腺相关病毒载体介导的RNAi抑制CTCF的表达

发布时间：2018-04-05 19:00

本文选题：RNA干涉　切入点：短发夹RNA　出处：《沈阳药科大学》2006年硕士论文

【摘要】：RNA干涉(RNA interference，RNAi)是指同源双链RNA所引发的特异序列的转录后基因沉默现象。它被证明是一项能有效而特异地抑制基因表达的新技术。腺相关病毒(adneo-associated virus，AAV)载体是一种有效的核酸递送载体，可将小片段DNA高效低毒的转入哺乳动物细胞。多功能转录因子CTCF(CCCTC-binding factor，CCCTC结合因子)是多锌指结构蛋白，许多报道表明其与肿瘤的发生及多个基因的调控有密切的关系。本课题旨在利用小干涉RNA(small interfering RNA，siRNA)抑制CTCF在HeLa细胞中的表达，进而研究CTCF改变所致调控网络的整体性变化，全面揭示CTCF的功能。本课题首先针对CTCF的mRNA设计RNAi的靶位，通过筛选确定2个靶位点。构建带有H1启动子的shRNA通用表达载体，分别针对2个靶位点构建抑制CTCF表达的病毒包装质粒，包装重组腺相关病毒。将重组病毒感染HeLa细胞后分别挑取能稳定表达shRNA的单细胞克隆，共得到29株细胞株。用实时荧光定量PCR法和Western印迹法检测细胞克隆的CTCF表达水平。结果发现在mRNA水平，以正常HeLa细胞为对照，GAPDH为内参，CTCF的表达最高可被下调76％，在蛋白水平，以正常HeLa细胞为对照，α-actinin为内参，CTCF的表达也被下调70％左右。在上述研究中，CTCF mRNA下降幅度，蛋白下降幅度与细胞生长抑制程度之间具有明显的相关性，，提示CTCF正常表达对细胞生长的重要性。同时本课题还对脂质体DC-chol／DOPE转染的方法进行了探讨，自己制备了脂质体DC-chol／DOPE，并且确定了转染实验所需的最适浓度和与转入DNA的最佳比例。利用此方法方便快速的大量制备重组腺相关病毒获得成功。本课题为整体性的研究CTCF基因抑制后相关表达谱的改变提供了重要的实验依据和必要的技术支持。
[Abstract]:RNA interference RNAi refers to post-transcriptional gene silencing of specific sequences induced by homologous double-stranded RNA.It has proved to be a new technique that can inhibit gene expression effectively and specifically.Adeno-associated virus (AAVV) vector is an effective nucleic acid delivery vector, which can transfer small fragments of DNA into mammalian cells with high efficiency and low toxicity.Multifunctional transcription factor (CTCF(CCCTC-binding factor-CCCTC binding factor) is a polyzinc finger structural protein, which has been reported to be closely related to tumorigenesis and the regulation of multiple genes.The purpose of this study was to inhibit the expression of CTCF in HeLa cells by using small interference RNA(small interfering siRNAs, and then to study the integrity changes of regulatory networks caused by CTCF changes, and to reveal the function of CTCF.In this paper, the target of RNAi is designed for mRNA of CTCF, and two target sites are determined by screening.The shRNA universal expression vector with H1 promoter was constructed, and the viral packaging plasmids which inhibited the expression of CTCF were constructed for two target sites respectively, and the recombinant adeno-associated virus was packaged.After the recombinant virus was infected with HeLa cells, 29 cell lines were isolated from the single cell clones which could stably express shRNA.The expression of CTCF was detected by real-time fluorescence quantitative PCR and Western blotting.The results showed that the expression of mRNA could be down-regulated by 76% at the level of normal HeLa cells, and 70% by 伪 -actinin at the protein level.In the above studies, there was a significant correlation between the decrease of CTCF mRNA, the decrease of protein and the degree of cell growth inhibition, suggesting the importance of normal expression of CTCF for cell growth.At the same time, the method of transfection of liposome DC-chol/DOPE was also discussed. The liposome DC-chol-DOPEwas prepared by itself, and the optimal concentration and the optimal proportion of the liposome into DNA were determined.The method is convenient and rapid to prepare recombinant adeno-associated virus.This study provides an important experimental basis and necessary technical support for the overall study of the changes of related expression profiles after CTCF gene suppression.
【学位授予单位】：沈阳药科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346

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