摘要
目的 应用小分子RNA干扰（siRNA）技术沉默PD模型中的勿动基因Nogo66,观察其对小胶质细胞激活诱导的PD模型的影响。 方法 SD乳鼠体外原代培养小胶质细胞,通过免疫组化检测小胶质细胞表面特异性标记物OX-42筛选出小胶质细胞并分成4组,分别为空白对照组、PD模型组,Nogo66 siRNA组及阴性对照组。空白对照组不做任何处理;PD模型组通过添加不同浓度（0.031、0.062、0.125、0.25、0.5、1μg/mL）LPS诱导小胶质细胞建立PD模型;Nogo66 siRNA组在建模的基础上通过RNA干扰技术沉默Nogo66基因;阴性对照组在建模的基础上转染随机顺序的siRNA。采用Western boltting、实时荧光定量PCR检测细胞中Nogo66蛋白及mRNA的表达,ELISA检测多巴胺的含量。 结果 成功筛选出小胶质细胞并诱导PD模型。Nogo66 siRNA转染组转染24h后,小胶质细胞Nogo66基因表达明显减少,与阴性对照组比较,Nogo66基因阻断效率达92.5％±6.8％;转染72 h后,Nogo66基因阻断效率为88.3％±6.2％,差异均有统计学意义（P＜0.05）。Nogo66蛋白表达呈现出相同的变化趋势。Nogo66 siRNA转染组转染24h、72h后多巴胺表达均明显升高,与PD组及阴性对照组比较,差异均有统计学意义（P＜0.05）。 结论 Nogo66基因可能是PD治疗的靶向基因。
Objective To observe the effect of Nogo gene （Nogo66） on microglia activation-induced Parkinson＇s disease （PD） models by small RNA interference （siRNA） silencing.Methods Primary microglias from SD suckling mice were cultured in vitro; immunohistochemical staining was employed to detect the microglia surface shape receptor （OX-42） so as to screen the microglias.Then,these cells were randomly divided into four groups：blank control group,PD model group,Nogo66 siRNA group,and negative control group; cells in the blank control group did not give any treatment,cells in the other three groups were added lipopolysaccharide （0.031,0.062,0.125,0.25,0.5 or 1 μg/mL） to induce PD cell models; besides that,cells in the Nogo66 siRNA group were performed RNA interference to silencing the Nogo66,and cells in the negative control group were transfected siRNA random sequences.Protein and mRNA expressions of Nogo66 were determined by Western blotting and real time fluorescence quantitative PCR,and ELISA was used to detect the dopamine （DA） levels.Results Microglias were successfully screened and PD models were induced.Twenty-four h after the Nogo66 siRNA transfection,Nogo66 gene expression was obviously decreased; as compared with that in the negative control group,the blocking efficiency in the Nogo66 siRNA group reached to 92.5%±6.8%; 72 h after the Nogo66 siRNA transfection,the blocking efficiency in the Nogo66 siRNA group reached to 88.3%±6.2%; the Nogo66 protein expression showed the same trend; all differences were statistically significant （P〈0.05）.Twenty-four and 72 h after the Nogo66 siRNA transfection,the DA levels in the Nogo66 siRNA group were significantly higher than those in the negative control group （P〈0.05）.Conclusion Nogo66 gene may be the targeted gene therapy for PD.
出处
《中华神经医学杂志》
CAS
CSCD
北大核心
2015年第2期131-135,共5页
Chinese Journal of Neuromedicine
基金
广东省自然科学基金（S2012010010242）
