摘要：抗真菌药的研究已将近百年的历史，但由于长期地使用，抗真菌药物的耐药程度愈加明显，因此，临床上急需一种新的抗真菌药物，而橙花醇作为一种挥发油，安全绿色，具有香气，同时具有抗菌的活性。本实验以线粒体为中心研究橙花醇诱导白色念珠菌(Candida albicans)细胞凋亡机制，以一定浓度的橙花醇药物处理C。albicans，通过DCFH-DA的检测，发现细胞内线粒体里的活性氧(ROS)累积。研究发现ROS的累积，导致线粒体内膜两侧跨膜形成的膜电位(MMP)超极化，使渗透压升高，引起细胞内线粒体释放细胞色素C(Cyt C)，同时线粒体膜电位的超极化又进一步作用于ROS的累积。用FITC-VAD-FMK染色在原位检测到细胞内Metacaspase的活性，发现由于橙花醇诱导C。albicans线粒体内的活性氧累积和细胞色素C的释放，进而激活Metacaspase的活性。通过TUNEL实验分析中，可以观察到细胞凋亡的典型特征：核凝结和断裂，从而证明橙花醇诱导C。albicans凋亡，并为开发新的抗菌药物提供了许多理论依据和实验数据支持。80354

毕业论文关键字：橙花醇、白色念珠菌、细胞凋亡

Apoptosis mechanisms of Candida albicans 

Abstract：Research of antifungal drugs has nearly a century of history, but due to long-term use, extent of the drug resistance of antifungal drugs more obvious, so clinical in urgent need of a new antifungal drugs and volatile oil of neroli as a security green, with aroma and has antibacterial activity。 This experiment centered on mitochondrial research nerol Candida albicans mechanism of apoptosis induced by according to the concentrations of nerol drug C。albicans through detection of DCFH-DA, found in the mitochondria of reactive oxygen species (ROS) accumulation。 Study found that the accumulation of ROS, resulting in cross-membrane formation on both sides of the inner mitochondrial membrane, the membrane potential (MMP) hyperpolarized, osmolality, causing the intracellular mitochondrial release of cytochrome c (Cyt C), while mitochondrial hyperpolarization of the membrane potential and further functions in the accumulation of ROS。 FITC-VAD-FMK detected by staining in situ Metacaspase activity inside the cell, found as nerol C。albicans induced by reactive oxygen species in mitochondria accumulate and Cyt C release, and then activate the Metacaspase activity。 By TUNEL analysis, typical characteristics of apoptosis could be observed: the nuclear condensation and break, nerol-induced apoptosis of C。albicans could be proved, and for the development of new antimicrobial agents to provide a number of theoretical and experimental data support。

Keywords: Nerol; C。albicans; Apoptosis

目录

前言 1

1 材料与方法 3

1。1供试材料 3

1。1。1原料 3

1。1。2主要试剂 3

1。1。3主要仪器 4

1。1。4培养基 4

1。1。5实验试剂的配制 5

1。2实验步骤 5

1。2。1 C。albicans菌种的活化与接种 5

1。2。2菌悬液的制备 5

1。2。3细胞内ROS活性检测 6

1。2。4线粒体膜电位的检测 6

1。2。5线粒体内Cyt C的释放 6

1。2。6 Metacaspase活性检测 7

1。2。7 TUNEL细胞凋亡原位检测