siRNA沉默P2X4表达对永生化小鼠小胶质细胞活化的抑制效应
Effect of P2X4siRNA on immortalized mice microglia
彭伟 1张成伟 1姚文龙 1邓乾 1伍源 1田玉科1
作者信息
- 1. 华中科技大学同济医学院附属同济医院麻醉科,湖北,武汉,430030
- 折叠
摘要
目的:探讨特异性P2X4siRNA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N control 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50 μmol/L ATP刺激,采用激光扫描共聚焦显微镜检测胞内钙离子浓度([Ca2+];);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.
Abstract
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 trans-feet group Ⅲ (transfection of siRNA-F1 screened by first experi-ment in microglia). The 3 groups were all stimulated by 50 μmol/L ATP, and then intracellular calcium concentratian ([Ca2+]i) and immunofluoreacance 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 [Ca2+]i released by group Ⅲ microglia was less than that in nor-real group Ⅰ and negative control group Ⅱ. Immunofluorescence double-labeling of P2X4R/OX42 indicated that activated microglia and resting microglia were detected in group Ⅲ, while only acti-vated microglia was detected in normal group Ⅰ and negative con-trol group Ⅱ. CONCLUSION: The activation of immortalized mice microglia by ATP is partly inhibited by siRNA-F1.
关键词
特异莹光染料Fluo-4/细胞内钙离子浓度/小胶质细胞/激光扫描共聚焦显微镜/RNA干扰/神经病理性痛Key words
Fluo-4/Intracellular calcium concentration/microglia/laser scanning confocal microscope/RNAinterference/neuropathic pain引用本文复制引用
基金项目
湖北省自然科学基金(2004ABA241)
出版年
2009
NETL
NSTL
维普
万方数据