【作者】 徐敏；

【导师】 张振中； 任雪玲；

【作者基本信息】 郑州大学 ， 药物分析学， 2009， 硕士

【摘要】 RNA干扰(RNA interference,RNAi)是指通过诱导同源mRNA降解导致特定基因发生转录后基因沉默。目前,RNAi技术已广泛应用于基因功能研究和基因治疗等领域。RNAi可以通过siRNA或shRNA来实现,shRNA普遍应用RNA聚合酶Ⅲ启动子来调控其转录,但此类启动子缺乏组织特异性,因此我们选择RNA聚合酶Ⅱ启动子来调控shRNA转录,从而实现RNAi的组织特异性。本研究采用RNA聚合酶Ⅱ启动子中的apoA-I启动子来调控shRNA在肝癌细胞中特异性表达,从而实现肿瘤基因治疗的肝靶向性。首先,本实验通过PCR扩增apoA-I基因启动子序列,并将其克隆至pEGFP的报告蛋白EGFP基因上游,构建肝靶向重组载体pAE。将其和阳性对照质粒pEGFP分别转染肝癌细胞和非肝癌细胞,以报告蛋白的表达为标志检测其组织特异性。结果表明apoA-I启动子能够特异性启动绿色荧光蛋白在肝癌细胞中表达,而在食管癌EC9706、乳腺癌MCF7、宫颈癌HeLa细胞中不表达,apoA-I启动子具有较强的肝细胞组织特异性,为肝组织靶向性基因治疗奠定了实验基础。其次,我们将肝特异性apoA-I启动子运用到RNAi载体构建中,期望实现肝癌的靶向性基因治疗,试验中用报告蛋白EGFP的表达下调情况来判定载体构建是否成功。由于apoA-I启动子属于PolⅡ启动子,它的转录调控机制有别于PolⅢ启动子,所以我们对shRNA的表达框进行结构修饰,添加了Ribozyme和MAZ结构,与未经修饰的质粒载体做对照,转染肝癌细胞,以报告蛋白的表达为标志检测其活性。实验结果表明,本研究构建的apoA-I启动子调控的RNAi载体不仅能够有效实现RNAi作用,同时可以有效降低脱靶效应,为PolⅡ启动子在RNAi中的应用奠定了实验基础。RNAi具有序列特异性的特点,因此碱基突变、缺失等都可能对RNAi效果产生一定的影响。在本研究中,对于PolⅡ启动子调控的shRNA序列中碱基缺失对RNAi效果的影响也进行了初步探索,在上步实验验证有RNAi作用的shRNA序列正义链3’端设计碱基缺失,和未缺失的质粒做对照,转染肝癌细胞,以报告蛋白的表达为标志检测其活性。研究结果发现,在PolⅡ启动子调控的shRNA正义链上进行碱基缺失,shRNA载体也具有一定的RNAi效果,为深入探索RNAi的干扰机制进行了初步的尝试。肿瘤的发生和发展是一种复杂的过程,涉及多种基因、多种因素的综合作用,因此联合基因治疗已成为肿瘤基因治疗研究的新方向。为了实现高效、稳定的双基因沉默,并预防RNAi逃逸,在本研究中,我们构建双基因RNA沉默载体pSAD-EGFP和pDAF-EGFP,共转染肝癌细胞,以报告蛋白的表达为标志检测其活性,尝试联合RNAi的可行性。结果显示两种质粒对EGFP都具有沉默效果,为肿瘤的联合RNAi基因治疗奠定了很好的实验基础。更多还原

【Abstract】 RNA interference (RNAi) is a post-transcriptional gene silencing process by inducing sequence-specific mRNA degradation. Nowadays, RNAi has been widely used in many areas, such as loss-of-function studies and gene therapy. RNAi may be achieved using short hairpin RNA (shRNA) as well as small interfering RNA (siRNA). Although Pol III promoters are commonly used to synthesize shRNA, they lack cell specificity. An alternative way is to use cell-specific Pol II-directed synthesis of shRNA. Here, a liver-specific RNAi system for hepatoma gene therapy was studied using the human apolipoprotein A-I promoter.First, the apoA-I promoter containing essential transcriptional regulatory elements was amplified by PCR and then inserted into upstream of the green fluorescent protein gene of pEGFP. The recombinant plasmid with apoA-I promoter and the control plasmid with CMV promoter were transfected into SMMC-7221 and EC9706, MCF7, HeLa, respectively. Cells generated using vector with liver-specific promoter expressed GFP only in the appropriate cell type. By contrast, when transfected with the control vector, cells gave similar levels of expression. The apoA-I promoter could obtain restricted expression of an exogenous gene only in liver cells.Second, apoA-I promoter was used to drive shRNA expression in a particular type of cell. It was showed that the apoA-I-driven small hairpin RNAs specifically depress the expression of the exogenous reporter - enhanced green fluorescent protein. To prevent the off-target effects, the gene-specific targeting sequence was cloned between the ribozyme cassette and the MAZ site. The cis-acting hammerhead ribozyme cassette was added to cut off the m~7G cap structure, and the MAZ site was added for Pol II transcriptional pausing. The study provides an efficient way in silencing a gene in a particular type of cell without interfering with other type of cells.RNAi has characteristic of sequence-specific. Gene deletion and gene mutation of shRNA will influence RNAi effect. We want to know the influence about a base deletion shRNA regulated by Pol II promoter. A shRNA with a base deletion in its sense strand was designed and inserted into downstream of the Pol II promoter, this vector was subsequently cotransfected with pEGFP into SMMC-7721 accompany with the comparison vector that have no gene deletion. The study demonstrated that the gene deletion shRNA controled by the Pol II promoter could also induce the RNAi effect, this make contribution to the in-depth exploration of RNAi mechanism.To achieve efficient, stable, simultaneous gene silencing, and to prevent RNAi escape, a single cell-specific RNAi expression system for silencing double targets is tested in this study. The system was transfected into SMMC-7721 to investigate the influence to RNAi of diffferent direction of the promoters. The silencing efficiency was identified by observing the expression of EGFP. The silencing efficiency of both vectors were comparable to each other. The study demonstrated that the liver-specific vectors construction of combined gene therapy is successful. This method is a promising therapeutic method in which gene silencing of a single cannot account for hepatoma gene therapy.更多还原