【作者】 李晶；

【导师】 王志平；

【作者基本信息】 兰州大学 ， 外科学， 2010， 博士

【摘要】 目的：膀胱癌术后复发率高是临床关注的焦点问题,但目前的药物治疗不能达到满意的疗效。因此,寻找一种安全有效的新型治疗药物迫在眉睫。本课题旨在研究一种新的饮食性黄酮类化合物漆黄素对膀胱癌细胞的抑制作用,并进一步探讨其体内外作用机制,为漆黄素作为一种高效无毒的天然抗癌药物治疗膀胱癌提供有效的理论依据。方法：不同浓度漆黄素处理膀胱癌细胞株后,采用MTT法和克隆形成实验观察细胞存活率。Hoechst 33258染色从形态学检测细胞凋亡。流式细胞术定量检测细胞周期变化和细胞凋亡率。RT-PCR检测了细胞中Bcl-2和Bax基因表达水平的变化。Western blot检测了细胞周期调控蛋白和凋亡调节蛋白的表达变化。另外,本研究建立了大鼠原位膀胱癌动物模型。HE染色观察了膀胱的病理学变化。给予漆黄素干预后,观察药物对肿瘤形成和发展的抑制作用。通过免疫组织化学法测定PCNA的表达和TUNEL末端标记法测定细胞凋亡,探讨漆黄素在体内的抑瘤作用机制。结果：MTT实验结果和克隆形成实验显示,漆黄素以剂量和时间依赖方式抑制人膀胱癌细胞系的增殖。研究结果表明,经不同浓度漆黄素(60-100μM)处理T24和EJ细胞48小时后,Hoechst 33258染色后荧光显微镜观察到癌细胞出现凋亡的形态学变化。流式细胞术表明漆黄素以浓度依赖方式影响细胞周期,并使细胞周期G0/Gl的比例增加而G2/M期的细胞减少,导致细胞周期停滞于G0/G1期。G1期的停滞是漆黄素通过下调细胞周期调节蛋白cyclin D1、cyclin E、CDK4/2的表达并上调p53和p21蛋白表达而实现的。随漆黄素作用浓度的增加,线粒体中Cyt c的含量减少,而胞液中Cyt c的含量则增多。此外,研究也发现,漆黄素下调了抗凋亡分子Bcl-2和Bcl-xL的表达,上调了促凋亡分子Bax和Bak的表达水平。Bax和Bcl-2分子表达的变化增加了Bax/Bcl-2比值。克隆形成实验模拟临床膀胱灌注的用药特点,采用大剂量漆黄素作用于膀胱癌细胞1小时,结果导致了细胞致命效应。体内实验仍模拟临床膀胱灌注用药特点。肿瘤模型组的肿瘤发生率为81.8%,而漆黄素治疗组肿瘤发生率15.4%。免疫组化检测PCNA结果表明,漆黄素治疗组的阳性细胞明显低于肿瘤模型组。TUNEL法检测显示,漆黄素治疗组膀胱癌细胞发生了明显的凋亡。各组的肝、脾、肺、心和肾未见明显损害。结论：本课题研究了漆黄素对膀胱癌细胞体内外增殖的抑制作用及其机制,为漆黄素用于膀胱癌的治疗提供了理论依据。研究表明,通过抑制细胞周期和诱导细胞凋亡,漆黄素抑制了膀胱癌细胞的生长。其机制可能是通过p53依赖的信号通路发挥作用,同时调节Bcl-2家族成员中的抗凋亡分子和促凋亡分子的结果；通过对线粒体通透性的影响,释放细胞色素c等促凋亡蛋白,导致细胞凋亡。此外,本课题成功构建了大鼠膀胱癌动物模型,该模型模拟了人类膀胱癌的疾病发展进程。同时我们模拟临床患者膀胱灌注治疗的特点,利用漆黄素溶液灌注膀胱内并且保留两小时的体内实验,探讨了漆黄素抑制大鼠膀胱癌形成和发展的作用机制。体内实验结果表明,漆黄素抑制和延迟大鼠膀胱癌细胞的形成与发展,同时诱导癌细胞凋亡。体内外实验结果预示了漆黄素是一种有效且安全的预防与治疗膀胱癌的天然植物药。更多还原

【Abstract】 Objective:Bladder cancer is one of the most common malignancies and tumor recurrence is a major clinical concern. Unfortunately, current chemotherapeutics are insufficient. Therefore, there is an obvious urgent need for novel, effective and safe therapies to prevent both recurrence and progression. The present study was carried out to investigate the effect of fisetin on cell cycle arrest and the induction of apoptosis in human bladder cancer cell lines and the mechanism of intravesical activity in a rat bladder tumor model.Methods:The MTT assay and clonal growth assay was used to determine cell viability. To determine whether the decrease in cell viability after fisetin treatment was the result of induction of apoptosis in bladder cancer cells, Hoechst 33258 fluorescence staining was performed to detect the morphology of cells. Meanwhile, apoptosis of the cancer cell lines was assessed by flow cytometric analysis after propidium iodide staining and the apoptotic-associated proteins were measured by western blot. RT-PCR was used to detect the expression of Bax and Bcl-xL mRNA. In addition, this study established a rat bladder carcinoma animal model induced by MNU. We observed the pathology changes of bladder by the HE staining. The tumor cell proliferation and apoptosis in a rat bladder carcinogenesis model were evaluated by proliferating cell nuclear antigen and terminal deoxyribonucleotide transferase-mediated nick-end labeling method. time-dependent inhibition of bladder cancer cell proliferation. Our data demonstrated that treatment of fisetin (60-100μM; 48 h) was found to bring about the induction of apoptosis in bladder cancer cells, as evidenced by Hochest 33258 staining and sub-G1 peak of apoptotic markers detection in T24 and EJ cells exposed to fisetin. Our results also clearly showed that, in fisetin-treated T24 and EJ cells, apoptosis was accompanied by an increase in the percent of cells in G0/G1 and reduction in G2/M indicating the cell cycle arrest in G0/G1. G1-phase arrest was associated with a marked decrease in the protein expression of cyclin D1, cyclin E, CDK4 and CDK2 and the increase in the protein expression of p53 and p21. There was also induction of mitochondrial release of cytochrome c into cytosol. In addition, the study also found that exposure of human bladder cancer cells to fisetin could upregulated the expression of Bax and Bak as well as downregulated the expression of Bcl-2, Bcl-xL in a dose-dependent manner. Changes in expression of Bax and Bcl-2 molecules resulted in an increase in the bax/bcl-2 ratio. Our in vitro study reveals that 400μM fisetin has been completely lethal to the 2 cell lines after a 1h incubation period. Based on these results, the same dose of fisetin and 2h dwell time to more closely resemble clinical instillation has been selected for our in vivo experiments. In the 11 MNU group was evident in the bladder of 9 for an incidence rate of 81.8%. Of the 13 MNU-Fis group fisetin-treated rats 2 had tumor for an incidence rate of 15.4%. Fisetin treatment significantly reduced the number of PCNA-positive cells in tumors from the MNU-Fis group as compared to the MNU group. In the MNU-Fis group treatment with fisetin resulted in a significant increase in the number of TUNEL-positive cells. After instillation of fisetin, heart, liver, kidneys, lungs and spleen are safe without damages.Conclusion:In summary, our present study showed the anticancer efficacy of fisetin against bladder cancer cells in vitro and in vivo. Our data in vitro demonstrated that fisetin inhibit the growth of bladder cancer cells both through retardation of the cell cycle and activation of the cellular apoptotic response in the cancer cells. Moreover, we successfully constructed the model of bladder cancer of rat by infusing bladder with MNU and the process of development of bladder tumor induced by MNU was similar with the development of human bladder cancer disease. Based on the in vitro results, the 2h dwell time to more closely resemble clinical instillation has been selected for our in vivo experiments. Our data reveals fisetin can result in significant inhibition and apoptosis in the progression of bladder cancer in rat tumor model. Our in vitro and in vivo results suggest that fisetin, a naturally dietary flavonoid, should be developed as a chemopreventive and chemotherapeutic agent against bladder cancer.更多还原