NgAgo-gDNA系统基因表达调控功能的验证及其抗肿瘤作用的初步研究

发布时间：2018-05-20 06:19

本文选题：NgAgo-gDNA系统 + 肿瘤细胞　；参考：《山西医科大学》2017年硕士论文

【摘要】：目的:在哺乳动物细胞中验证NgAgo-g DNA系统是否具有基因编辑和调控基因表达的功能;对利用NgAgo-g DNA系统靶向致癌突变基因杀灭肿瘤细胞进行初步探索研究。方法:提取肿瘤细胞基因组DNA,通过PCR的方式扩增基因突变序列。将扩增片段克隆入T载体中,测序突变序列,鉴定肿瘤细胞系突变位点。构建致癌突变基因与EGFP共表达载体,将共表达载体、NgAgo载体以及靶向突变部位或EGFP的g DNA通过Lipofectamine 2000瞬时转染到293FT细胞中,流式检测EGFP的表达。以EGFP荧光表达率为指标判断基因编辑的效果。之后,探究在不同血清浓度(10%、5%、2.5%)及不同效靶比(EGFP:NgAgo-g DNA=1:3、1:6、1:12、1:24)条件下的基因编辑效果。随后,我们对实验条件进行优化。在进行基因编辑时,我们常使用EGFP作为报告分子。但由于EGFP蛋白相对稳定,降低了其对微弱基因编辑效果的报告敏感性。为此,我们的研究尝试构建EGFP-PEST融合蛋白表达载体,利用PEST序列可通过泛素蛋白酶体途径降解蛋白的功能,加快EGFP的降解,从而使得EGFP能够更为灵敏的指示基因编辑效果,为实现基因编辑的可视化筛选奠定基础。因此,以NFATx6-m Pro-EGFP-PEST-YES为模板,PCR扩增EGFP-PEST序列,克隆入逆转录病毒载体p QCXIP-EGFP-N1构建p QCXIP-EGFP-PEST-N1,病毒包装后感染293FT细胞获得稳定细胞系。用蛋白酶体抑制剂(MG-132)处理细胞,Western Blot检测EGFP的表达变化。成功建立稳定表达p QCXIP-EGFP-PEST-N1的293FT后,将NgAgo的表达载体以及靶向EGFP的g DNA通过Lipofectamine 2000瞬时转染到293FT细胞中,流式检测EGFP的表达,分选出俩群差异表达EGFP的细胞,分别提取基因组DNA和RNA,PCR扩增基因突变部位,测序突变序列,逆转录RNA,利用q PCR检测RNA水平的表达。最后,利用脂质体Lipofectamine 2000将NgAgo以及靶向突变序列的g DNA转入到肿瘤细胞中,连续72小时监测肿瘤细胞增殖。结果:1.在我们所选择的肿瘤细胞系中,大部分细胞系均与文献报道的突变位点相符,仅H820细胞系并没有发现文献报道的T790M的突变。2.在不同血清浓度及不同效靶比条件下,NgAgo-g DNA系统靶向突变部位或EGFP后,EGFP的荧光表达率未见明显降低。3.瞬转靶向EGFP的NgAgo-g DNA到稳定表达EGFP-PEST的293FT细胞系中,通过流式分析发现EGFP的荧光表达强度有所降低,但基因序列分析未见DNA序列改变。4.NgAgo-g DNA系统可以靶向致癌突变抑制肿瘤细胞的增殖。结论:在哺乳动物细胞中,NgAgo-g DNA系统可以敲低基因表达,但基因编辑能力在本研究中未得到证实;基于NgAgo-g DNA系统治疗肿瘤值得进一步研究。
[Abstract]:Aim: to verify whether the NgAgo-g DNA system has the function of gene editing and gene expression regulation in mammalian cells and to explore the possibility of killing tumor cells by targeting oncogene with NgAgo-g DNA system. Methods: genomic DNA of tumor cells was extracted and gene mutation sequence was amplified by PCR. The amplified fragment was cloned into T vector and sequenced to identify the mutation site of tumor cell line. The coexpression vector of oncogene and EGFP was constructed. The expression of EGFP was detected by flow cytometry. The expression of EGFP was detected by flow cytometry (FCM). The expression of EGFP was detected by flow cytometry, and the expression of EGFP was detected by flow cytometry. The effect of gene editing was evaluated by EGFP fluorescence expression rate. After that, we explored the effect of gene editing under the condition of different serum concentrations of 10% and 5%) and EGFP: G Ago-g DNA 1: 3: 6: 6: 12: 12: 1: 24) and different effective target ratios (EGFP: NGAgo-g DNA 1: 3: 1: 6: 1: 12: 1: 24). Then, we optimize the experimental conditions. In gene editing, we often use EGFP as a reporter molecule. However, due to the relative stability of EGFP protein, its sensitivity to weak gene editing was decreased. Therefore, we try to construct the EGFP-PEST fusion protein expression vector, using PEST sequence can degrade the protein through the ubiquitin proteasome pathway, accelerate the degradation of EGFP, so that EGFP can be more sensitive to the indicator gene editing effect. It lays a foundation for the visual screening of gene editing. Therefore, NFATx6-m Pro-EGFP-PEST-YES was used as template to amplify the EGFP-PEST sequence, and was cloned into the retroviral vector p QCXIP-EGFP-N1 to construct pQCXIP-EGFP-PEST-N1. The stable cell line was obtained by infection of 293FT cells with pQCXIP-EGFP-PEST-N1. Western Blot was used to detect the expression of EGFP in the cells treated with proteasome inhibitor MG-132. After the stable expression of 293FT expressing p QCXIP-EGFP-PEST-N1 was successfully established, the expression vector of NgAgo and the g DNA targeting EGFP were transiently transfected into 293FT cells by Lipofectamine 2000. The expression of EGFP was detected by flow cytometry, and the two groups of cells expressing EGFP differently were selected. Genomic DNA and RNA-polymerase chain reaction were used to amplify the mutation site, sequencing the mutation sequence, reverse transcription RNAs, and using Q PCR to detect the expression of RNA. Finally, liposome Lipofectamine 2000 was used to transfer NgAgo and g DNA targeting mutated sequence into tumor cells, and the proliferation of tumor cells was monitored for 72 hours. The result is 1: 1. Most of the tumor cell lines we selected were consistent with the mutation sites reported in the literature. Only the H820 cell line did not find the mutation of T790M reported in the literature. The fluorescence expression rate of NGAgo-g DNA was not significantly decreased at different serum concentration and target ratio. The fluorescence expression rate of NGAgo-g DNA system was not significantly decreased after EGFP. The fluorescence expression intensity of EGFP was decreased by flow cytometry in the 293FT cell line which expressed EGFP-PEST stably, and the target NgAgo-g DNA of EGFP was transferred to 293FT cell line. However, no change of DNA sequence was found in gene sequence analysis. 4. NgAgo-g DNA system could inhibit the proliferation of tumor cells by targeting carcinogenic mutations. Conclusion: ngAgo-g DNA system can lower gene expression in mammalian cells, but the ability of gene editing has not been confirmed in this study. The treatment of tumor based on NgAgo-g DNA system is worthy of further study.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.5

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