【作者】 刘展；

【导师】 游潮；

【作者基本信息】 四川大学 ， 外科学， 2007， 博士

【摘要】 目的线粒体不仅是细胞内的能量供应单元，而且还参与多种细胞活动，包括蛋白质合成、细胞复制、细胞凋亡等。线粒体含有自己的DNA系统，可以进行独立于核DNA之外的复制以及转录参与电子转递过程的23种酶。和正常细胞不同的是，肿瘤细胞内的线粒体功能和结构发生了很多改变，其中包括线粒体DNA突变，电子传递链的中断，线粒体膜电位的变化以及以线粒体为中心的细胞凋亡过程的抑制等。同时，肿瘤细胞采用有氧糖酵解过程作为能量供应主要来源也是肿瘤细胞和正常细胞的主要差别之一，并且有氧糖酵解过程中产生的能量随着肿瘤级别的增加而增加。为了探讨胶质瘤治疗的新途经，探索可以活化正常细胞线粒体功能、增加氧化磷酸的药物对肿瘤细胞的作用，我们进行了本次研究。方法1．提取U251细胞中的线粒体DNA，设计ATP6ase和ND6基因相应的引物，对ATP6ase及ND6基因进行PCR扩增，对扩增产物进行测序。将测序结果和人类正常线粒体基因相比对，找出差别。2．使用U251、C6两种胶质瘤细胞系。观察终浓度为20、30、40μM的Idebenone对其生长曲线的影响。通过检测细胞内罗丹明和JC-1的荧光强度，观察不同浓度的Idebenone对线粒体膜电位的影响；通过检测DCFH-DA荧光强度，观察Idebenone对细胞内ROS生成和通过免疫组化方法检测Idebenone对PRDX3表达的影响；通过检测细胞和线粒体提取液中萤火虫荧光素酶强度，观察Idebenone对两者内ATP的影响。结果1．Blast比对显示U251 ATP6ase基因较正常基因相比存在A8660G改变，ND6较正常没有明显变化。2．我们发现Idebenone可以抑制两种肿瘤细胞的生长，且这种抑制作用随着药物浓度的增加而明显。Idebenone可以降低线粒体膜电位，但是这种作用有一定的时间期限。Idebenone增加细胞内ROS的生成，且ROS的浓度随着Ideenone的浓度增加而增加；Idebenone可以降低细胞内PRDX3的表达。Idebenone可以减少细胞内ATP浓度，同时这种作用随着药物浓度增加而增加；但是对两种细胞氧化磷酸化途经的ATP生成没有明显影响。结论1．U251细胞中ATP6ase基因存在点突变，且突变位点具有罕见多态性，提示A8860G可能与胶质瘤有关；同时分析显示出现氨基酸缺失，提示ATP6ase功能可能出现障碍，影响经线粒体途经的ATP合成。2．Idebenone在安全浓度范围内可以通过线粒体途经抑制肿瘤细胞的生长，其机制部分来源于增加了细胞内ROS生成，抑制PRDX3表达以及降低整体细胞内ATP浓度。更多还原

【Abstract】 ObjectMitochondrion is not only the power station of the cell, but also takes part in many other cellular activities, including protein synthesis, cellular duplication, and apoptosis. Mitochondrion has its idios DNA system, which is independent of nuclear DNA and has own cycle of duplication and transcription. Different from normal cell, structure and function of mitochondrion in tumorous cells have changed very much and they become the biological marker in the tumor research and treatment now. These differences include mitochondrial DNA mutation, disturbance of electron transport chain, upgrading of mitochondrial membrane potential and suppressing of mitochondrion-dependent apoptosis in tumorous cells. Additionally, energy production in the term of acrobic glycolysis is also the main difference between tumorous cell and normal cell. Energy produced by acrobic glycolysis is more in the maligent tumor commonly. To explore the new way of glioma therapy and observe the influence of Idebenone, which can activate mitochondrial oxidative phosphorylation and upgrading cellular ATP production in normal cells, on the glioma cells, we did this study.Materials and methods1. Extracting the mitochondrial DNA from U251 cells and amplified the gene ATP6ase and ND6 sequence by PCR, then sequencing the products and comparing them to normal human ATP6ase and ND6 sequences.2. U251 and C6 glioma cells were cultivated by normal ways. We observed the effects of Idebenone of 20, 30, and 40μM on their growing curve, mitochondrial membrane potential by observing the intensity of rhodamine and JC-1 fluorescence , reactive oxygen spaces (ROS) by observing the intensity of DCFH-AD flourescence, peroxiredoxin 3 (PRDX3) expression by immunochemistry, and cellular ATP and ATP from mitochondrial exact by observing the intensity of firefly luciferase.Results1. There was a point mutation in U251 ATP6ase (A8860G) and no changes of U251 ND6 sequence. Some anmino sequence of U251 ATP6ase is depleted.2. We found out that Idebenone could suppress the growth of glioma cells (P<0.05), lower the mitochondrial membrane potential (P<0.05), upgrade the cellular ROS (P<0.05), degrade the expression of PRDX3 (P<0.05), decrease the cellular ATP (P<0.05) and have no effect on ATP content from mitochondrial exact (P>0.05).Conclusion1. The changed base of U251 is a rare polymorphasms site and this change prompts that A8860G is associated with this entity. The depletion of the anmino sequence hints that the functional changes of ATP6ase and the disturbance of mitochondrial ATP production.2. Idebenoen can suppress the glioma growth in vitro. Its function was partly attributed to upgrading ROS, decreasing expression of PRDX3, lowering mitochondrial membrane potential and degrading the cellular ATP content.更多还原