化学合成siRNA抑制原代皮层神经元NgR的表达  

The effect of the chemically synthetic siRNA on the expression of NgR of primary cortical neuron in vitro

作　　者：沈剑虹[1] 罗其中[2] 包映晖[2] 江基尧[2] 童菊芳 

机构地区：[1]南通大学附属医院神经外科,226006 [2]上海第二医科大学附属仁济医院神经外科 [3]上海市消化疾病研究所

出　　处：《中华神经外科杂志》2007年第12期953-957,共5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：目的筛选能有效抑制神经元NgR表达的siRNA序列。方法根据siRNA靶序列设计原则,设计并化学合成针对大鼠NgR基因编码区的siRNA三对,并以一对无关序列的寡核苷酸作为阴性对照。原代培养的大鼠皮层神经元分为5组:siRNA-1组、siRNA-2组、siRNA-3组、无关序列组、非转染组,用阳离子脂质体TransMessenger Transfection Reagent转染。转染后48h行RT-PCR和免疫细胞化学检测NgR表达水平的变化。结果转染48h后,与序列2、3和无关序列转染以及未转染的细胞相比,序列1转染的神经元NgR mRNA水平明显下降,并且其NgR抗体免疫荧光染色的荧光强度也明显减弱。结论化学合成siRNA能有效地抑制原代皮层神经元NgR的表达。Objective To select the effective sequence of siRNA in inhibiting the expression of NgR of Neuron. Methods According to the principle of target sequence of siRNA, we designed three homologous sequences of the complete cds of rattus norvegicus NgR mRNA and synthesized siRNA chemically in vitro. A control siRNA which is independent of the genome of rat was also designed and chemically synthesized. Primary cortical neurons of rat were cultured and divided into 5 groups： siRNA-1 group, siRNA-2 group, siRNA-3 group, control siRNA group and untransfected group. Transfection was carried out using TransMessenger Transfection Reagent, a kind of cationic liposome. RT-PCR and immunocytochemistry were employed to detect the change of the expression of NgR 48h after transfection. Results Compared with the cells that were transfected with siRNA of sequence 2 or 3 or nonsense oligonucleotide and the cells that were not transfected, the cells which were transfected with siRNA of sequence 1 shows a lower level of NgR mRNA detected by RT-PCR and the fluorescence intensity of NgR immunofluorescence stain of them was also attenuated obviously. Conclusion Chemically synthetic siRNA can be used efficiently to inhibit the expression of NgR in primary cortical neuron in vitro.

关 键 词：神经元 RNA干扰 SIRNA NGR 神经细胞培养 

分 类 号：R651[医药卫生—外科学]