siRNA沉默P_2X_4表达对永生化小鼠小胶质细胞活化的抑制效应

Effect of P_2X_4siRNA on immortalized mice microglia

目的:探讨特异性P2X4 siRNA对永生化小鼠小胶质细胞活化的抑制效果.方法:体外培养永生化小鼠小胶质细胞,随机分为空白对照组、阴性对照组、转染siRNA-F1组.空白对照组未进行任何干预;阴性对照组转染Ncontrol siRNA;转染siRNA-F1组转染先前筛选出来的1条特异性siRNA.3组细胞分别用50μmol/LATP刺激,采用激光扫描共聚焦显微镜检测胞内钙离子浓度([Ca2+]i);P2X4R/OX42免疫荧光双标染色观察细胞形态.结果:转染siRNA-F1组ATP活化小胶质细胞后,胞内钙离子释放较阴性对照组和空白对照组减少;P2X4R/OX42免疫荧光双标染色显示转染siRNA-F1组细胞存在活化态小胶质细胞和静息态小胶质细胞,而空白对照组和阴性对照组只存在活化态小胶质细胞.结论:siRNA-F1能部分抑制永生化小鼠小胶质细胞的活化.

AIM： To investigate the inhibitory effect of P2X4siRNA on immortalized mice microglia. METHODS： Immortalized mice microglia cultured in vitro were randomly divided into 3 groups ： normal group Ⅰ（ no treatment ） , negative control group Ⅱ （transfection of N control siRNA in microglia） and transfect group Ⅲ （ transfection of siRNA-F1 screened by first experiment in microglia ）. The 3 groups were all stimulated by 50 μmol/L ATP, and then intracellular calcium concentration （ [ Ca^2＋ ]i ） and immunofluorescence double-labeling of P2X4R/ OX42 were detected by LSCM. RESULTS： After siRNA-F1 or transfection of N control siRNA in immortalized mice microglia, ATP activated the microglia in the 3 groups. LSCM indicated that [ Ca^2＋ ] i released by group Ⅲ microglia was less than that in normal group I and negative control group Ⅱ. Immunofluorescence double-labeling of P2 X4 R/OX42 indicated that activated microglia and resting microglia were detected in group Ⅲ, while only activated microglia was detected in normal group Ⅰ and negative control group Ⅱ. CONCLUSION： The activation of immortalized mice microglia by ATP is partly inhibited by siRNA-F1.