摘要
目的探讨长链非编码RNA核富集转录体I（NEAT1）在小胶质细胞活化中的作用及潜在机制。方法（1）体外常规培养小胶质细胞BV2，加入0、0．1、0．5、1μg／mL脂多糖（LPS）刺激6h后，RT—PCR检测细胞NEAT1mRNA的表达；用1μg/mL PS刺激BV2细胞0、6、12、24h后，RT—PCR检测细胞NEAT1mRNA的表达；（2）将特异性NEAT1siRNA（NEATI—si）转染BV2细胞，RT-PCR、Western blotting分别检测NEAT1-Si组和对照组细胞微管相关蛋白1轻链3（LC3）mRNA和蛋白的表达；（3）将细胞分对照组、NEAT1-si组、LPS组、NEAT1-si＋LPS组，RT-PCR检测细胞肿瘤坏死因子α（TNF-α）、诱导型一氧化氮合酶（iNOS）mRNA的表达，荧光倒置显微镜观察BV2细胞的形态变化；（4）将细胞分为对照组、Torin-1组和NEAT1-si＋Torin-1组，Western blotting检测细胞LC3蛋白的表达，RT-PCR检测细胞LC3、TNF-α、iNOSmRNA的表达。结果（1）0和0．1μg／mL LPS组、0．5μg／mL LPS组、1μg／mL LPS组BV2细胞NEAT1mRNA的表达依次增高，差异有统计学意义（P〈0．05）；0、6、12、24h组BV2细胞NEATlmRNA的表达依次增高，差异有统计学意义（P〈0．051；（2）NEAT1-si组BV2细胞LC3-II mRNA的表达低于对照组，差异有统计学意义（P〈0．05）。Western blotting检测显示NEAT1-si组BV2细胞LC3-II/I比值低于对照组（0．7vs 1．03）：（3）RT-PCR检测显示：与对照组比较，NEAT1-si组细胞TNF-α、iNOSmRNA的表达降低，与LPS组相比，NEAT1-si＋LPS组细胞TNF-α、iNOSmRNA的表达降低，差异均有统计学意义（P〈0．05）；（4）Western blotting检测显示对照组、Torin-1组、NEAT1-si＋Torin-1组细胞LC3-Ⅱ／I比值分别为0．7、1．14、0．97。RT-PCR结果显示：与对照组比较，Torin-1组Torin-1组LC3-II、TNF-α、iNOSmRNA的表达明显上调．差异有统计学意义（P〈0．05）；与Torin-1组相比，NEAT1-si＋Torin-1组LC3-II、TNF-α、iNOS mRNA的表达下调，差异�
Objective To investigate the role of long noncoding RNA nuclear paraspeckle assembly transcript 1 （NEA T1） in regulating the activation of microglias. Methods （1） Microglias BV2 were routinely cultured in vitro and NEA T1 mRNA expression was detected by real-time PCR （RT-PCR） after 0, 0.1, 0.5 and 1 μg/mL lipopolysaccharide （LPS） stimulation for 6 h; NEA T1 mRNA expression was detected by RT-PCR after 1 μg/mL LPS stimulation for 0, 6, 12 and 24 h. （2） NEA T1 siRNA （NEA T1-si） was transfected into BV2, and RT-PCR and Western blotting were employed to detect the LC3 mRNA and protein expressions. （3） The BV2 cells were divided into control group, NEATI-si group, LPS group and NEATI-si＋LPS group; RT-PCR was used to detect the tumor necrosis factor-α （TNF-α） and inducible nitric oxide synthase （iNOS） mRNA expressions; morphological changes of BV2 cells were observed under inverted microscope. （4） The BV2 cells were divided into control group, negative control＋Torin-1 group and NEATl-si＋Torin-1 group; Western blotting was used to detect the LC3 protein expression and LC3, TNF-α and iNOS mRNA expressions were detected by RT-PCR. Results （1） NEA T1 was significantly up-regulated in LPS-stimulated BV2 cells in dose- and time- d ependent manners; significant differences were noted between each two groups （P〈0.05）. （2） The LC3-11 mRNA expression in the NEATI-si group was significantly decreased as compared with that in the control group （P〈0.05）; LC3-II/I protein ratio （0.7） in the NEATI-si group was significantly lower than that in the control group （1.03, P〈0.05）. （3） As compared with those in the control group, the TNF-α and iNOS mRNA expressions in the NEATI-si group were decreased; As compared with those in the LPS group, the TNF-α and iNOS mRNA expressions in the NEA TI-si＋LPS group were significantly decreased （P〈0.05）. （4） LC3-II/I protein ratio in the control group, Torin-1 group and Torin-1＋NEAT1-si group were 0.7,
出处
《中华神经医学杂志》
CSCD
北大核心
2017年第9期886-892,共7页
Chinese Journal of Neuromedicine
基金
（1）基金项目：国家自然科学基金（81371397、81560220、81671240）（2）基金项目：江西省青年科学基金重点项目（20171ACB21054）（3）基金项目：江西省青年科学~（20151BAB215014）
关键词
核富集转录体1
细胞自噬
小胶质细胞
神经炎症
Nuclear paraspeckle assembly transcript 1
Autophagy
Microglia
Neuroinflammation
