XAGE-1b腺病毒干扰载体的构建及其功能研究与一种fiber缺失型重组腺病毒系统的构建
发布时间：2018-11-20 07:06
【摘要】： XAGE是通过生物信息学方法发现的新一类肿瘤-睾丸抗原(cancer/testisantigen,CT抗原)基因,其主要表达亚型为XAGE-1b,其功能及表达调控机制的研究刚刚起步,已有的研究表明该基因可能与肿瘤细胞的生长和转移相关.在本研究的第一部分中,构建了针对XAGE-1b进行RNA干扰的重组腺病毒载体Adv-Sh1和Adv-Sh2.半定量和定量RT-PCR显示Adv-Sh1比Adv-Sh2对XAGE-1b的干扰效率更高,细胞荧光干扰实验显示Adv-Sh1对XAGE-1b有显著的干扰作用.在细胞增殖和集落形成实验中,感染Adv-Sh1后的细胞增殖能力显著降低(P＜0.01,Student's t检验),形成的单克隆集落数也显著减少.研究结果显示:下调XAGE-1b基因表达对多种肿瘤细胞的增殖及转移能力均有一定的降低作用,提示XAGE-1b在肿瘤生长和转移过程中可能发挥重要作用.本研究为进一步深入研究XAGE-1b的功能和致癌机制打下了良好基础. 腺病毒无法靶向感染某种特定的细胞,这主要是由于其纤毛蛋白(fiber)受体广泛存在于各种细胞表面,因此限制了腺病毒作为基因治疗载体的应用.敲除腺病毒基因组中的fiber基因,替换为改造过的fiber基因是实现腺病毒靶向性的思路之一.在本研究的第二部分中,按照上述策略建立了一套新型的腺病毒靶向基因治疗系统.基于AdEasy腺病毒系统构建了敲除fiber基因的腺病毒载体,同时构建了稳定表达Ad5 fiber基因的HEK293细胞系作为该病毒载体的包装细胞系.结果表明该套系统产生的缺陷型病毒能够在该细胞系中完成包装并扩增,具有正常病毒的感染能力.该系统为进一步构建靶向腺病毒基因治疗系统打下了基础.
[Abstract]:XAGE is a new class of tumor-testicular antigen (cancer/testisantigen,CT) gene discovered by bioinformatics. The main expression subtype of XAGE-1b, is the function and regulation mechanism of XAGE-1b,. Previous studies have shown that the gene may be related to the growth and metastasis of tumor cells. In the first part of this study, we constructed recombinant adenovirus vectors Adv-Sh1 and Adv-Sh2. for RNA interference against XAGE-1b. Semi-quantitative and quantitative RT-PCR showed that Adv-Sh1 had higher interference efficiency to XAGE-1b than Adv-Sh2, and the cell fluorescence interference assay showed that Adv-Sh1 had a significant interference effect on XAGE-1b. In the experiment of cell proliferation and colony formation, the ability of cell proliferation was significantly decreased after Adv-Sh1 infection (P < 0.01), and the number of monoclonal colonies was also significantly decreased. The results showed that down-regulation of XAGE-1b gene expression could decrease the proliferation and metastasis of various tumor cells, suggesting that XAGE-1b might play an important role in tumor growth and metastasis. This study laid a good foundation for further study on the function and carcinogenic mechanism of XAGE-1b. Adenovirus can not be targeted to infect a specific cell, which is mainly due to the widespread presence of its ciliated protein (fiber) receptor on various cell surfaces, which limits the application of adenovirus as a gene therapy vector. Knockout of fiber gene in adenovirus genome and replacement with modified fiber gene is one of the ways to realize adenovirus targeting. In the second part of this study, a novel adenovirus targeting gene therapy system was established according to the above strategies. Adenovirus vector with fiber knockout was constructed based on AdEasy adenovirus system, and the HEK293 cell line stably expressing Ad5 fiber gene was constructed as the packaging cell line of the vector. The results showed that the defective virus produced by the system could be packaged and amplified in the cell line and had the ability to infect the normal virus. The system lays a foundation for further construction of target adenovirus gene therapy system.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R373
本文编号：2344184
[Abstract]:XAGE is a new class of tumor-testicular antigen (cancer/testisantigen,CT) gene discovered by bioinformatics. The main expression subtype of XAGE-1b, is the function and regulation mechanism of XAGE-1b,. Previous studies have shown that the gene may be related to the growth and metastasis of tumor cells. In the first part of this study, we constructed recombinant adenovirus vectors Adv-Sh1 and Adv-Sh2. for RNA interference against XAGE-1b. Semi-quantitative and quantitative RT-PCR showed that Adv-Sh1 had higher interference efficiency to XAGE-1b than Adv-Sh2, and the cell fluorescence interference assay showed that Adv-Sh1 had a significant interference effect on XAGE-1b. In the experiment of cell proliferation and colony formation, the ability of cell proliferation was significantly decreased after Adv-Sh1 infection (P < 0.01), and the number of monoclonal colonies was also significantly decreased. The results showed that down-regulation of XAGE-1b gene expression could decrease the proliferation and metastasis of various tumor cells, suggesting that XAGE-1b might play an important role in tumor growth and metastasis. This study laid a good foundation for further study on the function and carcinogenic mechanism of XAGE-1b. Adenovirus can not be targeted to infect a specific cell, which is mainly due to the widespread presence of its ciliated protein (fiber) receptor on various cell surfaces, which limits the application of adenovirus as a gene therapy vector. Knockout of fiber gene in adenovirus genome and replacement with modified fiber gene is one of the ways to realize adenovirus targeting. In the second part of this study, a novel adenovirus targeting gene therapy system was established according to the above strategies. Adenovirus vector with fiber knockout was constructed based on AdEasy adenovirus system, and the HEK293 cell line stably expressing Ad5 fiber gene was constructed as the packaging cell line of the vector. The results showed that the defective virus produced by the system could be packaged and amplified in the cell line and had the ability to infect the normal virus. The system lays a foundation for further construction of target adenovirus gene therapy system.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R373
【参考文献】
相关期刊论文 前1条
1 宋玉亮;彭思扬;王晨滔;朱乃硕;;XAGE-1b腺病毒表达载体的构建及其功能研究[J];复旦学报(自然科学版);2008年03期
，本文编号：2344184
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2344184.html
最近更新
教材专著
