摘要：细胞自噬是普遍存在于真核生物中的细胞内物质降解系统。酵母细胞中，错误折叠的蛋白质和受损的细胞器等通过细胞自噬途径于液泡中降解，实现了物质的循环，可以帮助细胞渡过逆境。高等生物中，细胞自噬还与多种生理和病理过程相关。V-ATPase是一种依赖于ATP的质子泵，能够将H+逆浓度地泵入细胞器，从而文持这些细胞器的酸性环境，保证了细胞各项生理活动的进行。酵母细胞中，液泡内部的酸化，以及蛋白酶在酸性环境中发挥作用是细胞自噬所必须的，这样酸性环境的文持主要依赖于V-ATPase的调控。本文探究了V-ATPase复合体亚基Vph1和Stv1单独或同时缺失后对液泡酸度和细胞自噬的影响。结果显示Vph1和Stv1的共存对液泡酸性的文持和液泡蛋白酶对自噬体的降解具有非常重要的作用。38757
毕业论文关键词：V-ATPase；细胞自噬；调控机制
Study on the Mechanism of V-ATPase Subunits Vph1 and Stv1 in Regulating Autophagy
Abstract： Autophagy is a conserved intracellular material degradation system that is commonly found in eukaryotes. In yeast cells, misfolding proteins, damaged organelles, and recycling nutrients are degraded in the vacuoles by the autophagy pathway, which could help the cells to pass through stress. In higher creatures, autophagy is involved in multiple physiological and pathological processes, such as development, immunity, programmed cell death, cancer and neurodegeneration and so on. V-ATPase is a highly conserved proton transport complexes in eukaryotes that is prevalent in all kinds of eukaryotic from yeast to mammals. V-ATPase is an ATP-dependent proton pump that can pump H+ into the intracellular organelles in order to maintain the acidic environment of these organelles and ensure the physiological activities of cells. The V-ATPase relies on the hydrolysis of ATP to produce energy as the driving force against the concentration gradient. In yeast cells, autophagosomes are degraded by the vacuolar hydrolase after fusing with the vacuole, meanwhile, the internal acidification of vacuoles and the activity of vacuolar hydrolase in acidic environments are necessary for autophagy. This study explored the effects of deletion of V-ATPase subunits Vph1 and/or Stv1 on the internal acidification of vacuoles and autophagy. Results showed that the coexistence of Vph1 and Stv1 are very important to maintain the acidification of vacuoles and the degradation of autophagosomes by vacuolar hydrolase.
Key words: V-ATPase; autophagy; regulation mechanisms
目  录

摘要1
关键词1
Abstract…1
Key words1
引言…1
1材料与方法…2
1.1实验材料 …3
1.1.1菌株和质粒3
1.1.2培养基3
1.1.3试剂3
1.1.4仪器3
1.2实验方法 …3
1.2.1基因敲除的方法3
1.2.2整合荧光蛋白4
1.2.3转化pcRosella质粒…5
1.2.4荧光观察…5
2结果分析…5
2.1 V-ATPase复合体亚基Vph1和/或Stv1缺失对细胞自噬过程中的蛋白酶Pep4运输的影响5
2.2 V-ATPase复合体亚基Vph1和/或Stv1缺失对细胞自噬过程中的Pep4和Atg8的运输和定位的影响…6
2.3 V-ATPase复合体亚基Vph1和/或Stv1缺失对液泡酸度和自噬体降解的影响8
3讨论与小结 9
3.1讨论 9
3.2小结 9
致谢…10
参考文献10
附录A…11
附录B…11
附录C…12
V-ATPase复合体亚基Vph1和Stv1调控细胞自噬的机制研究
引言
细胞自噬是普遍存在于真核生物中细胞内物质降解系统[1]。到目前为止，在酵母中发现有41种细胞自噬相关的Atg蛋白，其中在酿酒酵母中发挥作用的则有35种。
细胞自噬的标志性事件是产生一个叫做自噬体的双层膜结构。当因饥饿而引发细胞自噬时，会产生一个叫做隔离膜的膜泡，并且最终延伸成为一个封闭的自噬体。在这个过程当中，包括蛋白质和细胞器在内的细胞质的一部分被包裹于自噬体内，接着自噬体与液泡（或溶酶体）发生融合，最终内含物会被液泡内的水解酶降解[2]。 V-ATPase复合体亚基Vph1和Stv1调控细胞自噬的机制研究:http://www.751com.cn/shengwu/lunwen_37813.html