应用慢病毒载体介导RNA干扰在MCF-7细胞中剔降IKKα基因和制备转基因小鼠模型的研究

发布时间：2018-04-21 00:23

本文选题：NF-κB + IKKα　；参考：《第二军医大学》2006年博士论文

【摘要】：近年研究表明,乳腺肿瘤细胞中,IκB激酶-α(IκB kinase-α,IKKα)对激活NF-κB乃至其后的病变发挥着极其重要的作用。肿瘤坏死因子家族成员receptor activator of NF-κB ligand(RANKL)与其受体(RANK)结合,激活IKKα蛋白,释放NF-κB蛋白转位进入细胞核。在核内促使CyclinD 1的表达,若该表达持续进行将会导致乳腺上皮细胞的增生、癌变。该通路是经典的TNFR-IKKβ-NF-κB及LT-IKKα-NF-κB通路之外的另一重要信号转导途径,称之为RANKL-IKKα-NF-κB通路。它的异常表达是导致乳腺肿瘤形成的早期事件之一。本课题针对NF-κB信号转导通路中的重要枢纽因子IKKα,通过应用慢病毒载体(lentiviral vector)介导的RNAi效应,剔降(knockdown)乳腺肿瘤细胞MCF-7的IKKαm RNA,使其表达量下降,从而降低下游NF-κB蛋白的过度激活,以至恢复到正常水平,避免乳腺肿瘤的发生发展。此外,采用慢病毒载体感染受精卵方法,取代传统显微注射,建立了表达IKKα基因剔降的转基因小鼠模型,目前已繁殖至F2代。通过实时定量PCR技术验证,转基因小鼠血液中IKKα m RNA水平,明显低于野生型小鼠。该模型的建立为在体内开展IKKα基因的生物学功能及其与肿瘤发生关系的研究创造良好条件。上述研究工作在国内外尚未见文献报道。课题研究的内容包括以下四个主要部分: 1.pU6-IKKα-knockdown表达载体构建。 2.瞬时转染pU6-IKKα-knockdown表达载体剔降MCF-7细胞中IKKα的m RNA的研究。 3.IKKα-knockdown慢病毒载体的构建、稳定转染及用于乳腺肿瘤基因治疗的初步观察。
[Abstract]:Recent studies have shown that I 魏 B kinase- 伪, I 魏 B kinase- 伪 and IKK 伪 play an extremely important role in the activation of NF- 魏 B and subsequent lesions in breast tumor cells. Tumor necrosis factor family member receptor activator of NF- 魏 B ligand RANKL binds to its receptor RANKL, activates IKK 伪 protein and releases NF- 魏 B protein translocation into the nucleus. The expression of CyclinD 1 is promoted in the nucleus, which, if sustained, will lead to proliferation and canceration of breast epithelial cells. This pathway is another important signal transduction pathway in addition to the classical TNFR-IKK 尾 -NF- 魏 B and LT-IKK 伪 -NF- 魏 B pathways, known as the RANKL-IKK 伪 -NF- 魏 B pathway. Its abnormal expression is one of the early events leading to breast tumor formation. In this study, IKK 伪, an important pivotal factor in NF- 魏 B signal transduction pathway, was down-regulated by the RNAi effect mediated by lentiviral vector, and the expression of IKK 伪 m in breast cancer cells was reduced, thus reducing the overexpression of NF- 魏 B protein downstream. Even return to the normal level, to avoid the occurrence and development of breast tumors. In addition, a transgenic mouse model expressing IKK 伪 gene was established by using lentivirus vector to infect fertilized eggs instead of traditional microinjection, which has been propagated to F2 generation. The level of IKK 伪 m RNA in blood of transgenic mice was significantly lower than that of wild type mice. The establishment of the model provides favorable conditions for the study of the biological function of IKK 伪 gene and its relationship with oncogenesis in vivo. The above research work has not been reported in the literature at home and abroad. The content of the research includes the following four main parts: 1.pU6-IKK 伪 -knockdown expression vector was constructed. 2. Transient transfection of pU6-IKK 伪 -knockdown expression vector to degrade m RNA of IKK 伪 in MCF-7 cells. Construction of 3.IKK 伪 -knockdown lentivirus vector, stable transfection and preliminary observation of gene therapy for breast tumor.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R346;R-332

【引证文献】