【作者】 刘洁；

【导师】 庄乾元；

【作者基本信息】 华中科技大学 ， 外科学， 2009， 博士

【摘要】 第一部分葡萄籽提取物原花青素对人膀胱癌BIU87细胞的生长抑制作用目的探讨葡萄籽提取物原花青素（grape seed procyanidin extract，GSPE）对人膀胱癌BIU87细胞及人原代皮肤成纤维细胞生长的影响。方法体外培养的人膀胱癌BIU87细胞及人原代皮肤成纤维细胞与12．5、25、50、100、200μg／ml GSPE共同孵育24 h后，观察细胞形态学改变；并采用四甲基偶氮唑蓝（MTT）比色法测定其对人膀胱癌BIU87细胞及人原代皮肤成纤维细胞存活的影响。结果与对照组相比，50、100、200μg／ml GSPE可引起BIU87细胞形态的明显改变，各浓度GSPE对人原代皮肤成纤维细胞形态无明显影响。12．5、25、50、100、200μg／mlGSPE作用细胞24 h后，人膀胱癌BIU87细胞存活率分别为对照组的99．55±0．50％（n=18，P＞0．05），99．16±0．63％（n=18，P＞0．05），86．98±1．48％（n=18，P＜0．01），68．18±2．06％（n=18，P＜0．01）和51．42±1．78％（n=18，P＜0．01）；与对照组相比，50、100、200μg／ml GSPE可明显抑制BIU87细胞生长，但对人原代皮肤成纤维细胞生长无明显抑制作用。结论在一定浓度范围内，GSPE可呈剂量依赖性抑制人膀胱癌BIU87细胞生长，但对人原代皮肤成纤维细胞的生长无明显影响。第二部分葡萄籽提取物原花青素对人膀胱癌BIU87细胞周期的影响及其机制探讨目的探讨GSPE对人膀胱癌BIU87细胞周期的影响及其可能机制。方法将50、100、200μg／ml的GSPE作用于人膀胱癌BIU87细胞24 h后，采用流式细胞术检测细胞周期改变，采用RT-PCR及Western blot方法检测cyclin D1和CDK4mRNA及蛋白水平的表达变化。结果流式细胞分析表明，GSPE可使人膀胱癌BIU87细胞阻滞于G₁期，0、50、100、200μg／ml GSPE对应的阻滞于G₁期的肿瘤细胞平均比例分别为48．46、55．73、69．81和79．18％。RT-PCR及Western blot分析表明，随着GSPE浓度的增高，BIU87细胞cyclinD1和CDK4 mRNA及蛋白表达减少，半定量分析显示，各浓度组间差别有明显的统计学意义（P＜0．01）。结论GSPE可呈浓度依赖性阻滞BIU87细胞于G₁期。其机制可能与下调细胞周期相关因子cyclin D1和CDK4的表达有关。第三部分葡萄籽提取物原花青素对人膀胱癌BIU87细胞凋亡的影响及其机制探讨目的探讨GSPE对人膀胱癌BIU87细胞凋亡的影响及其可能机制。方法将50、100和200μg／ml的GSPE作用于人膀胱癌BIU87细胞24 h后，采用Hoechst 33258凋亡荧光染色观察细胞核形态改变，流式细胞术检测细胞凋亡率，RT-PCR及Western blot方法检测caspase-3和survivin mRNA及蛋白的表达变化。结果Hoechst 33258凋亡染色结果表明，GSPE可使BIU87细胞核呈现明显的凋亡形态学改变。流式细胞分析表明，GSPE可诱导BIU87细胞凋亡，0、50、100和200μg／mlGSPE对应的BIU87细胞的平均凋亡率分别为0．3、8．7、28．2和40．6％。RT-PCR及Western blot分析表明，随着GSPE浓度的增高，BIU87细胞caspase-3 mRNA及蛋白表达增多，survivin mRNA及蛋白表达减少，半定量分析显示各组间差别均有明显的统计学意义（P＜0．01）。结论GSPE可呈浓度依赖性诱导BIU87细胞凋亡。其机制可能与下调凋亡抑制基因survivin有关。第四部分丝裂霉素C联合葡萄籽提取物原花青素对人膀胱癌BIU87细胞的生长抑制作用目的探讨丝裂霉素C（mitomycin C，MMC）与GSPE联合应用对人膀胱癌BIU87细胞的生长抑制作用。方法采用四甲基偶氮唑蓝（MTT）比色法测定MMC与GSPE单独和联合应用时对人膀胱癌BIU87细胞生长的抑制作用，并应用Chou-Talalay联合指数（combination index，CI）法定量分析药物之间的相互作用效果。结果MMC和GSPE单独应用时，BIU87细胞生长的抑制作用随药物浓度的增加而增加，中效浓度分别为15．422μg／ml和195．865μg／ml。MMC与GSPE联合应用时，作用效果表现为在低浓度（0＜fa＜37％）时呈协同作用（CI＜1），高浓度（37％＜fa＜100％）时呈拮抗作用（CI＞1），中效浓度为109．496μg／ml（其中MMC为8．111μg／ml，GSPE为101．385μg／ml）。结论MMC与GSPE相互作用的效果在低浓度时呈协同作用，高浓度时呈拮抗作用。更多还原

【Abstract】 PartⅠEffect of growth inhibition of grape seed procyanidinextract on human bladder cancer BIU87 cellsObjective To investigate the effect of growth inhibition of grape seed procyanidinextract（GSPE） on human bladder cancer BIU87 cells and human primary skin fibroblasts.Methods BIU87 cells and human primary skin fibroblasts were cultured with differentconcentrations of GSPE（12.5, 25, 50, 100 and 200μg/ml） for 24 h. The morphologicalchanges of BIU87 cells and human primary skin fibroblasts were observed, the cellviabilities were determined by 3-（4,5-dimethylthiazol-2-y1）-2,5-diphenyltetrazoliumbromide（MTT） assay.Results Compared with the control group, 50, 100 and 200μg/ml of GSPE causedcharacteristic morphological changes in BIU87 cells. The human primary skin fibroblastsshowed no morphological changes in all groups. The viability of the BIU87 cells incubatedwith GSPE at concentrations of 12.5, 25, 50, 100 and 200μg/ml for 24 h was 99.55±0.50%（n=18, P＞0.05）, 99.16±0.63% （n=18, P＞0.05）, 86.98±1.48%（n= 18, P＜0.01）, 68.18±2.06%（n=18, P＜0.01） and 51.42±1.78% （n=18, P＜0.01） of the control value, respectively. 50, 100and 200μg/ml of GSPE inhibited the growth of BIU87 cells, while no effect on humanprimary skin fibroblasts.Conclusions GSPE, in some concentration extent, inhibites the growth of human bladdercancer BIU87 cells, and has no influence on the growth of human primary skin fibroblasts. PartⅡInhibition of cell cycle by grape seed procyanidin extractin human bladder cancer BIU87 cellsObjects To study the effect of grape seed procyanidin extract （GSPE） on cell cycle inhuman bladder cancer BIU87 cells and investigate its molecular mechanism.Methods BIU87 cells were treated with different concentrations（50, 100 and 200μg/ml） ofGSPE and cultured for 24 h in vitro while untreated group as control, flow cytometry wasused to evaluate cell cycle, and RT-PCR and Western blot were used to detect the mRNAand protein expression of cyclin D1 and CDK4.Results We found that GSPE inhibited the cell growth through cell cycle arrest at G₁ phraseby a dose-dependent manner. Semiquantitated RT-PCR and Western blot analyses indicatedthat GSPE decreased cyclin D1 and CDK4 expression significantly （P＜0.01）.Conclusions GSPE induces cell cycle arrest in BIU87 cells in vitro, and the effect mayberelated with its down-regulation of cyclin D1 and CDK4. PartⅢGrape seed procyanidin extract induces apoptosis inhuman bladder cancer BIU87 cellsObjects To study the effect of grape seed procyanidin extract （GSPE） on cell apoptosis inhuman bladder cancer BIU87 cells and investigate its molecular mechanism.Methods BIU87 cells were treated with different concentrations（50, 100 and 200μg/ml） ofGSPE and cultured for 24 h in vitro while untreated group as control, MTT[3-（4,5-dimethylthiazol-2-y1）-2,5-diphenyltetrazolium bromide] assay, hoechst 33258 stainning,flow cytometry, RT-PCR and Western blot were used to detect the apoptotic induction effectof GSPE on BIU87 cells.Results We found that GSPE induced cell apoptosis in BIU87 cells by a dose-dependentmanner. Semiquantitated RT-PCR and Western blot analyses indicated that GSPE increasedthe expression of caspase-3 and decreased the expression of survivin （P＜0.01）.Conclusions GSPE induces apoptosis in BIU87 cells in vitro, and the effect maybe relatedwith its down-regulation of survivin. PartⅣInhibitive effect of combining mitomycin C with grapeseed procyanidin extract on human bladder cancer BIU87 cellsObjective To explore the anti-cancer effects of mitomycin C （MMC） and grape seedprocyanidin extract （GSPE） on human bladder cancer BIU87 cells.Methods The 3-[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium（MTT） method wasused to detect the inhibition rates of MMC and GSPE single or combined, the combinationindex（CI） method of Chou-Talalay was used to analyze the effect of combined applicationquantitatively.Results The anti-cancer effects of MMC and GSPE were enhanced with the increasing ofdrug concentration when used alone. The media-effect concentrations were 15.422 and195.865μg/ml respectively. MMC was synergetic with GSPE （CI＜1） at low concentrations（0＜fa＜37%）, while antagonist （CI＞1） at high concentrations （37%＜fa＜100%） when usedcombined. The media-effect concentration was 109.496μg/ml （MMC 8.111μg/ml andGSPE 101.385μg/ml）.Conclusions The effect of combination between MMC and GSPE is synergetic at lowconcentrations, while antagonist at high concentrations.更多还原