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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。
Objective To explore the relationship between Nucleolin (NCL) and two known α-Synuclein and Park7 (Parkinson disease) genes related to Parkinson Disease (PD) in SH-SY5Y cells exposed to rotenone protein 7, also known as DJ-1, interacts with DJ-1 on α-synuclein (α-Syn). Methods MTT assay was used to determine the effect of rotenone (20, 100 and 500 nmol / L) on the cell viability of SH-SY5Y cells 24 h and 3 d. Immunofluorescent staining (IF) The proteins were observed under a confocal microscope and the spatial relationships among the three proteins were analyzed. Immunoprecipitation (IP) and Western Blot (WB) were used to detect the nucleolus Interaction with DJ-1 and α-Synuclein, respectively. Results The survival rate of SH-SY5Y cells treated with rotenone (20, 100 and 500 nmol / L) for 24 h was not significantly different from that of the control group (P> 0.05) After 20 nmol / L exposure, there was no significant difference in cell survival between the control group and the control group (P> 0.05), but there was significant difference between the 100 and 500 nmol / L groups (P <0.05). Laser scanning confocal analysis showed that DJ-1 and nucleolin were located in the nucleus and cytoplasm of SH-SY5Y cells, while α-Synuclein was located in the cytoplasm. Both nucleolin and nucleotides showed co-localization with DJ-1 and α-Synuclein. Before and after rotenone exposure to SH-SY5Y cells, nucleolin was co-immunoprecipitated with DJ-1 and α-Synuclein, respectively. Immunoblotting showed that both nucleolin and protein could form protein complexes. CONCLUSIONS: Nucleoprotein has a protein-protein interaction with both DJ-1 and α-Synuclein in SH-SY5Y cells. The interaction of nucleolin with DJ-1 and α-Synuclein after exposure to rotenone did not disappear, suggesting that nucleolus Supramids may be Parkinson’s disease-related proteins.