siRNA抑制神经元NgR mRNA的时效关系和毒性研究  

The effect of RNA interference on the NgR mRNA of primary cortical neuron in vitro

作　　者：沈剑虹[1] 罗其中[1] 包映晖[1] 江基尧[1] 童菊芳 杨绍峰[1] 

机构地区：[1]上海交通大学医学院附属仁济医院神经外科,200127 [2]上海市消化疾病研究所

出　　处：《中华神经外科杂志》2006年第11期695-698,共4页Chinese Journal of Neurosurgery

基　　金：国家自然科学基金(30371456);上海市科委科技攻关项目(034119822)

摘　　要：目的了解化学合成小干扰RNA(small interference RNA,siRNA)对原代培养神经元Nogo受体(Nogo receptor,NgR)mRNA作用的时效关系和毒性作用。方法化学合成一段针对大鼠NgR基因编码区的siRNA,用阳离子脂质体转染大鼠皮层神经元。分别于转染前、转染后24h、48h、72h、96h行RT-PCR检测NgR mRNA水平。碘化丙啶(PI)染色检测转染后各时间点神经细胞的存活情况。结果转染siRNA后24h和48h NgR mRNA水平明显下降,到72h明显回升,转染后96h NgR mRNA水平与转染前差异无统计学意义。PI染色显示转染后24h和48h,用脂质体转染的细胞(包括转染siRNA、无关序列Oligo和单纯脂质体组)其PI阳性率与非转染对照组无统计学意义,而转染后72h和96h两者差异有统计学意义,但在各时间点,脂质体转染的各组间PI阳性率差异无统计学意义。结论化学合成siRNA能有效促进原代皮层神经元的NgR mRNA降解,并于短期内维持于低水平。该段siRNA转染的毒性作用主要来自于转染试剂,与序列本身无明确关系。Objective To reveal the time-effect relationship between the chemically synthesized siRNA and the primary cortical neurons and make clear the toxic effect of the synthesized siRNA on the cultured neurons. Methods SiRNA directed to the complete cds of rattus norvegicus NgR mRNA was chemically synthesized and transfected into the primary cortical neurons of rat using TransMessenger Transfection Reagent. RT-PCR was employed to detect the change of NgR mRNA at several time points after transfection （0h,24h,48h,72h,96h）. PI dyeing was used to detect the survival status of the ceils after transfection. Results The level of NgR mRNA decreased notably at 24h and 48h after siRNA transfection and recovered obviously at 72h after transfection. By the time of 96h after transfection, the level of NgR mRNA had no significant difference compared with that before transfection. There was not obvious difference between the PI positive rates of liposome-transfected cells and non-transfected cells at both 24h and 48h after transfection. But the difference became significant at 72h and 96h. At the fourth time points, there was no obvious difference of the PI positive rates among the three liposome-transfected groups. Conclusion Chemi cally synthetic siRNA can inhibit the NgR mRNA in primary cortical neuron and the effect can be maintained for a short period. The toxic effect of siRNA transfection principally comes from the transfection reagent.

关 键 词：RNA干扰 神经元 细胞培养 NGR 

分 类 号：R346[医药卫生—基础医学]