RNA干扰抑制Nogo-A基因表达对PC12细胞多巴胺释放及细胞凋亡的影响

发布时间：2018-07-25 16:32

【摘要】： 第一部分Nogo-A短发夹样RNA真核表达载体的构建目的构建靶向Nogo-A基因的短发夹样RNA(short hairpin RNA,shRNA)真核表达载体。方法根据Gene bank中提供的Nogo-A基因核苷酸序列,设计并化学合成2条shRNA,退火后克隆到pGenesil-1质粒的U6启动子下游从而构建重组载体,行限制性内切酶酶切和基因测序进行鉴定。结果序列分析结果表明,两个重组质粒的shRNA编码序列均成功插入到预定位置。结论成功构建靶向Nogo-A的shRNA真核表达载体,为进一步研究Nogo-A蛋白的功能及轴突再生抑制的基因治疗提供了新的方法。第二部分短发夹样RNA抑制PC12细胞Nogo-A基因表达的研究目的研究Nogo-A shRNA对PC12细胞Nogo-A基因表达的抑制作用,为下一步探索Nogo-A的功能奠定基础。方法经脂质体2000将前期构建的两个pGenesil-1/Nogo-A shRNA质粒转染PC12细胞,通过荧光显微镜和流式细胞仪观察EGFP的表达,分别于转染后48h后应用逆转录聚合酶链反应(RT-PCR)及Western blot法检测Nogo-A mRNA及蛋白表达水平。结果与对照组相比,空载体组的Nogo-A mRNA及蛋白表达水平无显着性差异(p0.05),而转染pGenesil-1-/Nogo-A组的Nogo-A mRNA及蛋白表达水平均明显下降(p0.05)。结论构建的pGenesil-1-/Nogo-A shRNA重组质粒能有效抑制Nogo-A基因在PC12细胞的表达。第三部分RNA干扰抑制Nogo-A基因表达对PC12细胞多巴胺释放的影响目的研究抑制Nogo-A基因表达对PC12细胞多巴胺释放的影响,探讨Nogo-A在神经内分泌细胞表达的意义。方法经脂质体将Nogo-A shRNA真核表达质粒转染到PC12细胞,分别于转染后24h、48h采用高效液相色谱仪(high performance liquid chromatography, HPLC)检测不同处理组细胞的多巴胺释放量。结果与对照组相比,转染48h后Nogo-A shRNA组细胞多巴胺释放量明显减少(P0.05),而转染空载体组多巴胺释放量无明显变化(P0.05)。结论Nogo-A基因沉默可以抑制PC12细胞多巴胺的释放,Nogo-A可能参与多巴胺的释放调节。第四部分RNA干扰抑制Nogo-A基因表达对PC12细胞凋亡的影响目的研究Nogo-A基因沉默对PC12细胞凋亡的影响。方法应用脂质体将Nogo-A shRNA真核表达载体转染到PC12细胞,在转染后不同时间应用四甲基偶氮唑盐(MTT)法检测细胞增殖活性,原位末端标记(TUNEL)法及流式细胞仪检测细胞凋亡率。结果与对照组相比,Nogo-A siRNA处理组细胞的增殖活性明显增加(P0.05),凋亡率明显下降(P0.05)。结论Nogo-A基因可能与肿瘤细胞凋亡有关。
[Abstract]:Part I: construction of Nogo-A short hairpin RNA eukaryotic expression vector objective to construct a short hairpin RNA (short hairpin RNA-shRNA-targeting eukaryotic expression vector targeting Nogo-A gene. Methods according to the nucleotide sequence of Nogo-A gene provided in Gene bank, two shRNAs were designed and chemically synthesized. After annealing, two shRNAs were cloned into the downstream of U6 promoter of pGenesil-1 plasmid to construct recombinant vector. Restriction endonuclease digestion and gene sequencing were performed to identify the recombinant vector. Results the sequence analysis showed that the shRNA coding sequences of the two recombinant plasmids were successfully inserted into the predetermined position. Conclusion the successful construction of shRNA eukaryotic expression vector targeting Nogo-A provides a new method for further study of the function of Nogo-A protein and gene therapy of axon regeneration inhibition. The second part: short hairpin RNA inhibits the expression of Nogo-A gene in PC12 cells objective to study the inhibitory effect of Nogo-A shRNA on Nogo-A gene expression in PC12 cells and to lay a foundation for further exploring the function of Nogo-A. Methods two previously constructed pGenesil-1/Nogo-A shRNA plasmids were transfected into PC12 cells by liposome 2000. The expression of EGFP was observed by fluorescence microscope and flow cytometry. The expression of Nogo-A mRNA and protein were detected by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot after 48 hours of transfection. Results compared with the control group, the expression of Nogo-A mRNA and protein in the empty vector group was not significantly different (p0.05), but the expression level of Nogo-A mRNA and protein in the transfected pGenesil-1-/Nogo-A group was significantly decreased (p0.05). Conclusion the constructed pGenesil-1-/Nogo-A shRNA recombinant plasmid can effectively inhibit the expression of Nogo-A gene in PC12 cells. The effect of RNA interference on dopamine release in PC12 cells objective to study the effect of inhibiting Nogo-A gene expression on dopamine release in PC12 cells and to explore the significance of Nogo-A expression in neuroendocrine cells. Methods the eukaryotic expression plasmid of Nogo-A shRNA was transfected into PC12 cells by liposome. The dopamine release was detected by high performance liquid chromatograph (high performance liquid chromatography, HPLC) at 24 h and 48 h after transfection. Results compared with the control group, the dopamine release of Nogo-A shRNA group decreased significantly (P0.05) after 48 h transfection, but the dopamine release of empty vector group did not change significantly (P0.05). Conclusion Nogo-A gene silencing can inhibit the release of dopamine in PC12 cells. Nogo-A may be involved in the regulation of dopamine release. The effect of RNA interference on the apoptosis of PC12 cells by inhibiting the expression of Nogo-A Gene objective to study the effect of Nogo-A gene silencing on the apoptosis of PC12 cells. Methods Nogo-A shRNA eukaryotic expression vector was transfected into PC12 cells by liposome. The cell proliferation activity was detected by tetramethylazolium (MTT) method at different times after transfection, and the apoptosis rate was detected by in situ end labeling (TUNEL) assay and flow cytometry. Results compared with the control group, the proliferation activity of the cells treated with Nogo-A siRNA was significantly increased (P0.05), and the apoptosis rate was significantly decreased (P0.05). Conclusion Nogo-A gene may be related to apoptosis of tumor cells.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R651;R346

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