细胞应激状态下的线粒体

论文摘要

DIP2B是由DIP2B基因编码的DIP2蛋白家族中的一员。对于DIP2B在细胞应激反应中的作用还不明确。本研究的主要目的是探究DIP2B是否参与线粒体应激。在本论文中,使用到了 293T细胞、DIP2B沉默质粒、红色线粒体荧光探针和药物巴弗洛霉素A1以及流式细胞术,用于研究DIP2B在细胞应激中对线粒体的作用。通过细胞学分析研究293T细胞的形态,从而发现DIP2B和线粒体之间可能的功能联系。并通过在无血清的培养基中添加二甲基-α-酮戊二酸,提高了细胞的AcCoA水平,从而抑制自噬。我们的发现对于研究DIP2B的功能提供了进一步的依据。

论文目录

• Abstract
• 摘要
• Abreviations
• 1. Introduction
•   1.1 Stress
•   1.2 Mitochondria in Cell stress
•   1.3 Factors responsible for Mitochondrial stress
•   1.4 Mitochondrial stress response
•   1.5 Role of Mitochondria in a Cell starvation
•   1.6 The energy transformation in Mitochondria
•   1.7 Some basic biology and Evolutionary history of the mitochondrion
•   1.8 Mitochondrial structure function and Mitochondrial dysfunction
•   1.9 DIP2B Protein
•   1.10 Types of DIP2B Protein
•   1.11 The DIP2B Gene
•   1.12 DIP2B Gene Expression
•   1.13 DIP2B Domains
•   1.14 Fluorescence microscopy
• 2. MATERIALS AND METHODS
•   2.1 Plasmid constructs
•   2.2 Polymerase chain reaction （P.C.R）
•   2.3 Western Blotting
•   2.4 Cell culture and Transfection
•   2.5 Fluorescence Microscopy
•   2.6 Flow Cytometry
•   2.7 Mito-Tracker in 293T cells to check their signal strength
•   2.8 Transfection of DIP2B shRNA and Scramble PLL 3.7
•   2.9 Bafilomycin A1
•   2.10 Statistical analysis
• 3. Results
•   3.1 Human DIP2B shRNA construct designed based on U6 promoter
•   3.2 Human DIP2B shRNA Construct by Polymerase chain reaction （P.C.R.）
•   3.3 Transfection of DIP2B shRNA into 293Tcells
•   3.4 Knockdown efficiency of DIP2B shRNA in 293T cells checked by Western Blottingtechnique
•   3.5 Staining cells with Mito-tracker Red checked by Fluorescence Microscopy
•   3.6 The signal strength of Mito-Tracker detected by Flow cytometry
•   3.7 DIP2B shRNA Mito
•   3.8 Comparison between DIP2B shRNA 400 and Scramble PLL3.7 stained along withMito-Tracker checked by Flow cytometry
•   3.9 Statistical analysis of Knock down effect of DIP2B shRNA on Mitochondria
•   3.10 Different concentration of Bafilomycin A1 used along with mito-tracker to check theeffects on 293T cells
•   3.11 The signal strength of Mito-Tracker along with different concentration BafilomycinA1 into 293T cells by Flow cytometry
•   3.12 Transfection of DIP2B shRNA and Scramble PLL 3.7 stained with Mito-Trackeralong with 100 n M. Bafilomycin A1
•   3.13 Comparison between DIP2B shRNA and Scramble PLL3.7 along with Mito-Trackerand Bafilomycin A1 Drug by Flow cytometry
•   3.14 Starvation Induces Depletion of Acetyl CoA and addition of DMKG increasesAcetyl CoA
• Discussion
•   We used 293T cell line in our study due to following reasons
•   Why Flow Cytometry is used in this field of study?
•   General Principles
• Conclusion and Novelty
• References
• Acknowledgements

文章来源

类型: 硕士论文

作者: Abbasi Abdul Rahman

导师: Xiaojuan Zhu

关键词: 红色线粒体荧光探针,流式细胞术,巴弗洛霉素

来源: 东北师范大学

年度: 2019

分类: 基础科学

专业: 生物学

单位: 东北师范大学

分类号: Q25

总页数: 54

文件大小: 5757K

下载量: 65