摘要
目的使用载体介导的RNA干扰(RNA interference,RNAi)方法抑制MN9D细胞中的α-synuclein基因表达,并检测其对细胞增殖和活力的影响。方法在α-synuclein的开放读码区选择两段19个核苷酸的片断,并选择一段与小鼠基因无同源性的阴性对照序列,分别设计合成单链寡核苷酸,退火融合成双链寡核苷酸。将双链寡核苷酸克隆入pENTR/H1/TO质粒载体,鉴定正确后,转染MN9D细胞,通过筛选获得稳定的细胞克隆。使用RT-PCR、Real-timePCR、免疫细胞化学染色和Westernblot方法检测细胞内的α-synuclein的表达水平,鉴定RNAi的效率。利用生长曲线和MTT法分别检测比较各组细胞的增殖和活力。结果转染pSH2/α-SYN的细胞与正常和转染pSH/CON的MN9D细胞相比,α-synuclein mRNA及蛋白水平均明显减少;而转染pSH1/α-SYN的细胞α-synuclein mRNA及蛋白水平与两组对照细胞没有明显差别。抑制α-synuclein表达不影响细胞增殖,但可以降低细胞活力。结论结果表明,SH2/α-SYN是有效的小干扰RNA(small interfering RNA,siRNA)序列,通过载体介导的RNAi方法可以有效抑制MN9D细胞中的α-synuclein表达,这为进一步研究α-synuclein蛋白的生理功能及其在帕金森病(Parkinson’s disease,PD)发病中的作用提供了良好的细胞模型;α-synuclein在维持MN9D细胞活力中起到重要作用,α-synuclein功能缺失会导致细胞直接或间接的损伤。
Objective To silence the expression of α-synuclein in MN9D dopaminergic cells using vector mediated RNA interference （RNAi） and examined its effects on cell proliferation and viability. Methods We identified two 19-nucleotide stretches within the coding region of the α-synuclein gene and designed three sets of oligonucleotides to generate doublestranded （ds） oligos. The ds oligos were inserted into the pENTR^TM/Hl/TO vector and transfected into MN9D dopaminergic cells, α-Synuclein expression was detected by RT-PCR, real-time PCR, immunocytochemistry staining and Western blot. In addition, we measured cell proliferation using growth curves and cell viability by 3-（4, 5）-dimethylthiahiazo （-z-y 1）-3, 5-diphenytetrazoliumromide （M＇FF）. Results The mRNA and protein levels of α-synuclein gene were significantly down-regulated in pSH2/α-SYN-transfected cells compared with control MN9D and pSH/CON-transfected MN9D cells, while pSHI/α-SYN- transfected cells showed no significant difference. Silencing α-synuclein expression does not affect cell proliferation but may decrease cell viability. Conclusion Our results demonstrated pSH2/α-SYN is an effective small interfering RNA （siRNA） sequence and potent silencing of mouse α-synuclein expression in MN9D cells by vector-based RNAi, which provides the tools for studying the normal function of α-synuclein and examining its role in Parkinson＇s disease （PD） pathogenesis. α-Synuclein may be important for the viability of MN9D cells, and loss of α-synuclein may induce cell injury directly or indirectl
