【作者】 白宇；

【导师】 谢立平；

【作者基本信息】 浙江大学 ， 外科学， 2008， 博士

【摘要】 第一部分白藜芦醇对人膀胱癌T24细胞增殖和细胞周期的影响目的研究白藜芦醇在体外对人膀胱癌细胞系T24细胞增殖和细胞周期的影响及其可能机制。方法采用MTT法观察不同浓度白藜芦醇(0,25,50,100,150,200和250μM)在不同的作用时间(12,24,和48h)后,T24细胞增殖的变化。收集不同浓度白藜芦醇(0,50,100,150和200μM)作用24小时后的T24细胞,碘化丙锭(PI)染色后经流式细胞仪检测膀胱癌T24细胞的周期变化。Western blotting法检测与细胞周期调节相关蛋白cyclin D1、CDK4和WAF-1/p21在不同浓度白藜芦醇(0,50,100,150和200μM)作用24小时后的表达。结果1.白藜芦醇能明显抑制T24细胞增殖,并随药物浓度升高(0-250μM)和作用时间的延长(12-48h)而抑制作用增强,呈显著浓度和时间依赖性。2.在白藜芦醇(0-250μM)作用12h后,T24细胞生存率范围为49.8%-97.0%,在作用24和48 h后,细胞生存率范围分别为27.7%-98.0%和20.6%-95.7%。3.经流式细胞仪检测,不同浓度白藜芦醇(50,100,150和200μM)作用24小时能明显使处于G1期T24细胞增多,细胞明显阻滞于G1期。4.白藜芦醇(0,50,100,150和200μM)作用24小时后,随着作用浓度的增加,T24细胞Cyclin D1和CDK4的表达逐渐降低,而WAF-1/p21表达逐渐增强,呈显著的浓度依赖性。结论白藜芦醇能显著抑制T24细胞增殖,其抑制作用呈时间和剂量依赖性。白藜芦醇可以T24细胞明显阻滞于G1期,其分子机制可能是抑制T24细胞中Cyclin D1和CDK4的表达,并增强了WAF-1/p21蛋白的表达。第二部分白藜芦醇对人膀胱癌T24细胞凋亡的影响目的研究白藜芦醇在体外对人膀胱癌细胞系T24细胞凋亡的诱导作用,并探讨其诱导凋亡的相关分子机制。方法采用流式细胞仪(PI和annexin V双染法)检测T24细胞凋亡的变化。采用Western blotting方法检测不同浓度白藜芦醇(0,50,100,150和200μM)作用24小时后,T24细胞中PI3K/Akt、ERK1/2和p38三条信号通路的改变,同时研究了Bcl-2家族、caspase-3和caspase-9等蛋白在白藜芦醇作用后表达的变化。结果1.不同浓度白藜芦醇(0,50,100,150和200μM)作用于T24细胞24小时后能明显诱导细胞凋亡,凋亡的诱导作用呈显著剂量依赖性。2.光学显微镜显示不同浓度白藜芦醇(0,50,100,150和200μM)作用于T24细胞24小时后使细胞密度呈剂量依赖性降低,细胞形态改变也呈剂量依赖性,同时观察到凋亡细胞所具有的形态学改变。3.通过流式细胞仪(PI和annexin V双染法)检测发现白藜芦醇作用于T24细胞24小时后凋亡细胞数量呈剂量依赖性增加。白藜芦醇(0-200μM)作用24小时后产生的早期凋亡细胞为4.0%-14.1%;晚期凋亡细胞分别为2.6%-36.7%,总的凋亡细胞为6.6%-47.5%。4.白藜芦醇(0-200μM)作用24小时后,随着作用浓度的增加,T24细胞phospho-Ser473-Akt与phospho-ERK1/2的表达逐渐降低,但t-Akt与t-ERK1/2未见改变;phospho-p38表达逐渐增强,t-p38表达未见改变,三种信号蛋白表达变化均呈显著的浓度依赖性。5.白藜芦醇(0-200μM)作用24小时后,随着作用浓度的增加,T24细胞中Bcl-2与Bcl-xL的表达逐渐降低,而Bax表达逐渐增高,呈显著的浓度依赖性。6.白藜芦醇(0-200μM)作用24小时后,随着作用浓度的增加,T24细胞中Bax/Bcl-2的比例逐渐增高,呈显著的浓度依赖性。7.白藜芦醇(0-200μM)作用24小时后,T24细胞procaspase-3与procaspase-9表达降低,而caspase-3与PARP激活片段表达逐渐升高。结论白藜芦醇能诱导T24细胞凋亡,凋亡的诱导作用呈显著剂量依赖性。白藜芦醇能显著抑制T24细胞PI3K/Akt信号通路的激活,调节MAPK通路的信号变化(ERK1/2与p38),进而引起Bcl-2家族蛋白变化,激活caspase-3,最终诱导凋亡发生。第三部分白藜芦醇对人膀胱癌T24细胞侵袭转移的影响目的研究白藜芦醇在体外对人膀胱癌细胞系T24细胞侵袭转移能力的影响及其可能机制。方法MTT法检测白藜芦醇对T24细胞粘附纤维连接蛋白(fibronectin)能力的影响,Transwell小室进行人工重组基底膜侵袭实验检测白藜芦醇对T24细胞侵袭能力的影响,划痕愈合试验检测白藜芦醇对T24细胞迁移能力的影响。采用Western blotting方法检测白藜芦醇(0,10,25,和50μM)作用24小时后的T24细胞中MMP-9蛋白的表达。结果1.白藜芦醇(0-200μM)作用90分钟后,随着作用浓度的增加,T24细胞粘附纤维连接蛋白能力逐渐降低,呈显著的浓度依赖性。2.白藜芦醇(0-50μM)作用不同时间(0,8,16和24小时)后,T24细胞的迁移能力逐渐降低,呈显著的浓度和时间依赖性。3.体外的侵袭试验显示白藜芦醇(0-50μM)作用24小时后,随着作用浓度的增加,T24细胞侵袭能力逐渐降低,呈显著的浓度依赖性。4.白藜芦醇(0-50μM)作用24小时后,随着作用浓度的增加,T24细胞的MMP-9蛋白表达逐渐降低,呈显著的浓度依赖性。结论白藜芦醇能显著抑制T24细胞的粘附、迁移和侵袭能力。白藜芦醇这种侵袭转移抑制能力可能与降低MMP-9的表达有关。第四部分白藜芦醇抗肿瘤作用的体内实验研究目的研究白藜芦醇对裸鼠膀胱癌移植瘤生长的抑制作用及其可能机制。方法无菌条件下,裸鼠右后背侧部皮下接种膀胱癌T24细胞悬液,接种后,裸鼠在无菌、恒温和恒湿条件下饲养,待肿瘤直径长成约3mm大小时,采用随机数字法随机将24只Balb/C裸鼠分为3组,A组不予任何处理;B组腹腔内注射0.1ml丙二醇;C组腹腔内注射白藜芦醇(溶于0.1ml丙二醇,30 mg/kg)。每日给药1次,持续4周,每2日测量一次肿瘤体积。第28天处死裸鼠后取下标本,免疫组织化学法观察肿瘤组织CD34的变化,RT-PCR法检测肿瘤组织相关基因VEGF和FGF-2的表达情况。结果1.持续观察4周后,A组与B组移植瘤生长情况无明显区别,肿瘤体积变化无明显区别;实验前十天,C组移植瘤生长情况与另外两组无明显区别,从实验第十二天,C组肿瘤体积开始明显小于另外两组。2.通过免疫组织化学法观察肿瘤组织中CD34的变化,发现白藜芦醇处理组中,肿瘤标本CD34表达明显降低。3.RT-PCR法分析肿瘤组织基因后发现,白藜芦醇处理组中,VEGF和FGF-2的表达明显降低。结论白藜芦醇可以明显抑制裸鼠膀胱癌移植瘤的生长,这种作用可能与白藜芦醇降低肿瘤组织中CD34、VEGF和FGF-2的表达有关。更多还原

【Abstract】 Part 1 Effects of resveratrol on proliferation of human bladder cancer T24 cellsObjective The present study was undertaken to examine the effects of resveratrol on growth inhibition,cell cycle and corresponding molecular mechanism of T24 cells.Methods The effect ofresveratrol on human bladder cancer T24 cells was evaluated with varying concentration of resveratrol(0,50,100,150,200 and 250μM) treatment for 12,24 and 48 hours by MTT assay.The cell cycle arrest effect of resveratrol on human bladder cancer T24 cells was determined with the indicated concentration ofresveratrol treatment for 24 hours by flow cytometry.The cell cycle modulating protein WAF-1/p21,cyclin D1 and CDK4 was determined by western blotting analysis.Results1.Using the T24 human bladder cancer cell line,we found that resveratrol treatment exerted a significant cytotoxic effect upon T24 cells in a dose-dependent(0-250μM)and time-dependent(0-24 hours)manner.2.The cell viability with resveratrol treatment at concentrations of 0-250μM after 12 hours ranged from 49.8%-97.0%,whereas after 24 and 48 hours ranged from 27.7%-98.0%and 20.6%-95.7%respectively.3.Compared with the vehicle-treated controls,the resveratrol treatment resulted in an appreciable arrest of T24 cells in G1 phase of cell cycle.It was observed that the G1 phase population of the untreated cells was 47.6%and the percentage of cells in G1 phase was significantly increased(74.7%,78.2%,78.3%and 73.5% cells at 50,100,150,and 200μM concentrations of resveratrol,respectively)after 24 hours of the treatment.4.Using immunoblot analysis,we found that resveratrol treatment resulted in a significant dose-dependent activation of WAF-1/p21.Also,the results revealed that resveratrol treatment of T24 cells leads to a dose-dependent decrease in protein expression of cyclin D1 and CDK4.Conclusions Our data suggested resveratrol treatment resulted in a significant doseand time-dependent Inhibition in the growth of T24 Cells.Our study also suggests that resveratrol causes cell cycle arrest in G1 phase from the modulation of WAF-1/p21,cyclin D1 and CDK 4.Part 2 Resveratrol induced apoptosis of human bladder cancer T24 cellsObjective The present study was undertaken to examine the effects of resveratrol on apoptosis of human bladder cancer T24 cells and to identify the altered signaling pathway(s)underlying the response to resveratrol exposure.Methods We evaluated the effects of resveratrol on apoptosis of T24 cell by annexin V-FITC and flow cytometry.The extent of apoptosis was quantified by flow-cytometric analysis of resveratrol-treated cells labeled with PI and annexin V. Alterations in signaling events were determined in Western blot analysis probing for phosphorylated MAPK(ERK1/2 and p38)and Akt proteins,indicative of activation. The role of caspase-3,caspase-9,PARP,Bcl-2 family proteins in apoptosis was also analyzed by Western blotting.Results 1.Using the T24 human bladder cancer cell line,we found that resveratrol treatment resulted in induction of apoptosis in dose-dependent manner(0-200μM).2.After the treatment for 24 hours,it was observed that treatment of T24 cells with 0-100μM of resveratrol increased the number of early apoptotic cells from 4%to 14.1%,which decreased from 14.1%to 10.0%further after 100μM resveratrol treatment.The number of late apoptotic cells increased from 2.6%to 36.7%.The total percent of apoptotic cells was directly related to resveratrol concentration increasing from 6.6%to 47.5%3.Western blot analysis indicated that treatment of T24 cells with resveratrol resulted in a dose-dependent activation of caspase-3,caspase-9 and PARP proteins 24 hours after resveratrol treatment.4.After 24 hours treatment ofresveratrol,the phosphorylation of Akt and ERK1/2 was decreased,while the phosphorylation of p38 was increased and the results suggested that the levels of both phosphorylated Akt,ERK1/2 and phosphorylated p38 are dose-dependent in response to resveratrol.However,the total Akt,ERK1/2 and p38 expression were not affected in T24 cells.5.Resveratrol treatment of T24 cell lines resulted in a decrease in antiapoptotic Bcl-2 and Bcl-xL protein levels,whereas a concomitant increase in proapoptotic Bax proteins.The levels of these proteins are dose-dependent in response to resveratrol.6.The ratio of Bax/Bcl-2 was significantly increased in a dose-dependent manner with resveratrol treatment.Conclusions Our data suggested that treatment of resveratrol on T24 cells could leads to modulation of Akt and MAPK(ERK1/2 and p38)pathways that,in turn, results in modulations in Bcl-2 family proteins,such as Bcl-2,Bax and Bcl-xL.Then, caspases are activated in such a way that the apoptosis of T24 cells is initiated. Based on these studies,we suggest that resveratrol could be developed as an agent for the management of bladder cancer. Part 3 Effects of resveratrol on invasion and metastasis of human bladder cancer T24 cellsObjective The present study was undertaken to examine the effects of resveratrol on invasion and metastasis of human bladder cancer T24 cells and to identify the possible mechanisms.Methods To evaluate the antimetastatic activity of resveratrol,we first assessed the inhibitive effect of resveratrol on the adhesion of T24 cells to fibronectin with MTT assay.Wound healing assay and the in vitro invasion assay were used to investigate the effect of resveratrol on the migration ability and invasion ability of T24 cells.The role of MMP-9 protein levels was analyzed by Western blotting.Results1.Usingthe T24 human bladder cancer cell line,we found that resveratrol treatment resulted in dose-dependent(0-200μM)inhibition of the adhesion activities.2.The cellular migration ability was evidently inhibited in dose-dependent(0-50μM)and time-dependent(0-24 hours)manner by resveratrol.3.Similarly,the results of the in vitro invasion assay also displayed that resveratrol was able to inhibit invasion ability of T24 cells in dose-dependent manner(0-50μM).4.Western blot analysis indicated that treatment of T24 cells with resveratrol resulted in a dose-dependent(0-50μM)decrease in MMP-9 protein levels.Conclusions resveratrol elicited a significant inhibition of in vitro cell adhesion, migration,and invasion in human bladder carcinoma T24 cells.The inhibition of invasion ability of T24 cells by resveratrol was shown to may be attributed to decreases of the expression of MMP-9.Thus,clinical application of resveratrol may contribute to the potential benefit for suppression of bladder cancer invasion and metastasis. Part 4 Inhibition effects of resveratrol on bladder cancer xenografts in vivoObjective Evaluate the effects of resveratrol on bladder cancer xenografts in vivo and investigate the underlying mechanism.Methods T24 cells were injected subcutaneously on the right flank of Male BALB/c-nude mice.At a tumor size of approximately 30 mm~3 the mice were divided into three groups.Group A received no treatment.Groups B and C were treated with intraperitoneal injection of propylene glycol(vehicle,0.1 ml),30 mg/kg of resveratrol (in 0.1 ml ofpropylene glycol),once daily for 4 weeks,respectively.CD34 expression of xenografts was evaluated by immunohistochemistry.VEGF and FGF-2 mRNA level were analyzed by RT-PCR.Results1.The tumor growth in the resveratrol group was significantly slower than that in the vehicle and control groups.In the first 10 days,tumor volume was not different among three groups.In contrast,tumor volume in group A and group B were significant larger than group C from days 12 to 28.2.By immunohistochemistry,CD34 expression of xenografts was significantly reduced in the resveratrol group.3.VEGF and FGF-2 mRNA level was decreased significantly in the resveratrol group analyzed by RT-PCR.Conclusions In the nude mice xenograft model,resveratrol were capable of inhibiting tumor growth.The inhibition effects of resveratrol may be attributed to decreases of the expression of CD34,VEGF and FGF-2.更多还原