Notch1特异的RNAi慢病毒载体的构建及其对神经母细胞瘤细胞增殖的影响

发布时间：2018-02-26 21:55

本文关键词： 神经母细胞瘤 Notch-1信号通路 RNAi 慢病毒　出处：《福建医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的: 构建针对Notch-1基因的RNAi慢病毒表达载体,从转录后水平抑制Notch-1基因表达,为研究Notch-1信号通路提供技术工具;并利用构建的RNAi慢病毒感染神经母细胞瘤细胞SH-SY5Y,筛选出稳定转染细胞株,观察抑制Notch-1信号通路对神经母细胞瘤细胞SH-SY5Y增殖的影响。方法: 设计合成两对针对Notch-1 mRNA不同位点的shRNA编码序列,克隆到慢病毒穿梭质粒中,然后通过Gateway重组构建重组RNAi慢病毒表达载体;在293FT细胞中包装并进行病毒滴度测定,获得高滴度的病毒上清;慢病毒感染神经母细胞瘤细胞SH-SY5Y,RT-PCR和Western Blot检测构建的RNAi慢病毒对Notch-1表达的抑制效果;利用RNAi慢病毒抑制Notch-1表达,探讨Notch-1信号通路对神经母细胞瘤细胞SH-SY5Y增殖的影响。结果: 1、成功构建了针对Notch-1基因两个不同位点的RNAi慢病毒表达载体,经PCR和测序鉴定干扰片段的碱基序列及插入位点完全正确; 2、在293FT细胞中包装,获得高滴度慢病毒上清;病毒液滴度在1.5×10~5TU -2.3×10~5TU之间,可满足后续实验需要; 3、构建的两个RNAi慢病毒感染SH-SY5Y细胞后可使Notch-1mRNA表达量分别下降80%和66%; 4、慢病毒感染SH-SY5Y后进行MTT检测,RNAi慢病毒组与无义干扰组和对照组相比,细胞增殖率明显下降(p0.05或p0.01)。结论: 构建Notch-1特异的RNAi慢病毒表达载体能有效抑制Notch-1基因的表达,可以作为研究Notch-1信号通路的重要技术手段;Notch-1信号通路被抑制后对神经母细胞瘤细胞SH-SY5Y的增殖有重要影响。
[Abstract]:Objective:. The expression vector of RNAi lentivirus targeting Notch-1 gene was constructed to inhibit the expression of Notch-1 gene at post-transcriptional level, which provides a technical tool for the study of Notch-1 signaling pathway. Using the constructed RNAi lentivirus to infect the neuroblastoma cell line SH-SY5Y, a stable transfected cell line was screened, and the effect of inhibiting the Notch-1 signaling pathway on the SH-SY5Y proliferation of neuroblastoma cells was observed. Methods:. Two pairs of shRNA coding sequences targeting different sites of Notch-1 mRNA were designed and synthesized and cloned into lentivirus shuttle plasmids. Then recombinant RNAi lentivirus expression vector was constructed by Gateway recombination, and then packaged in 293FT cells and detected by virus titer. High titer virus supernatant was obtained, RNAi lentivirus constructed by RT-PCR and Western Blot was used to detect the inhibitory effect of RNAi lentivirus on Notch-1 expression, RNAi lentivirus was used to inhibit Notch-1 expression, SH-SY5YT RT-PCR and Western Blot were used to detect the inhibitory effect of lentivirus on Notch-1 expression. To investigate the effect of Notch-1 signaling pathway on SH-SY5Y proliferation of neuroblastoma cells. Results:. 1. The expression vector of RNAi lentivirus targeting two different sites of Notch-1 gene was successfully constructed. The base sequence and insertion site of the interfering fragment were identified by PCR and sequencing. (2) High titer lentivirus supernatant was obtained by packaging in 293FT cells, and the titer of the virus was between 1.5 脳 10 ~ (5) TU and 2.3 脳 10 ~ (5) TU, which could meet the needs of subsequent experiments. 3After the two RNAi lentiviruses were infected with SH-SY5Y cells, the expression of Notch-1mRNA decreased by 80% and 66, respectively. 4. After lentivirus was infected with SH-SY5Y, the cell proliferation rate of lentivirus group was significantly lower than that of unsense interference group and control group (P 0.05 or p 0.01). Conclusion:. The construction of Notch-1 specific RNAi lentivirus expression vector can effectively inhibit the expression of Notch-1 gene. It can be used as an important technical means to study the Notch-1 signaling pathway. The inhibition of Notch-1 signaling pathway has an important effect on the proliferation of SH-SY5Y in neuroblastoma cells.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R346

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