【作者】 武冬梅；

【导师】 袁星；

【作者基本信息】 东北师范大学 ， 环境科学， 2009， 博士

【摘要】 本文以MCF-7细胞和PC-3细胞为模型细胞,采用电化学法、HPLC法、化学计量学法测定了细胞的伏安行为,分析了细胞电化学活性物质的种类和含量,研究和揭示了细胞电化学伏安响应机理;通过对比细胞分泌液和细胞质的伏安行为,建立了基于细胞质伏安行为的电化学检测环境雌激素活性新方法,使环境雌激素的筛选更有效、简单、快速和灵敏。研究结果概括如下:1、揭示了细胞电化学响应机理。研究了活细胞悬浮液和细胞质的电化学行为,发现活细胞悬浮液的电化学信号来源于细胞分泌物中鸟嘌呤和黄嘌呤的氧化反应,而不是通常人们推测的来源于活细胞本体或酶的电化学响应。本文采用了超声波和加热灭活法破碎活细胞得到细胞质,并检测了其电化学信号,发现细胞质的电化学信号远高于活细胞悬浮液,并且加热灭活法获得的细胞质的伏安信号更强,这是由于该方法获得的细胞质中鸟嘌呤和黄嘌呤的含量较高。2、建立了电化学法评价细胞活性系统。为了揭示细胞电化学信号与细胞活性的关系,本文采用了经典天然抗癌药物紫杉醇和系列非强效天然抗癌药物黄酮类化合物作为细胞活性抑制剂,使用电化学法研究了药物对细胞活性的影响。结果发现,随着药物作用时间和剂量的增加,细胞活性下降,细胞质中鸟嘌呤和黄嘌呤的浓度下降,细胞质电化学信号也随之下降,并具有时间和剂量依赖性。采用MTT和流式细胞仪常规检测法验证电化学法的可靠性,结果发现,两者的检测结果与电化学法基本一致,说明基于细胞质伏安行为建立的电化学评价细胞活性方法具有高灵敏性和可靠性。揭示了细胞质电化学行为与核苷酸代谢之间的关系。3、建立了细胞电化学检测环境雌激素活性方法。比较了去激素MCF-7细胞与正常培养MCF-7细胞电化学行为及其细胞质中电活性物质成分的区别。结果发现,去激素MCF-7细胞质中的黄嘌呤和鸟嘌呤浓度降低,使细胞分泌黄嘌呤和鸟嘌呤能力降低,导致细胞和细胞质的伏安信号减弱,说明去激素MCF-7细胞的活性下降;研究了标准雌激素雌二醇、环境雌激素壬基酚和双酚A对去激素培养的MCF-7细胞增殖的影响,发现其均存在剂量-效应关系和时间-效应关系,并且雌激素效应由大到小的顺序为:雌二醇＞壬基酚＞双酚A;同时使用3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)法验证了电化学法评价雌激素效应的可靠性。结果发现,两者的检测结果基本一致,说明细胞电化学法检测环境雌激素活性是可行的。更多还原

【Abstract】 This paper studies the voltammetric response of the MCF-7 cells and the PC-3 cells as the model cell by using electrochemical method, HPLC method and chemometric analysed the kinds and the concentration of the electrochemical active specises of the cells, and explained the mechanism on the origin of the voltammetric response; By comparison of the electrochemical behaviors of the cell secretion and the cytoplasm, the electrochemical method of measuring active of the environmental estrogens based on the electrochemical behaviors of the cytoplasm was built. The electrochemical method are rapid, highly sensitive, and inexpensive. The results are as follows:1. The mechanism on the origin of the voltammetric response of the cells. The electrochemical behaviors of the living cell suspension and the cytoplasm were studied, and the results were found that the electrochemical response of the living cell suspension was given by the oxidation of guanine and xanthine in the cell secretion, not by the cells. Ultrasonication and heat treatment were carried out for obtaining the cytoplasm, whose voltammeric response was stronger than that of the cell suspension. The voltammeric response of the cytoplasm obtained by heat treatment was stronger than that by ultrasonication due to that the concentrations of guanine and xanthine in the cytoplasm obtained heat treatment were higher than that by ultrasonication.2. Building the system of evaluating the active of the cells by the electrochemical method. To suggest the relation between the electrochemical signal and the cell viability, the effect of cytotoxicity of taxol, luteolin, quercetin, isoquercitin and rutin on the cell viability was studied by electrochemical method. The result obtained by using the electrochemical method displayed cytotoxic tendencies similar to those by the conventional in vitro drug MTT and flow cytometry sensitivity tests, suggesting that the method of recording voltammetric measurements described above was credible. The altered response was driven by the decreased concentration of xanthine and guanine in the cytoplasm. The relation between the voltammeric behavior of cells and intracellular catabolism was interprected.3. Establishment the method of measuring environmental estrogens based on the cell electrochemistry. Comparative studies of electroactive components and voltammetric behaviors different between the MCF-7 cell without estrogen and the MCF-7 cell with estrogen showed that the concentration of the guanine and xanthine bases in the cytoplasm decreased, which resulted in the decrease of secretion ability of the MCF-7 cell without estrogen decreased, and these result in the decreasing of the voltammetric response of the cells, impling that the cell vaibility decreases. The effect of estradio. nonylphenol.bisphenol on the proliferation of the MCF-7 cells without estrogen.The results showed that dosage-dependen and time-dependen was present and the order of the estrogen effect is as follow: estradio>nonylphenol>bisphenol; The effect of environmental estrogens on the proliferation of the MCF-7 cells was studied by the electrochemica method, and the result above was similar to those by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method, suggesting that voltammetric measurements was credible.更多还原