本文選題：NgAgo-gDNA系統(tǒng) + 腫瘤細胞　； 參考：《山西醫(yī)科大學》2017年碩士論文

【摘要】：目的:在哺乳動物細胞中驗證NgAgo-g DNA系統(tǒng)是否具有基因編輯和調(diào)控基因表達的功能;對利用NgAgo-g DNA系統(tǒng)靶向致癌突變基因殺滅腫瘤細胞進行初步探索研究。方法:提取腫瘤細胞基因組DNA,通過PCR的方式擴增基因突變序列。將擴增片段克隆入T載體中,測序突變序列,鑒定腫瘤細胞系突變位點。構(gòu)建致癌突變基因與EGFP共表達載體,將共表達載體、NgAgo載體以及靶向突變部位或EGFP的g DNA通過Lipofectamine 2000瞬時轉(zhuǎn)染到293FT細胞中,流式檢測EGFP的表達。以EGFP熒光表達率為指標判斷基因編輯的效果。之后,探究在不同血清濃度(10%、5%、2.5%)及不同效靶比(EGFP:NgAgo-g DNA=1:3、1:6、1:12、1:24)條件下的基因編輯效果。隨后,我們對實驗條件進行優(yōu)化。在進行基因編輯時,我們常使用EGFP作為報告分子。但由于EGFP蛋白相對穩(wěn)定,降低了其對微弱基因編輯效果的報告敏感性。為此,我們的研究嘗試構(gòu)建EGFP-PEST融合蛋白表達載體,利用PEST序列可通過泛素蛋白酶體途徑降解蛋白的功能,加快EGFP的降解,從而使得EGFP能夠更為靈敏的指示基因編輯效果,為實現(xiàn)基因編輯的可視化篩選奠定基礎。因此,以NFATx6-m Pro-EGFP-PEST-YES為模板,PCR擴增EGFP-PEST序列,克隆入逆轉(zhuǎn)錄病毒載體p QCXIP-EGFP-N1構(gòu)建p QCXIP-EGFP-PEST-N1,病毒包裝后感染293FT細胞獲得穩(wěn)定細胞系。用蛋白酶體抑制劑(MG-132)處理細胞,Western Blot檢測EGFP的表達變化。成功建立穩(wěn)定表達p QCXIP-EGFP-PEST-N1的293FT后,將NgAgo的表達載體以及靶向EGFP的g DNA通過Lipofectamine 2000瞬時轉(zhuǎn)染到293FT細胞中,流式檢測EGFP的表達,分選出倆群差異表達EGFP的細胞,分別提取基因組DNA和RNA,PCR擴增基因突變部位,測序突變序列,逆轉(zhuǎn)錄RNA,利用q PCR檢測RNA水平的表達。最后,利用脂質(zhì)體Lipofectamine 2000將NgAgo以及靶向突變序列的g DNA轉(zhuǎn)入到腫瘤細胞中,連續(xù)72小時監(jiān)測腫瘤細胞增殖。結(jié)果:1.在我們所選擇的腫瘤細胞系中,大部分細胞系均與文獻報道的突變位點相符,僅H820細胞系并沒有發(fā)現(xiàn)文獻報道的T790M的突變。2.在不同血清濃度及不同效靶比條件下,NgAgo-g DNA系統(tǒng)靶向突變部位或EGFP后,EGFP的熒光表達率未見明顯降低。3.瞬轉(zhuǎn)靶向EGFP的NgAgo-g DNA到穩(wěn)定表達EGFP-PEST的293FT細胞系中,通過流式分析發(fā)現(xiàn)EGFP的熒光表達強度有所降低,但基因序列分析未見DNA序列改變。4.NgAgo-g DNA系統(tǒng)可以靶向致癌突變抑制腫瘤細胞的增殖。結(jié)論:在哺乳動物細胞中,NgAgo-g DNA系統(tǒng)可以敲低基因表達,但基因編輯能力在本研究中未得到證實;基于NgAgo-g DNA系統(tǒng)治療腫瘤值得進一步研究。
[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.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R730.5

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1 袁龍;NgAgo-gDNA系統(tǒng)基因表達調(diào)控功能的驗證及其抗腫瘤作用的初步研究[D];山西醫(yī)科大學;2017年

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本文編號：1913518