摘要
目的探讨使用慢病毒(LV-P2rx4)沉默P2X4嘌呤受体(P2X4R)对活化状态小胶质细胞释放肿瘤坏死因子α(TNF-α)的影响。方法常规建立脂多糖(LPS)和三磷酸腺苷(ATP)双信号诱导的小胶质细胞活化模型,随机分为(1)空白对照组(常规DMEM培养液培养);(2)活化模型组(细胞种板后待密度接近80%后先给予LPS 1μg·m L-1处理6 h后弃原培养液,再加ATP 2 mmol·L-1处理3 h);(3)目的基因阴性对照组(种板1 d,先加入病毒处理12 h后弃培养液,再加入常规培养液,间隔12 h更换培养液);(4)目的基因阴性对照+活化模型组(种板1 d后,先加入病毒处理12 h后弃培养液,再加常规培养液,期间每隔12 h更换培养液,待细胞密度达80%后,予LPS 1μg·m L-1处理6 h后弃原培养液,再加ATP 2 mmol·L-1处理3 h);(5)空载病毒阴性对照组(处理方法同目的基因阴性对照组);(6)空载病毒阴性对照+活化模型组(处理方法同目的基因阴性对照+活化模型组)。各组均n=6。细胞处理后每组取3孔分别提取核糖核酸(RNA)和蛋白,应用Real time PCR和Western blot法分别检测NLRP3、TNF-α的mRNA及蛋白的相对表达。结果与活化模型组和空载病毒阴性对照+活化模型组比较,目的基因阴性对照+活化模型组TNF-αmRNA相对表达水平明显减少(P <0. 001,F=195. 4),TNF-α蛋白相对表达水平显著降低(P <0. 001,F=60. 10)。结论沉默小胶质细胞中的P2X4R基因,可下调细胞中TNF-α表达,继而可能削弱小胶质细胞的炎症反应。
Aim To investigate the effect of lentivirus( LV-P2rx4) silencing P2X4 receptor( P2X4R) on the release of tumor necrosis factor alpha( TNF-α) in activated microglia. Methods The models of microglia activation induced by dual-signaling of lipopolysaccharide( LPS) and adenosine triphosphate( ATP) were routinely established,and randomly divided into the following groups:(1)Blank control group( n = 6,conventional DMEM culture medium);(2)Activation model group( n = 6,when cell seed plate was close to 80%,first treated with 1 μg·m L^-1 LPS for 6 h,discarded the original culture solution,and then ATP 2 mmol·L^-1 was added for 3 h);(3)Target gene negative control group( n = 6;seed plate for 1 day,first treated with virus for 12 h,then discarded the culture solution,and then added the conventional culture solution,changed the culture solution at intervals of 12 h;(4)Target gene negative control plus activation model group( n = 6,after seeding for 1 day,first treated with virus for 12 h,then discarded the culture solution,and then added the normal culture solution,and changed the culture solution every 12 h,until the cell density reached 80%,treated with 1 μg·m L^-1 LPS for 6 h,then discarded the original culture solution,and treated with 2 mmol·L^-1 ATP for 3 h;(5)Empty virus negative control group( n = 6,the treatment method is the same as the target gene negative control group);(6)Empty virus negative control plus activation model group( n = 6,the treatment method is the same as the target gene negative control plus activation model group). After the treatment,RNA and proteins were extracted from each well by 3 wells. The relative expression of NLRP3 and TNF-α mRNA and protein were detected by real time PCR and Western blot. Results Compared with the activation model group and the empty virus negative control plus activation model group,the relative expression level of TNF-α mRNA in the target gene negative control plus activation model group was significantly decreased( P < 0. 001,F =195. 4),and the relative expression level of TNF-α protein significantly decreased( P < 0. 001,F =60. 10). Conclusion Silencing the P2X4R gene in microglia can down-regulate TNF-α expression in cells,which in turn may impair the inflammatory response of microglia.
作者
许文帅
薛莉
蔡敏
谢安木
XU Wen-shuai;XUE Li;CAI Min;XIE An-mu(Department of Neurology,the Affiliated Hospital of Qingdao University,Qingdao 266003,China;Department of Medicine,the Affiliated Hospital of Qingdao University,Qingdao 266003,China)
出处
《中国临床神经科学》
2019年第1期1-8,共8页
Chinese Journal of Clinical Neurosciences
基金
国家自然科学基金(编号:81571225)
