摘要
目的探讨阻断CXC趋化因子10(CXCL10)在神经母细胞瘤细胞(N2a)氧糖剥夺(OGD)模型中对toll样受体4(TLR4)/核因子κB(NF-κB)通路调节炎症的影响。方法应用脂质体转染法将CXCL10基因转染至小鼠神经瘤母细胞,采用实时定量荧光PCR(RT-PCR)与Western blot法检测转染后CXCL10的基因和蛋白表达水平;实验分为未传染组、OGD/R组、OGD/R+NC组、OGD/R+CXCL10 siRNA组、OGD/R+CXCL10siRNA+LPS组、OGD/R+TAK242组,细胞转染24 h后建立OGD模型,3 h后予以复氧;复氧24 h后,RT-PCR法检测肿瘤坏死因子-α、白细胞介素-1β、白细胞介素-2基因表达水平,应用Western-blot法检测细胞CXCL10、TLR4、NF-κB p65、p-NF-κBp65、cleaved-caspase3蛋白表达水平。结果 RT-PCR与Western blot结果显示,OGD损伤细胞后,TLR4被激活,进而激活NF-κB信号通路,导致炎症因子表达。CXCL10基因沉默降低了相关炎症因子的表达,并抑制了N2a细胞的TLR4、P-NF-κBp65和caspase3的蛋白表达。沉默CXCL10基因加入TLR4激动剂可以促进炎症因子表达及相关信号通路的蛋白表达。结论 CXCL10对OGD损伤有保护作用,可能通过TLR4/NF-κB信号通路实现。
Objective To investigate the effect of blocking CXC chemokine 10(CXCL10) on regulating inflammation with toll-like receptor 4(TLR4)/nuclear factor kappa B(NF-κB) in neuroblastoma cell(N2 a) oxy-sugar deprivation(OGD) model.Methods Liposome transfection method was used to transfect CXCL10 gene into mouse neuroblastocytes.Real-time quantitative fluorescence PCR(RT-PCR) and Western blot were used to detect the gene and protein expression levels of CXCL10 after transfection.The experiments were divided into the control group,OGD/R group,OGD/R+NC group,OGD/R+CXCL10 siRNA group,OGD/R+CXCL10 siRNA+LPS group and OGD/R+TAK242 group.After 24 hours of reoxygenation,the expression levels of tumor necrosis factor-α,interleukin-1β and interleukin-2 genes were detected by RT-PCR,and the expression levels of CXCL10,TLR4,NF-κB p65,p-NF-κB p65 and cleaved-caspase3 were detected by Western-blot.Results RT-PCR and Western blot results showed that after OGD damaged the cells,TLR4 was activated and then activated the NF-κB signaling pathway,leading to the expression of inflammatory factors.Silence of CXCL10 gene reduced the expression of related inflammatory factors and inhibited the protein expressions of N2 a cells such as TLR4,P-NF-κB p65 and caspase3.Silencing CXCL10 gene by adding TLR4 agonist could promote the expression of inflammatory cytokines and proteins of related signaling pathways.Conclusion CXCL10 has protective effect on OGD injury,possibly through the TLR4/NF-κB signaling pathway.
作者
仇靖
李岩松
王敏
姚奔驰
郑军
QIU Jing;LI Yan-song;WANG Min;YAO Ben-chi;ZHENG Jun(Department of Neurology,General Hospital of Northern Theater Command,Shenyang 110016, China)
出处
《临床军医杂志》
CAS
2019年第8期776-780,共5页
Clinical Journal of Medical Officers
