CAJ | 학술논문

目的探讨坏死性凋亡信号通路中受体相瓦作用蛋白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(RIPI)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)处理小同时间点,western blot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RIP1,检测胞浆和胞核中RIP3的蛋白水平变化以及相应的RIP1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(胞浆分另0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±0.02,0.57±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RIP1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,RIP1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.
목적 탐토배사성조망신호통로중수체상와작용단백3(RIP3)적포내정위이급기재해신호통로중적작용,병진일보연구RIP3핵이위시부여수체상호작용단백1(RIPI)존재상관성.방법 SD대서원대피질신경원세포배양12d,zVAD예처리30min.(1)종류배사인자(TNFα)처리소동시간점,western blot검측포질、포핵중RIP3단백수평,결합면역형광관찰RIP3적포내정위.(2)응용Nec-1급만병독간우RIP1,검측포장화포핵중RIP3적단백수평변화이급상응적RIP1표체수평적변화,병검측각조배양기중유산탈경매(LDH)함량변화.결과 (1)재배사성조망신호통로중,수착TNF작용시간적연장,RIP3단백함량축점증가,포질중단백함량재8h체고봉,수후하강,동시포핵중단백수평재12h출현핵전위적고봉(포장분령0위0.45 ±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;포핵분별위0.07±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),차이유현저성(P<0.01).(2)응용Nec-1이급만병독간우RIP1표체후,발생핵전위적RIP3감소,차LDH수평교전강저(1.00±0.05,0.39±0.03,0.50±0.03),차이유현저성(P<0.01).결론 정상세포중RIP3존재우포질,배사성조망시발생핵이위,RIP1시RIP3발생핵전위필불가소적상관단백,차조단RIP3핵이위가이대항세포조망.
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 pre-treated with zVAD-fmk(20μ,mol/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 Nec-1 and shRlPl ,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 a-chieved 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 necrostatin-1 and shRIPl , 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 cy-tolymph 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.