存活率的影响;采用免疫荧光染色法(Immunofluorescent staining,IF)分别染色3种蛋白,在共聚焦显微镜下观察蛋白的亚细胞定位,分析3种蛋白在空间上的关系;采用免疫沉淀法(Immunoprecipitation,IP)和免疫蛋白印记(Western Blot,WB)法检测染毒前后核仁素分别与DJ-1和α-Synuclein的相互作用。结果用鱼藤酮(20、100和500 nmol/L)染毒SH-SY5Y细胞24 h后,各个染毒组细胞存活率改变与对照组比较差异没有统计学意义(P>0.05);染毒3 d后20 nmol/L染毒组细胞存活率改变与对照组比较差异没有统计学意义(P>0.05),而100和500 nmol/L染毒组细胞存活率改变与对照组比较差异有统计学意义(P<0.05)。激光共聚焦分析显示DJ-1和核仁素分布于SH-SY5Y细胞核和细胞质中,而α-Synuclein分布于细胞质中,核仁素与DJ-1和α-Synuclein都有共定位。鱼藤酮染毒SH-SY5Y细胞前后,核仁素分别与DJ-1和α-Synuclein免疫共沉淀后,免疫蛋白印迹结果显示核仁素与二者均可形成蛋白复合体。结论在SH-SY5Y细胞中核仁素与DJ-1和α-Synuclein都存在蛋白-蛋白相互作用,染毒鱼藤酮后核仁素与DJ-1和α-Synuclein的相互作用并没有消失,提示核仁素可能是帕金森病相关蛋白。更多还原

【Abstract】 Objective To identify nucleolin as a protein interacting with α-Synuclein and DJ-1,two critical proteins involved in PD pathogenesis by SH-SY5 Y celluar model exposed to rotenone. Methods The effect of cell proliferation were detected by MTT,following rotenone( 20,100,500 nmol / L) exposure for 24 h and 72 h in SH-SY5 Y cells. The protein interaction and collocation were identified by Immunofluorescence staining( IF) and Immunoprecipitation / Western Blot( IP / WB) respectively. Results After exposure to rotenone in100 and 500 nmol / L for 72 h,the cell proliferation had a significant change compared with control( P < 0. 05),however,It had not been found the significance in 20 nmol / L group exposure for 72 h and in 20,100,500 nmol / L exposure for 24 h,comparatively. The results of IP / WB and IF indicate that nucleolin could form protein complex with DJ-1,as well as α-Synuclein. Conclusion Nucleolin has interaction with α-Synuclein and DJ-1 in both SH-SY5 Y cells and rotenone-treated celluar model of PD. This undefined protein may be related with PD.更多还原