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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 inhibit the expression of α-synuclein gene in MN9D cells by using vector-mediated RNA interference (RNAi) method and to detect its effect on cell proliferation and viability. METHODS: Two 19-nucleotide segments of α-synuclein were selected from the open reading frame of α-synuclein and a negative control sequence with no homology to the mouse gene was selected. Single-stranded oligonucleotides were synthesized and annealed to Double stranded oligonucleotides. The double-stranded oligonucleotide was cloned into the pENTR / H1 / TO plasmid vector. After being identified correctly, MN9D cells were transfected and stable cell clones were obtained by screening. The expression of α-synuclein in cells was detected by RT-PCR, Real-time PCR, immunocytochemistry and Western blotting to identify the efficiency of RNAi. Growth curve and MTT method were used to detect the proliferation and viability of the cells in each group. Results Compared with normal and transfected pSH / CON MN9D cells, the expression of α-synuclein mRNA and protein in pSH2 / α-SYN transfected cells was significantly decreased. However, the expression of α-synuclein mRNA in pSH1 / α-SYN transfected cells and There was no significant difference in protein level between the two groups of control cells. Inhibition of α-synuclein expression does not affect cell proliferation, but can reduce cell viability. CONCLUSIONS: SH2 / α-SYN is an effective small interfering RNA (siRNA) sequence. The vector-mediated RNAi can effectively inhibit the expression of α-synuclein in MN9D cells. Protein and its role in the pathogenesis of Parkinson’s disease (Parkinson’s disease, PD) provides a good cell model; α-synuclein plays an important role in maintaining the vitality of MN9D cells, α-synuclein function loss will lead to direct cell Or indirect damage.