受体相互作用蛋白3在大鼠皮质神经元坏死性凋亡信号通路中的作用  

Role of RIP3 in necroptosis signaling pathways of cortical neurons

作　　者：陈巍巍[1] 张翠翠[1] 程言博[1] 徐兴顺[1] 耿德勤 

机构地区：[1]苏州大学第一附属医院 [2]附属医院神经内科

出　　处：《中华行为医学与脑科学杂志》2011年第6期481-484,共4页Chinese Journal of Behavioral Medicine and Brain Science

基　　金：国家自然科学基金资助项目（30700245）;江苏省政府重点实验室开放课题资助项目（Jsb10703）

摘　　要：目的探讨坏死性凋亡信号通路中受体相互作用蛋白3（RIP3）的胞内定位以及其在该信号通路中的作用，并进一步研究RIP3核移位是否与受体相互作用蛋白1（RIP1）存在相关性。方法SD大鼠原代皮质神经元细胞培养12d，zVAD预处理30min。（1）肿瘤坏死因子（TNFα）处理不同时间点，westernblot检测胞质、胞核中RIP3蛋白水平，结合免疫荧光观察RIP3的胞内定位。（2）应用Nec-1及慢病毒干扰RIPl，检测胞浆和胞核中RIP3的蛋白水平变化以及相应的RIPl表达水平的变化，并检测各组培养基中乳酸脱氢酶（LDH）含量变化。结果（1）在坏死性凋亡信号通路中，随着TNF作用时间的延长，RIP3蛋白含量逐渐增加，胞质中蛋白含量在8h达高峰，随后下降，同时胞核中蛋白水平在12h出现核转位的高峰（胞浆分别为0．45±0．03，0．41±0．02，0．73±0．03，0．90±0．01，1．15±0．04，1．30±0．02，0．99±0．03，0．63±0．03；胞核分别为0．07±0．02，0．26±0．02，0．574-0．02，0．68±0．02，0．80±0．01，0．92±0．02，1．28±0．03，0．87±0．02），差异有显著性（P〈0．01）。（2）应用Nec-1以及慢病毒干扰RIPl表达后，发生核转位的RIP3减少，且LDH水平较前降低（1．00±0．05，0．39±0．03，0．50±0．03），差异有显著性（P〈0．01）。结论正常细胞中RIP3存在于胞质，坏死性凋亡时发生核移位，RIPl是RIP3发生核转位必不可少的相关蛋白，且阻断RIP3核移位可以对抗细胞凋亡。Objective To investigate the location of receptor interacting protein 3 （RIP3） in Necroptosis and its function in this signal passage, and explore the relationship between receptor interacting protein 1 （ RIP1 ） and RIP3 in nuclear translocation. Methods Primary cerebrocortical neurons were cultured for 12 days, then pretreated with zVAD-fmk（20μmol/L） for half an hour to block apoptosis. ①Extracting nuclear and cytoplasmic protein after neurons were exposed to TNF for different time , then protein levels of RIP3 were analyzed by western blot and immunofluorescence for qualitative observation;②In the following research, the neurons were treated with Nee-1 and shRIP1 , then the protein level of RIP1 and RIP3 with western blot were analyzed, cell viability were determined by measuring LDH levels. Results ①In signaling pathways of necroptosis, the protein level of RIP3 in cytoplasmic decreased gradually with prolonged TNF exposure, to the corresponding it rolled up in nucleus and achieved the peak in 12 hours of TNF treatment（ Cytoplasmic 0.45 ±0.03,0.41±0.02,0.73 ± 0.03,0.90 ± 0.01,1.15 ±0.04,1.30± 0.02,0.99 ±0.03,0.63 ±0.03 ; Nucleus 0.07±0.02,0.26±0.02,0.57 ± 0.02,0. 68 ±0. 02,0.80 ± 0.01,0.92 ± 0.02,1.28 ± 0.03,0.87 ± 0.02） （P 〈 0.01 ）. ②Blocking the relationship between RIP1 and RIP3 with neerostatin-I and shRIP1, nuclear translocation of RIP3 decreased and caused a great increase in cell viability（1.00±0. 05,0.39 ±0.03,0.50±0.03） （P〈0.01）. Conclusion RIP3 mainly locates in cytolymph of normal cells,it translocates into nucelus as necroptosis takes place. RIP1 function with RIP3 in nuclear translocation. Block nuclear translocation of RIP3 is a potential way to protect cells.

关 键 词：受体相互作用蛋白3 受体相互作用蛋白1 坏死性凋亡 

分 类 号：R743.3[医药卫生—神经病学与精神病学]