【作者】 于海宁；

【导师】 沈生荣；

【作者基本信息】 浙江大学 ， 茶学， 2004， 博士

【摘要】 据调查，在欧美，人类因患前列腺癌引起的死亡率现已位居第二，仅次于肺癌。据流行病学调查、细胞和动物实验研究初步证实EGCG具有一定的预防、治疗前列腺癌的作用。铜离子是人体必需的微量元素，过量积累或缺乏会导致多种疾病的发生，包括癌症。金属离子与儿茶素的相互作用是近年来茶叶生物化学和生物无机化学的研究热点，而铜离子与EGCG的相互作用及其细胞生物学行为至今国内外少有报道。 本文利用前列腺癌细胞（包括激素依赖型前列腺癌细胞LNCaP和非激素依赖型前列腺癌细胞PC-3）培养体系研究了EGCG、Cu²⁺对前列腺癌细胞生长的影响和损伤机理；EGCG与Cu²⁺共存对前列腺癌细胞生长的影响，及两者生物学活性的变化；细胞培养条件下EGCG含量变化速度；EGCG、Cu²⁺存在及两者共存时，F-12培养液发光强度的动态变化和自由基的产生情况。 MTT法实验结果发现，EGCG与Cu²⁺均能抑制LNCaP细胞和PC-3细胞的生长，并与剂量呈正相关，EGCG与Cu²⁺抑制作用的最佳作用时间均为24小时；加入Cu²⁺，只有EGCG与Cu²⁺的相对浓度较高时，EGCG对Cu²⁺诱导的LNCaP细胞毒性有一定的抑制作用，否则促进细胞死亡；在PC-3细胞培养体系中，Cu²⁺存在下，EGCG对Cu²⁺诱导的PC-3细胞的毒性明显减弱；LNCaP细胞培养体系中，当后加入Cu²⁺时，Cu²⁺对EGCG诱导的细胞毒性有极显著促进作用，在同样水平下，Cu²⁺对EGCG诱导的PC-3细胞毒性无显著影响。增加PC-3细胞体系中EGCG的浓度，Cu²⁺对EGCG诱导的PC-3细胞毒性有促进作用。而在EGCG存在下，Cu²⁺对LNCaP细胞的毒性显著降低。PC-3细胞体系中，低浓度EGCG对Cu²⁺诱导的细胞毒性明显减弱，但高浓度EGCG对低浓度Cu²⁺诱导的PC-3细胞的毒性无影响，只有高浓度Cu²⁺诱导的PC-3细胞的毒性才能被抑制。当后加入EGCG时，只有EGCG与Cu²⁺浓度比达到一定值时Cu²⁺对LNCaP细胞的毒性被抑制，而PC-3细胞体系中，EGCG对Cu²⁺诱导的毒性无影响。凝胶电泳及流式细胞仪测定结果表明，坏死是导致LNCaP细胞和PC-3细胞生长受到抑制的主要原因。利用透射电镜观察，发现处理后的前列腺癌细胞多数呈坏死症状，细胞膜有不同程度的损伤，细胞膜是EGCG、Cu²⁺或两者相互作用时对细胞损伤的主要作用位点。 HPLC分析结果表明，在细胞培养条件下，F一12培养液EGCG单体浓度快速下降，且培养液中EGCG含量变化与其在不含细胞的培养液中的变化不同，另外，不同的细胞株，EGCG含量变化也不同。在PC一3细胞培养体系中，EGCG6小时后完全消失，而在LNCaP细胞培养体系中，6小时后仍可检测到微盆的EGCG单体，EGCG与前列腺癌细胞可能发生相互作用:在F一12培养液中加入cu2+后，cuz+明显影响EocG的变化速度，其影响程度与c矿+的加入顺序和加入浓度有关.在细胞培养体系中，CuZ+存在下，6小时EGCG全部消失，这可能与EGCG较易被PC一3细胞吸收，通过胞内、胞外两种机制发挥作用有关。EGCG与CuZ十相互作用导致细胞膜破裂，单体EGCG或EGCG的结构重排物可能进入细胞在胞内起作用，而EGCG的各种金属络合物也许是胞外作用的重要物质。 化学发光结果表明，EocG、Cuz+均能增强F一12培养液的发光强度，但增加幅度与加入的浓度和时间有关.当EGCG与Cuz+共存时，F-12培养液的发光强度均较单独作用时不同，变化的程度与加入浓度、加入顺序和作用时间有关。EsR测定结果显示，C了十加入后，F一12培养液产生.OH自由基，Cu2+与EGCG共存时，无论加入顺序如何均可产生.OH自由基。但随着加入顺序改变，.OH自由基信号强度发生变化，表明EGCG与C矿+的相互作用与两者加入次序有关。更多还原

【Abstract】 As reports, the mortality of human body caused by prostate cancer has been stood the second place among all diseases, and that is next to the lung cancer. Depending on the epidemicology, cell scanning tests and animal verification, it is primarily confirmed that EGCG could act as the prevention and treatment from prostate cancer. Copper ion is necessary for the normal growth of mankind. After exceeding or lacking of copper ion in the body, it could result in many kinds of diseases including cancer. Recently, it has been becoming the focusing point for the scientists to investigate the interactions of metal ions with catechins extracted from green tea in the field of tea biochemistry and inorganic biochemistry. However, up to now it has been unclear in the world how to interact between copper ion and EGCG, and their behavior in cell biology.In this dissertation using PC-3 and LNCaP prostate cancer cells culture system, we investigated the effects of EGCG, Cu2+ and their coexistence on the growth of prostate cancer cell and their mechanism; the change of EGCG content in the cell culture system, and the dynamic of changes of chemiluminescence density of F-12 medium; the generation of free radicals in the presence of EGCG, copper ion or their coexistence.MTT assay demonstrated that EGCG or Cu2+ treatment of androgen-insensitive PC-3 and androgen-sensitive LNCaP PCA cells resulted in a maximum loss of cell viability at 24 h. When added EGCG after treatment with different concentrations of Cu2+, EGCG accelerated Cu2+-induced death of LNCaP cell unless the concentration ratio of EGCG/Cu2+ was enough high. However, EGCG inhibited the toxicity of Cu2+ to PC-3 cell in all groups. When Cu2+ were put into F-12 medium lastly, the toxicity of EGCG sharpened significantly in LNCaP cell culture system but in PC-3 cell, and EGCG-induced death of PC-3 cell was not accelerated unless the concentration of Cu2+ was enough high. Pretreated with EGCG, Cu2+-induced toxicity was decreased in LNCaP cell culture system. Cu2+-induced mortality of PC-3 cell was decreased in the presence of low concentrations of EGCG, and high concentrations of EGCG couldinhibit high concentrations of Cu2+-induced toxicity but low concentrations of Cu2+-induced toxicity to PC-3 cell. When EGCG was added into the system lastly, damages of LNCaP cell induced by Cu2+ were not inhibited unless the concentration ratio of EGCG to Cu2+ was enough high, and EGCG could not decreased Cu2+-induced toxicity in PC-3 cell culture system no matter what its concentrations. Also, results of flow cytometry and gel electrophoresis showed that necrosis was the main reason for inhibition of growth of PC-3 cell and LNCaP cell, and apoptosis was hardly detected. We found characters of necrosis and damages of cytoplasm membrane in PCA cells culture system, and cytoplasm membrane might be the important target point of EGCG, Cu2+ and their complex.EGCG content in F-12 medium with or without PC-3 cells was evaluated by HPLC. Without PC-3 cells, EGCG content of F-12 medium declined quickly at 37℃, 5.0%CO2. The change tendency of EGCG content was different in the presence or absence of PCA cells. EGCG was disappeared at 6h with PC-3 cells no matter what its concentration, and in a very small amount with LNCaP cells. We postulated that PCA cells might interact with EGCG Cu2+ could affect the change of EGCG content of F-12 medium, which was relative to added concentration and added order of Cu2+. EGCG disappeared at 6h with PCA cells in all groups, which probably had something to do with absorption of PC-3 to EGCG EGCG might exert its action in and out cells. Interaction of EGCG with Cu2+ was responsible for damages of cytoplasm membrane of PCA cells, and EGCG or its derivates might enter PCA cells and affect the growth of PCA cells. But derivates of EGCG, especially its complexs with metal ions, still were important components affected the growth of PCA cells.From chemiluminescence tests, chemiluminescence density of F-12 medium was increased after added EGCG or Cu2+,更多还原