莲房原花青素诱导ROS积蓄介导HepG2细胞氧化应激损伤及其机制研究

发布时间：2018-05-12 19:59

  本文选题：莲房原花青素 + 氧化应激　； 参考：《江苏大学》2017年硕士论文

【摘要】：肝癌是我国最常见的恶性肿瘤之一,其死亡率位居全球第三,且仍呈上升趋势,每年新增约60万病例,依然严重威胁人类健康。大量研究报道已证实氧化应激可作为一种有效的抗肿瘤手段,能够通过多途径多靶点诱导细胞死亡。本课题组发现莲房原花青素(lotus seedpod procyanidins,LSPCs)具有抗氧化、抗辐射、抗肿瘤等多种生物活性。前期研究证实,LSPCs能够刺激人肝癌HepG2细胞产生大量活性氧(reactive oxygen species,ROS),诱导细胞氧化应激损伤。但并未对ROS种类、作用途径及机制进行深入研究。基于此,本论文致力于研究LSPCs通过ROS积蓄介导HepG2细胞氧化应激对线粒体功能、细胞内钙稳态以及胞内蛋白质组的影响,力求阐明LSPCs通过诱导HepG2细胞氧化应激的抗肿瘤机制。具体研究内容和结果如下:1.LSPCs诱导ROS积蓄介导HepG2细胞氧化应激对线粒体功能的影响(1)ROS的种类、分布和来源。利用H_2O_2敏感的荧光探针和O·2-的特异性荧光探针、线粒体特异性荧光探针以及线粒体呼吸链复合物(MRCC)抑制剂检测给予LSPCs处理前后,胞内ROS的种类、分布和主要来源。结果表明,LSPCs诱导HepG2细胞产生ROS的种类并非是单一的,至少存在H_2O_2和O·2-两种,且ROS主要来源于MRCC I和III。(2)LSPCs对线粒体主要特性的影响。经25、50和100μg/m L LSPCs处理HepG2细胞12 h后,线粒体膜电位水平、胞内ATP水平分别下降到对照组的0.86-0.49倍和0.83-0.45倍;MPTP开放度增加到对照组的0.52-0.87倍;显著降低了线粒体内GSH、SOD、SDH、CAT四种抗氧化酶的活性,且呈现剂量依赖关系,四种酶活力分别下降到对照组的0.91-0.57倍、0.77-0.40倍、0.81-0.35倍和0.83-0.30倍;MDA含量升高到对照组的1.24-4.16倍;线粒体内Ca~(2+)含量是对照组的1.21倍;MRCC I和III酶活性分别下降到对照组的0.80-0.37倍和0.78-0.33倍;MRCC I(subunit NDUFS1)和MRCC III(subunit UQCRC1)的蛋白表达量分别下降到对照组的0.84-0.37倍和0.86-0.35倍;Cyt-c释放至胞浆的量逐渐增加。经ROS抑制剂(NAC)预处理后,上述所有指标均得到不同程度的改善。以上表明,LSPCs通过诱导HepG2细胞积蓄ROS严重损伤线粒体功能和特性,而NAC能够有效改善上述现象,减轻线粒体损伤。2.LSPCs诱导ROS积蓄介导HepG2细胞氧化应激对细胞钙稳态的影响经50μg/m L LSPCs处理HepG2细胞12 h后,ROS水平达到最大值,荧光强度是对照组的1.22倍;处理24 h后,Ca~(2+)水平达到最大值,荧光强度是对照组的1.39倍,而且升高的Ca~(2+)是胞内钙库释放和胞外钙内流共同作用的结果。经25、50、75、100和150μg/m L LSPCs处理HepG2细胞1、3、6、12、24和36 h后,ROS和Ca~(2+)相关系数r分别是0.862、0.937、0.968、0.940和0.931。以上表明,LSPCs能诱导HepG2细胞内ROS积蓄和Ca~(2+)超载,而且两者之间存在显著的正相关性。3.LSPCs诱导ROS积蓄介导HepG2细胞氧化应激对细胞蛋白质组的影响采用蛋白质组学技术对LSPCs处理HepG2细胞前后总蛋白表达谱进行分析及鉴定。结果表明,LSPCs处理前后共鉴定出22个显著差异表达蛋白,其中,17个蛋白为下调蛋白,5个蛋白在LSPCs处理组消失。这些蛋白主要与应激反应、氧化损伤、细胞骨架、细胞凋亡、迁移、细胞增殖分化、信号传导等功能相关。因而推测LSPCs诱导HepG2细胞氧化应激可能是通过抑制这些蛋白的表达水平而达到抑制肿瘤细胞增殖的目的。
[Abstract]:Liver cancer is one of the most common malignant tumors in China. Its mortality rate ranks third in the world, and it is still on the rise. About 600 thousand cases are added every year. It is still a serious threat to human health. A large number of research reports have proved that oxidative stress can be used as an effective anti-tumor means and can induce cell death through multiple channels and multiple targets. It was found that lotus seedpod procyanidins (LSPCs) has many biological activities, such as antioxidant, anti radiation, and anti-tumor. Earlier studies have confirmed that LSPCs can stimulate a large number of reactive oxygen species (reactive oxygen species, ROS) and induce oxidative stress damage in human hepatocellular carcinoma cells, but not to ROS species, the pathway and mechanism. Based on this, this paper aims to study the effect of LSPCs on mitochondrial function, intracellular calcium homeostasis and intracellular protein group through ROS accumulation in HepG2 cells, and to clarify the anti-tumor mechanism of LSPCs by inducing oxidative stress in HepG2 cells. The specific content and results are as follows: 1.LSPCs induced ROS accumulation The effects of oxidative stress on HepG2 cells (1) the species, distribution and origin of ROS, using H_2O_2 sensitive fluorescent probe and O 2- specific fluorescence probe, mitochondrial specific fluorescence probe and mitochondrial respiratory chain complex (MRCC) inhibitors detected by LSPCs treatment, the species, distribution and main of intracellular ROS The results showed that the type of ROS induced by LSPCs in HepG2 cells was not single, at least there were two kinds of H_2O_2 and O. 2-, and ROS mainly originated from the effect of MRCC I and III. (2) LSPCs on the main characteristics of mitochondria. After 25,50 and 100 micron, the level of the mitochondrial membrane potential decreased to the control, respectively. The 0.86-0.49 times and 0.83-0.45 times of the group increased to the 0.52-0.87 times of the control group; the activity of four antioxidant enzymes in the mitochondria, GSH, SOD, SDH, CAT, was significantly reduced, and the activity of the four enzymes was dosed, and the activity of the enzyme decreased to the 0.91-0.57 times of the control group, the 0.77-0.40 times, the 0.81-0.35 and the 0.83-0.30 times, and the content increased to the control. The content of Ca~ (2+) in mitochondria was 1.21 times as high as that of the control group; the activity of MRCC I and III enzyme decreased to 0.80-0.37 times and 0.78-0.33 times of the control group, and the expression of MRCC I (subunit NDUFS1) and the protein expression in the control group decreased respectively to the control group. Increase. After pretreatment with ROS inhibitor (NAC), all of the above indexes were improved in varying degrees. The above indicated that LSPCs could seriously damage the mitochondrial function and properties by inducing the accumulation of ROS in HepG2 cells, and NAC could effectively improve the above phenomenon and reduce the mitochondrial damage.2.LSPCs induced ROS accumulation to mediate the oxidative stress of HepG2 cells to cell calcium. After 50 g/m L LSPCs treatment of HepG2 cell 12 h, the level of ROS reached the maximum, the fluorescence intensity was 1.22 times that of the control group; after 24 h, the level of Ca~ (2+) reached the maximum, the fluorescence intensity was 1.39 times that of the control group, and the elevated Ca~ (2+) was the result of the interaction of intracellular calcium library release and extracellular calcium influx. Through 25,50,75100 and 15 After 0 g/m L LSPCs treatment of HepG2 cells 1,3,6,12,24 and 36 h, ROS and Ca~ (2+) correlation coefficient r are 0.862,0.937,0.968,0.940 and 0.931., which can induce accumulation and overload, and there is a significant positive correlation between them. The effect of the mass group was analyzed and identified by proteomics technology before and after LSPCs treatment of HepG2 cells. The results showed that 22 significant differentially expressed proteins were identified before and after LSPCs treatment, of which 17 proteins were down regulated proteins and 5 proteins disappeared in LSPCs treatment group. These proteins were mainly induced by stress reaction and oxidative damage. The functions of cytoskeleton, cell apoptosis, migration, cell proliferation and differentiation, and signal transduction are related. Therefore, it is presumed that LSPCs induced oxidative stress in HepG2 cells may inhibit the proliferation of tumor cells by inhibiting the expression level of these proteins.

【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285

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