兰州大学机构库 >生命科学学院
程序性坏死诱导系统的建立
Alternative TitleThe establishment of inducible necroptosis system
甘晓丽
Thesis Advisor牟长军
2018-04-01
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Keyword程序性坏死 RIPK3 DOX Idimer p-MLKL
Abstract

背景:传统意义上,细胞坏死被认为是由极端的物理和化学因素等诱发的细胞死亡方式。然而,近年来的研究表明,细胞坏死并不只是被动的不受调控的。在机体内,某些细胞发生坏死受特定的蛋白激酶RIPK3和MLKL所调控,被称为程序性坏死。迄今为止,对程序性坏死信号通路的研究并不十分明确,有待进一步的分析和研究。目的:基于以上考虑,首先需要构建能快速发生程序性坏死的诱导系统,将程序性坏死关键基因RIPK3与FK506结合蛋白结构域(FKBP)进行融合,通过“Tet-On”系统调控RIPK3的表达,构建可诱导表达RIPK3-2×FKBP的Hela、A549和HT-29稳定细胞系,为深入研究程序性坏死机理等工作提供有力的工具。
结果:通过western blot和免疫荧光实验检测发现DOX诱导细胞中RIPK3表达,且1 µg/mL DOX就有很好的诱导效果,随DOX诱导时间的延长RIPK3的表达量逐渐升高。加入Idimer后0 h、3 h、9 h,细胞死亡程度加剧且caspase-8抑制剂能够促进DOX和Idimer对细胞的杀伤力。而后通过免疫荧光实验发现DOX诱导RIPK3在Hela细胞质中表达,非变性聚丙烯酰胺凝胶电泳实验能够检测到细胞中RIPK3二聚体/寡聚体和MLKL寡聚体形成。进一步的Western blot实验表明DOX和Idimer能够诱导Hela、HT-29和A549细胞三种细胞中的MLKL磷酸化。与对照组DMSO处理的细胞相比,DOX和Idimer处理的Hela,HT-29和A549细胞,最早0.5 h就能检测到MLKL磷酸化。同时,用上述细胞构建NOD/SCID小鼠皮下何瘤模型,通过western blot和组织免疫荧光实验,发现DOX能诱导小鼠肿瘤组织中RIPK3表达和p-MLKL升高。

Other Abstract

Background: Traditionally, cell necrosis is thought to be a form of cell death induced by extreme physical and chemical factors. However, recent studies have shown that cell necrosis is not a just passive and unregulated cell death way. In the body, necrosis of certain cells is regulated by specific protein kinases RIPK3 and MLKL, which is called necroptosis. So far, the study of necroptosis signaling pathway is not very clearly and it needs further analysis and research.Objective: Based on the above considerations, we firstly need to construct an inducible system that can rapidly process necroptosis in which the key gene RIPK3 of necroptosis fused with the two copies of FK506-binding protein domain (FKBP) to regulate the expression of RIPK3-2×FKBP through the “Tet-On” system. We generated Hela, A549 and HT-29 cell lines in which RIPK3-2×FKBP proteins expressed inductively by adding DOX to the culture medium, which provide a powerful tool for further study of the mechanism of programmed necrosis.
Results: Western blot and immunofluorescence assays demonstrated that DOX induced RIPK3 expression in cells, and 1 μg/mL DOX could reach ideal induction effect. With the increase of DOX induction time, RIPK3 expression gradually increased. At 0 h, 3 h, and 9 h after adding Idimer, the degree of cell death increased and caspase-8 inhibitors promote lethality to cells of DOX and Idimer. Afterwards, DOX induced expression of RIPK3 in the cytoplasm of Hela cells was detected by immunofluorescence experiments. Non-denaturing polyacrylamide gel electrophoresis experiments showed that the formation of RIPK3 dimers/oligomers and MLKL oligomers in cells. Further Western blot experiments showed that DOX and Idimer could induce MLKL phosphorylation in three kinds of cells including Hela, HT-29 and A549 cells. Compared to cells treated with DMSO, MLKL phosphorylation was detected in Hela, HT-29 and A549 cells as early as 0.5 h after DOX and Idimer treatment. At the same time, using the above cell lines to construct a subcutaneous tumor model of NOD/SCID mice. Western blot experiments and tissue immunofluorescence experiments showed that DOX could induce the expression of RIPK3 and increase of p-MLKL in mouse tumor tissues.

URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/221632
Collection生命科学学院
Recommended Citation
GB/T 7714
甘晓丽. 程序性坏死诱导系统的建立[D]. 兰州. 兰州大学,2018.
Files in This Item:
There are no files associated with this item.
Related Services
Recommend this item
Bookmark
Usage statistics
Export to Endnote
Altmetrics Score
Google Scholar
Similar articles in Google Scholar
[甘晓丽]'s Articles
Baidu academic
Similar articles in Baidu academic
[甘晓丽]'s Articles
Bing Scholar
Similar articles in Bing Scholar
[甘晓丽]'s Articles
Terms of Use
No data!
Social Bookmark/Share
No comment.
Items in the repository are protected by copyright, with all rights reserved, unless otherwise indicated.