【作者】 吴宜林；

【导师】 林秋华；

【作者基本信息】 中南大学 ， 妇产科学， 2008， 博士

【摘要】 宫颈癌是妇科最常见的恶性肿瘤之一。传统的治疗方法以手术和放疗为主,新辅助化疗是近十年来兴起的治疗手段,有效地改善了局部晚期患者的生活质量及预后。但仍有20%左右患者出现耐药现象,是临床治疗上棘手的问题。泛素—蛋白酶体途径是细胞内蛋白质降解的重要途径,研究表明蛋白酶体抑制剂不仅本身具有诱导细胞凋亡的作用,而且可增加化疗药物的敏感性,逆转耐药细胞株的耐药性。目前,蛋白酶体抑制剂已应用于多发性骨髓瘤的治疗,取得了令人满意的效果,被认为是具有实用价值的肿瘤治疗的新型靶向药物。然而,对宫颈癌的研究才刚起步,其诱导肿瘤细胞凋亡和增强肿瘤细胞对化疗耐药性的机制尚不清楚,对其功能的研究极具理论和临床意义。MG132是一种可逆性蛋白酶体抑制剂,在多种肿瘤细胞研究中广泛应用,为探讨MG132是否具有抗宫颈癌活性及能否增加顺铂敏感性,为蛋白酶体抑制剂辅助治疗宫颈癌的临床应用的可能性提供实验和理论依据,本研究分为三部分:（一）MG132对Hela细胞增殖凋亡的影响;（二）MG132增强Hela细胞对顺铂敏感性及其治疗裸鼠成瘤模型的疗效观察;（三）MG132逆转HCE1多细胞球体对顺铂天然耐药的研究。第一部分目的:研究蛋白酶体抑制剂MG132对人宫颈癌细胞株Hela细胞增殖和凋亡的作用,并观察MG132对Hela细胞中p27蛋白表达的影响。方法:1 MTT法检测MG132对Hela细胞的生长抑制率.（1）不同终浓度MG132培养Hela细胞24h,MTT比色法检测细胞生长抑制率和计算IC₅₀值;（2）不同终浓度MG132分别培养Hela细胞48h、72h,MTT比色法检测细胞生长抑制率。2 MG132干预后Hela细胞凋亡的形态学变化（1）倒置显微镜观察活细胞生长情况:（2）丫啶橙（AO）联合溴化乙锭（EB）双染:荧光显微镜观察对照组及MG132终浓度为10μmol/L处理组Hela细胞培养48h后细胞形态和染色变化。（3）电镜观察超微结构:观察对照组及MG132终浓度为10μmol/L处理组Hela细胞培养48h后的超微结构。3 DNA裂解片段凝胶电泳:对照组及MG132终浓度为10μmol/L处理组Hela细胞培养48h后,抽提Hela细胞DNA,行DNA裂解片段凝胶电泳。4流式细胞仪:检测MG132干预后Hela细胞凋亡率及细胞周期改变.（1）不同终浓度MG132培养Hela细胞48h后,流式细胞仪定量检测不同浓度组的细胞凋亡率.（2）经MG132终浓度为10μmol/l处理Hela细胞24h、48h、72h,流式细胞仪检测细胞凋亡率,并分析细胞周期改变。5免疫组化法检测不同终浓度MG132处理Hela细胞48h后,p27的蛋白表达。结果:1 MTT法检测MG132干预Hela细胞后的抑制率:经MG132终浓度分别为2.5μmol/L、5μmol/L、10μmol/L、20μmol/L、40μmol/L、80μmol/L、160μmol/L处理HeLa细胞24h后,细胞抑制率分别为14.13%±0.95%、21.50%±1.01%、31.33%±2.37%、33.40%±1.01%、35.77%±1.11%、79.23%±1.10%、89.62%±0.89%,IC₅₀值为62.65μmol/L;经MG132终浓度分别为2.5μmol/L、5μmol/L、10μmol/L、20μmol/L、40μmol/L处理HeLa细胞48h后,细胞抑制率分别为49.13%±1.20%、70.40%±2.17%、80.40%±1.25%、82.90%±1.71%、85.80%±1.28%;作用72h后,细胞抑制率分别为58.93%±1.63%、89.37%±1.03%、94.33%±2.06%、95.30%±1.65%、95.73%±1.75%。随MG132作用浓度升高Hela细胞抑制率增高,不同浓度MG132组之间HeLa细胞生长抑制率有显著性差异（F=3569.301,P＜0.001）;随MG132作用时间延长,HeLa细胞抑制率明显上调,且各组之间的差异有统计学意义（F=6155.339,P＜0.001）,MG132处理浓度与处理时间之间具有交互作用（F=272.722,P＜0.001）.2倒置显微镜观察:MG132处理后凋亡细胞明显增多。凋亡细胞形态为:细胞缩小变圆,细胞膜出泡等。3 AO/EB双染显示:对照组Hela细胞呈绿色均匀荧光,细胞圆形均匀。实验组可见部分Hela细胞核染色质凝聚、固缩或断裂,呈致密浓染的黄绿色荧光,或黄绿色颗粒,部分带弱桔红荧光,呈凋亡细胞表现,偶见强橘红色死细胞。4透射电镜观察:实验组部分出现细胞凋亡的典型形态学特征:细胞体积变小,细胞膜皱缩,胞核固缩,染色质固化、边集化,密度增高,帽状附于核膜,可见细胞凋亡小体。5 DNA裂解片段凝胶电泳分析:对照组DNA条带集中在样品孔附近,实验组出现明显“阶梯状”条带。6流式细胞仪检测:（1）MG132终浓度为2.5μmol/L、5μmol/L、10μmol/L、20μmol/L分别作用于HeLa细胞48h后,流式细胞仪检测细胞凋亡率分别为7.47%±0.16%、12.15%±0.52%、19.35%±0.77%、28.94%±0.18%,不同浓度MG132组与对照组的早期凋亡率有显著差异（F=1182.745,P＜0.001）。（2）MG132终浓度为10μmol/L处理Hela细胞24h、48h和72h细胞凋亡率分别为6.86%±0.40%、19.35%±0.77%、57.30%±3.68%,随时间延长凋亡率明显升高,差别均有统计学意义（F=357.090,P＜0.001）;且随药物作用时间延长,细胞周期被阻滞于G2/M期（F=49.206,P=0.001）。7免疫组化检测:MG132 10μmol/L处理Hela细胞24小时,实验组各组及对照组相互比较,HeLa细胞中p27蛋白表达明显上调,差别有统计学意义（PearsonX²=19.167,P＜0.05）。结论:（1）特异性蛋白酶体抑制剂MG132能有效抑制人宫颈癌Hela细胞的增殖,并促进其凋亡,其作用呈剂量—时间依赖关系;（2）MG132诱导细胞凋亡的作用可能与其上调p27蛋白表达有关。第二部分目的:1观察MG132能否增强Hela细胞对顺铂的敏感性,同时检测Hela细胞内NF-kB（p65）和GST-π的表达。2观察蛋白酶体抑制剂MG132联合顺铂对人宫颈癌Hela细胞的裸鼠移植瘤的治疗效果.方法:1倒置显微镜观察药物干预细胞24小时后细胞形态学变化并摄片:对照组:1640培养基;顺铂组:顺铂2.5μg/ml加DMSO处理;MG132组:MG132 5μmol/L;MG132联合顺铂组:顺铂2.5μg/ml加MG132 5μmol/L处理。2四甲基偶氮唑盐法（MTT法）检测Hela细胞的抑制率:对照组1:1640培养基处理24、48、72小时;顺铂组:分别采用不同浓度的顺铂处理Hela细胞24、48、72小时;对照组2:等体积浓度的DMSO处理24、48小时;MG132组:用蛋白酶体抑制剂MG132 5μmol/L处理Hela细胞24、48小时;MG132联合顺铂组:用不同浓度的顺铂分别加蛋白酶体抑制剂MG132 5μmol/L干预Hela细胞24、48小时。3流式细胞仪分析药物干预细胞48小时后Hela细胞凋亡率及细胞周期:对照组:DMSO处理;顺铂组:顺铂2.5μg/ml加DMSO处理;MG132组:MG132 5μmol/L处理;MG132联合顺铂组:顺铂2.5μg/ml加MG132 5μmol/L处理。4 Western blot分析测定药物干预细胞48小时后Hela细胞中NF-κB（p65）表达的变化.对照组:1640培养基加DMSO;顺铂组:顺铂2.5μg/ml加DMSO;MG132组:MG132 5μmol/L;MG132联合顺铂组:MG132 5μmol/L加顺铂2.5μg/ml.5免疫组化方法检测药物干预细胞24小时后Hela细胞内GST-π表达的变化。对照组:1640培养基加DMSO;顺铂组:顺铂2.5μg/ml加DMSO处理;MG132联合顺铂组:顺铂2.5μg/ml加MG132 5μmol/L处理.6人宫颈癌裸鼠移植瘤模型的建立（1）复苏Hela细胞,传代培养两代后,取对数生长期细胞,0.25%胰酶消化,用不含血清的1640培养基悬浮细胞,计数后定容至细胞浓度约为4×10⁷/ml.（2）将制备好的细胞悬液接种于20只裸鼠右前肢背侧皮下,每只0.2ml,观察裸鼠成瘤情况。7荷瘤鼠的分组及药物干预待裸鼠肿瘤长至40至70mm³,用随机区组的分组方法将荷瘤鼠分成4组,每组5只.分别给予腹腔注射,对照组:生理盐水0.2ml,第1天至第7天,每天一次.MG132组:MG132,5mg/kg小鼠体重,0.2ml,第1天至第7天,每天一次.顺铂组:顺铂3mg/kg小鼠体重,0.2ml,第3,5,7天,每天一次.MG132联合顺铂组:MG132,5mg/kg小鼠体重,0.2ml,第1天至第7天,每天一次,顺铂3mg/kg小鼠体重,0.2ml,第3,5,7天,每天一次.8裸鼠移植瘤的观察及相关指标的测定（1）瘤体测量绘制瘤体生长曲线:以给药的当天记第1天,以后每三天测量瘤体长,短径,计算瘤体积,绘制瘤体生长曲线。（2）计算抑瘤率和瘤体抑制率:给药后第22天断颈处死小鼠,剥出瘤体,称瘤重.计算抑瘤率及瘤体抑制率.比较各组荷瘤鼠瘤体积和瘤重的差异.（3）瘤体病理学检查.结果:1倒置显微镜观察细胞形态学变化:对照组细胞生长状态良好,细胞伸展、透亮,呈不规则对角形,细胞接触紧密;顺铂组细胞数目减少,胞浆内出现空泡,体积缩小,变圆,细胞间隙增大;MG132联合顺铂组细胞数目减少更加明显,视野内可见大量悬浮的凋亡细胞。2四甲基偶氮唑盐法（MTT法）检测Hela细胞的抑制率:与对照组1比较,顺铂组（不同浓度）的抑制率分别为:（24h）8.79%±0.49%、16.56%±0.93%、20.13%±1.22%、29.82%±1.34%;（48h）19.55%±1.23%、30.76%±0.66%、40.11%±0.99%、65.02%±0.83%;（72h）25.27%±1.19%、40.02%±0.76%、54.63%±0.58%、80.19%±0.69%;随顺铂作用浓度升高Hela细胞抑制率增高,不同浓度顺铂组HeLa细胞增殖抑制率有显著性差异（P＜0.001）;随顺铂作用时间延长,HeLa细胞细胞增殖抑制率明显上调,且各组之间的差异有统计学意义（P＜0.001）,顺铂处理浓度与处理时间之间具有交互作用（P＜0.001）;与对照组2比较,MG132组的抑制率为:（24h）21.50%±1.01%,（48h）70.40%±2.17%;MG132联合顺铂组的抑制率分别为:（24h）63.21%±0.98%、66.98%±0.78%、70.21%±1.56%、87.19%±1.77%;（48h）78.88%±0.89%、86.69%±0.54%、92.76%±1.36%、95.28%±1.21%,MG132联合顺铂组对Hela细胞的增殖抑制率显著高于顺铂组和MG132组,差异有统计学意义（P＜0.001）。3流式细胞仪分析Hela细胞凋亡率:对照组为0.96%±0.25%;顺铂组为38.77%±1.17%;MG132组为12.15%±0.52%;MG132联合顺铂组为90.12%±0.95%。MG132与顺铂联合能有效诱导Hela细胞凋亡,其诱导的凋亡率显著高于对照组、顺铂组及MG132组,差异有统计学意义（P＜0.001）。MG132联合顺铂组S期细胞百分数显著增加,各组细胞周期分布差异具有统计学意义（P＜0.001）。4 Western blotting分析测定Hela细胞中p65表达的变化:与对照组相比,顺铂组的p65表达增高,MG132组以及MG132联合顺铂组表达明显降低.各组相对灰度值进行q检验,MG132组以及MG132联合顺铂组p65的表达没有统计学差异.其他各组两两比较均有统计学差异（p＜0.05）.5免疫组化方法检测Hela细胞内GST-π表达的变化:MG132联合顺铂组与对照组、顺铂组比较,HeLa细胞中GST-π的表达明显下调,且有统计学意义（P＜0.001）。6接种的20只裸鼠全部成瘤,成瘤率100%.瘤体组织病理学检查符合低分化腺癌.7各干预组荷瘤鼠的瘤体积的变化接种的20只裸鼠全部成瘤,在接种的第7天接种部位可见粟粒大小结节,接种的第10天裸鼠瘤体体积大小为约40至70mm³.接种的第10天开始给药,记为给药的第1天.给药后第22天时断颈处死小鼠,结束时荷瘤鼠仍为20只,无一例死亡.给药第1,4,7,10,13,16,19,22天测量各组荷瘤鼠瘤体体积,自第7天开始各组荷瘤鼠瘤体积两两比较均有显著性差异（P＜0.001）.MG132组,顺铂组以及MG132联合顺铂组的瘤体生长较对照组明显缓慢.8处死荷瘤鼠时各干预组瘤重和体积的比较（1）荷瘤鼠瘤重分别为:对照组:0.86±0.12g,MG132组:0.74±0.13g,顺铂组:0.49±0.08g,MG132联合顺铂组:0.27±0.04g.各组间两两比较均差异有显著性（P＜0.001）。各干预组的荷瘤鼠瘤重比对照组明显较轻,MG132联合顺铂组瘤重较单用MG132和单用顺铂组明显轻。（2）荷瘤鼠瘤体积:对照组:1004.79±239.83 mm³,MG132组:855.75±192.52mm³,顺铂组:551.76±143.60mm³,MG132联合顺铂组:281.72±71.64 mm³.各组间两两比较均有显著性差异（P＜0.001）.各干预组的荷瘤鼠瘤体积比对照组明显较小.MG132联合顺铂组较单用MG132及单用顺铂组明显小.9处死荷瘤鼠时抑瘤率及瘤重抑制率MG132组,顺铂组,MG132联合顺铂组荷瘤鼠抑瘤率为:15%,48%,81%.MG132组,顺铂组,MG132联合顺铂组荷瘤鼠瘤重抑制率为:9%,43%,69%.MG132联合顺铂较单用MG132及单用顺铂组抑瘤率及瘤重抑制率明显增高.结论:1.MG132在体外能增加Hela细胞对顺铂化疗的敏感性.2 MG132增加Hela细胞对顺铂化疗的敏感性的作用可能与阻断NF-κB（p65）的激活和降低Hela细胞内GST-π的表达有关.3 MG132能抑制裸鼠体内Hela细胞移植瘤的生长,并增强顺铂对裸鼠Hela细胞移植瘤生长的抑制作用.第三部分目的:研究蛋白酶体抑制剂MG132及DDP对人宫颈鳞癌细胞株HCE1单层细胞及多细胞球体的增殖和凋亡作用及差异,观察HCE1多细胞球体对顺铂的天然耐药现象,探讨MG132是否可以逆转多细胞球体天然耐药以及与NF-κB（p65）及bcl-2表达的关系。方法:（一）运用液体重叠法和旋转培养法建立宫颈鳞癌细胞株HCE1多细胞球体模型观察其的生长、组织学结构并绘制球体生长曲线。（二）分别采用不同浓度的蛋白酶体抑制剂MG132（1.5、2.0、2.5、5.0、10.0、20.0umol/l）及顺铂（5.0、10.0、15.0mg/l）干预HCE1单层细胞48h,台盼兰拒染法检测细胞抑制率,分别计算MG132的IC10值及顺铂的IC50值。（三）药物干预及实验1.实验分组:按受试药物将实验分为四组,对照组:除加入同等浓度的DMSO外,均不加任何干预因素;MG132组:采用MG1322.0umol/l为受试浓度;顺铂组:采用顺铂6.0mg/ml为受试浓度;MG132+顺铂组:采用顺铂6.0mg/ml+MG132 2.0umol/l为受试浓度。2.台盼兰拒染法:各组药物干预HCE1单层细胞及多细胞球体48小时（1）光学显微镜下观察药物干预后HCE1单层细胞及球体细胞生长情况（2）台盼兰拒染法计算各组药物干预后HCE1单层细胞及多细胞球体细胞抑制率。（3）流式细胞仪检测各组药物干预后HCE1单层细胞及多细胞球体细胞周期分布及凋亡。3.HCE1单层细胞及各组药物干预多细胞球体24小时后（1）Western-Blot检测HCE1单层细胞及各组药物干预后多细胞球体NF-κB（p65）的表达。（2）免疫组化检测HCE1单层细胞及各组药物干预后多细胞球体bcl-2的表达。结果:（一）多细胞球体模型的建立:1.HCE1细胞生长状态:显微镜下见HCE1细胞生长状态良好,旋转培养24小时可见细胞聚集成团,并随培养时间的延长,结构更加紧密,直径逐渐增大。2.HCE1多细胞球体组织学结构:球体贴壁后可见中央区坏死核心、球体周围细胞生长晕;HE染色下见多细胞球体核心部位无结构红染区,周围细胞可见核分裂。3.HCE1多细胞球体生长曲线:HCE1多细胞球体培养至第10天球体生长进入平台期,球体倍增时间为4.43±0.47天。（二）台盼兰拒染法检测MG132和顺铂对HCE1单层细胞的抑制率:1.蛋白酶体抑制剂MG132浓度分别为:1.5、2.0、2.5、5.0、10.0、20.0umol/l干预HCE1单层细胞48h,细胞抑制率分别为5.89±0.48%,10.00±0.33%,20.33±1.21%,50.00±1.76%,61.67±5.00%,78.56±4.16%IC10值为2.0umol/L。2.顺铂浓度分别为:5.0、10.0、15.0ug/ml干预HCE1单层细胞48h,细胞抑制率分别为39.22±6.20%,80.34±1.53%,93.89±3.47%;IC50值6.1ug/mL。（三）药物干预及相关实验:1.各组药物干预HCE1单层细胞及多细胞球体48小时:（1）形态学观察:①对照组:HCE1单层细胞生长状态良好,胞浆透亮,细胞伸展为不规则对角形;HCE1多细胞球体生长状态良好,细胞呈圆形、细胞间连接紧密,胞浆透亮。②MG132组:HCE1单层细胞中少数细胞胞膜皱缩、变圆并脱壁,贴壁细胞间隙增宽;HCE1多细胞球体开始解聚,散落细胞增加,细胞皱缩,边缘折光增强。③顺铂组:HCE1单层细胞中角多量细胞胞膜皱缩、变圆并脱壁,贴壁细胞进一步减少;HCE1多细胞球体结构仍紧密,未见明显解聚现象。④MG132+顺铂组:HCE1单层细胞中几乎未见正常贴壁细胞,绝大多数细胞脱壁、漂浮;HCE1多细胞球体明显解聚,见大量散落细胞,边缘折光增强。（2）各组药物干预后HCE1单层细胞及多细胞球体抑制率:①对照组:HCE1单层细胞抑制率及多细胞球体抑制率均为1.00±0.00%。②MG132组:HCE1单层细胞及多细胞球体抑制率分别为11.67±2.34%、10.78±1.17%,（P＞0.05）。③顺铂组:HCE1单层细胞及多细胞球体抑制率分别为45.00±7.44%、9.45±5.98%（P＜0.05）,即HCE1多细胞球体对顺铂产生天然耐药。④MG132+顺铂组:HCE1单层细胞及多细胞球体抑制率为92.67±2.52%、91.33±2.18%,（P＞0.05）。（3）流式细胞学检测各组药物干预后细胞周期分布及凋亡:①对照组:（A）HCE1单层细胞G1/G0期细胞较HCE1多细胞球体G1/G0期细胞比例低,有显著性差异（P＜0.05）。（B）HCE1单层细胞S期细胞比例较HCE1多细胞球体S期细胞比例为高,有显著性差异（P＜0.05）。（C）HCE1单层细胞G2/M期细胞比例较HCE1多细胞球体G2/M期细胞比例稍高,两组间无显著性差异（P=0.21）。②MG132组:HCE1单层细胞及多细胞球体均出现明显G2/M期阻滞,凋亡细胞比例分别为8.14%、5.97%。③顺铂组:HCE1单层细胞出现明显S期阻滞,凋亡细胞比例为33.6%;HCE1多细胞球体未出现明显周期阻滞,凋亡细胞比例为0.88%。④MG132+顺铂组:HCE1单层细胞及多细胞球体均出现明显凋亡峰,凋亡细胞比例分别为99.0%、95.2%。2.检测HCE1单层细胞及各组药物干预多细胞球体24小时后NF-κB（p65）、bcl-2的表达:（1）western-blot检测NF-κB（p65）的表达:①HCE1多细胞球体表达NF-κB（p65）较HCE1单层细胞明显增高（P＜0.05）。②在HCE1多细胞球体中:（A）MG132组NF-κB表达显著低于对照组（P＜0.05）。（B）顺铂组NF-κB表达显著高于对照组（P＜0.05）。（C）MG132+顺铂组NF-κB（p65）表达显著低于对照组（P＜0.05）,但较MG132组无显著性差异（P＞0.05）。（2）免疫组化检测bcl-2的表达:①单层细胞低表达bcl-2,多细胞球体表达bcl-2明显增强。②在多细胞球体中:（A）MG132组bcl-2表达强度低于对照组。（B）顺铂组bcl-2表达强度高于对照组。（C）MG132+顺铂组bcl-2表达强度低于对照组。经统计学分析:各组较对照组有统计学差异（P＜0.05）,MG132组较MG132+顺铂组无显著性差异（P＞0.05）。结论:1、HCE1多细胞球体模型对顺铂可产生天然耐药现象,是体外研究宫颈癌细胞株HCE1肿瘤耐药的良好模型。2、MG132对HCE1多细胞球体有抑制增殖诱导凋亡作用,可部分逆转HCE1多细胞球体对顺铂的天然耐药性。3、MG132干预后HCE1多细胞球体对顺铂天然耐药性的部分逆转可能与NF-κB（p65）、bcl-2表达下调有关。更多还原

【Abstract】 Cervical cancer is the one of the most common gynecological malignancy.Traditional therapy was based on surgery and radiotherapy. Neoadjuvant Chemotherapy is a new treatment rising in the past 10 years,which can effectively improve the quality of life and prognosis for the patient with locally advanced cervical cancer.However there are still about 20%of the patients having a drug-resistant phenomenon,to become a formidable problem in our clinical therapy.Ubiquitin-proteasome pathway plays a key role in the intracellular protein degradation.Research shows that proteasome inhibitors can not only induce the apoptosis of tumor cells but also increase the sensitivity of the chemotherapeutics and reverse the chemoresistance of drug-resistant cell lines.At present,the proteasome inhibitors have been used in multiple myeloma treatment and got satisfactory therapeutic efficacy.Given this important role the proteasome represents a novel target for chemotherapy with practical value.Nevertheless,the study of the proteasome inhibitor on the solid tumor is just at its starting stage and its mechanism of action is not yet clear,so it is of theoretical and clinical significance to investigate its function.MG132,a reversible proteasome inhibitor,has been used for the study of several tumors.To probe whether MG132 can enhance the sensitivity of cisplatin to cervical cancer cells and offer an experimental and theoretical basis on the possibility for the clinical application of proteasome inhibitor adjuvant therapy to cervical cancer this study which contains three parts:（1）The effect of MG132 on cell proliferation and apoptosis of human cervical Hela cells.（2）The observation whether MG132 could sensitize the Hela cells to cisplatin and inhibit the xenografts of Hela cells on the nude mice.（3）The research of MG132 on reversal of natural drug-resistance to cisplatin of HCE1/MCS Part OneObjectives:To investigate the effect of MG132 on cell proliferation and apoptosis of human cervical Hela cells in vitro and observe the influence of MG132 on the expression of p27.Methods:1 Detection of the cellular growth inhibition ratio by MTT assay.（1） HeLa cells were cultured and treated by MG132 with different concentrations for 24 hours respectively.Cellular viability and IC₅₀ were measured by MTT assay.（2）HeLa cells were cultured and treated by MG132 with different final concentration for 48 and 72 hours and the cellular growth inhibition ratio were detected by MTT assay respectively.2 The morphological features of the apoptosis of HeLa cells induced by MG132（1） we observed the morphological features of apoptotic cells induced by MG132 with inverted microscope.（2） Acridine orange combined with Ethidium bromide staining:Hela cells were treated with 10μmol/L MG132.Afterwards they were collected 48 hours later and observed after acridine orange combined with EB staining by fluorescence microscope.（3） Transmission electron microscope was used to observe the morphological alteration of Hela cells treated with 10μmol/L MG132 for 48 hours.3 The biochemistry feature of HeLa cells treated with MG132: After treated with MG132 10μmol/L for 48 h,Hela cells were collected and DNA was abstracted for gel electrophorosis.4 Analysis of apoptosis rate and cell cycle of HeLa cells through flow cytometry been exposed to MG132.（1） HeLa cells were cultured and treated by MG132 with disdinct concentrations for 48h,the apoptotic rate of HeLa cells were analyzed by flow cytometry after PI staining respectively.（2）Treated with MG132 10μmol/1 for 24,48 and 72 hours,the apoptotic rate and cell cycle distribution were examined through flow cytometry after PI staining.5 Immunocytochemistry was explored to detect the expression of p27 protein of different groups in which HeLa cells treated by MG132 with various final concentrations for 24 hours respectively.Results:1 After treated with MG-132,the growth of Hela cells were inhibited obviously.There were statistically significant diffferences of the proliferative inhibition among different experimental groups （F=3569.301,P＜0.001）,and there were also statistically significant differences of the proliferative inhibition rates for different duration of treatment（F=6155.339,P＜0.001）.There was an interaction between different concentrations and different duration by MG132（F=272.722, P＜0.001）.2 Hela cells became round,small,cell shrinkage,membrane blebbing and so on after being exposed to MG132.3 Acridine orange combine with Ethidium bromide staining showed that most of the cells in control group appeared fluorescent green uniform.But in experement group,most cells were yellow-green fluorescence.4 DNA ladder exist in experiment group.5 The typical ultrastructure of apoptosis cell was found by electron microscope after the cells treated with MG132:cell shrinkage,nucleus pycnosis,chromatin margination and apoptotic body can be seen.6 Quantification of the apoptosis effect by flow cytometric analysis showed that it was in a good correspondence with the loss in cell viability induced by MG132.There were statistically significant differences among experimental groups with different density of MG132 （F=1182.745,P＜0.001）;Meanwhile,treated with MG132 10μmol/l for 24,48 and 72 hours,the apoptotic rate of HeLa cells advanced obviously, and there were significant differences among experimental groups （F=357.090,P＜0.001） Cell cycle arrested at G2/M phase when the duration of treatment prolonged.（F=49.206,P=0.001）.7 In contrast to control group,The p27 protein expression level obvious up-regulated in MG132 group.（P＜0.05）.Conclusions:1 These observations suggested that the proteasome inhibitor MG132 could actively suppress the proliferation and promote apoptosis of human cervical carcinoma cell line HeLa cells in a dose and time-dependent manner. 2 The effect of apoptosis induced by MG132 in cervical cancer cells were proposed to be followed by p27-overexpressing. Part TwoObjective 1 To observe whether proteasome inhibitor MG132 can enhance the sensitivity of cisplatin to HeLa cell and detect the expression of GST-πand NF-κB.2 To observe the therapeutic efficacy of proteasome inhibitor MG132 combined with cisplatin on the xenografts of Hela cells on the nude mice.Methods 1 Inverted microscope was used to observe the morphological features and take photos of HeLa cells:HeLa cells were divided into four groups:the first group was just cultured by culture medium for 24h;the second group was treated by cisplatin2.5μg/ml for 24hours;the third group was treated by MG132 5μmol/l for 24hours;the forth group was treated by MG132 5μmol/l combined with cisplatin 2.5μg/ml for 24hours.2 MTT assay was used to detect the growth inhibition rate of HeLa cells:The first control group were only treated by culture medium; cisplatin group were treated by different concentrations of cisplatin for 24、48、72 hours respectively;the second control group were treated by DMSO which have the same concentrations with MG132 group;MG132 group were treated by MG132 5μmol/1 for 24、48 hours;MG132 combined with cisplatin group were treated by different concentrations of cisplatin respectively combined with MG132 5μmol/1 for 24 and 48 hours.3 PI staining flow cytometry was used to confirmed the apoptotic rates and cell cycles of HeLa cells:HeLa cells were divided into four groups:the first group was just treated by DMSO for 48hours;the second group was treated by cisplatin2.5μg/ml and DMSO for 48hours;the third group was treated by MG132 5μmol/l for 48hours;the forth group was treated by cisplatin2.5μg/ml combined with MG132 5μmol/l for 48hours.4 Western blotting was used to detect the expression of p65 of HeLa cells:HeLa cells were divided into four groups:the first group was just cultured by culture medium;the second group was treated by MG132 5μmol/l for 48hours;the third group was treated by cisplatin2.5μg/ml for 48hours;the forth group was treated by MG132 5μmol/l combined with cisplatin2.5μg/ml for 48hours.5 Strep tavidin-peroxidase conjugated method（SP） of immunocytochemistry was used to detect the expression of GST-πin HeLa cells:HeLa cells were divided into three groups:the first group was just cultured by DMSO for 24hours;the second group was treated by cisplatin2.5μg/ml and DMSO for 24hours;the third group was treated by MG132 5μmol/l combined with cisplatin2.5μg/ml for 24hours.6 Establishment of the xenografts model of Hela cells on the nude mice. （1） Cells were cultured to logarithm phrase and digested with EDTA and then adjusted density of cells to 4×10⁷/ml.（2） Inoculability in nude mice:We inoculated the cells on the right anterior limbs of the nude mice by hypodermic injection under the sterile condition and observed the growth state of xenografts on the nude mice.7 The grouping and drug treatment of the bearing cancer mice.When the volum of the xenografts reached 40-70mm³,we divided the nude mice into four stochastic groups.There were 5 mice in every group and managed by the way there in after.1) control group:0.2ml 0.9%saline,i.p,2)MG132 group:MG132 5mg/kg,0.2ml,i.p,3)cisplatin group:cisplatin 3mg/kg,0.2ml i.p,4) MG132 with cisplatin group: MG132 5mg/kg,0.2ml and cisplatin 3mg/kg,0.2ml i.p.8 Inspection of xenograft’s growth and detection of the correlated index.（1）Measurement of xenograft and draw growth curve:to mark the day starting treated as the first day,then measure the diameter of the xenograft per trid,calculate the volume and draw growth curve.（2） Calculation of tumor inhibiting rate and inhibiting rate of the weight of xenograft:we measured the weight of the 22th-day mice xenograft,calculated tumor inhibiting rate and inhibiting rate of the weight of xenograft and compared the differences of the volum and weight of xenograft in every group.（3） Inspection of pathology and histology:some of xenografts were fixed by 10%formaldehyde.Then they were dehydrated and embed. After slicing up and HE stain.They were observed by light microscope.Results 1 Morphological features was observed by inverted microscope:compared with culture medium group and cisplatin group, we observed that the cells in the MG132 combined with cisplatin group have changed a lot,most cells turned round and small and had balloon inside.2 MTT assay:there statistical significant diffferences of the proliferative inhibition rates among the groups with different concentration of cisplatin（P＜0.001） and there were also statistically significant differences of the proliferative inhibition rates among cisplatin groups with different durations（P＜0.001）.The effect of different concentrations and different durations of cisplatin on cell growth was interactive（P＜0.001）.There were also statistically significant diffferences of the proliferative inhibition rates among the cisplatin, MG132,MG132 combined with cisplatin groups（P＜0.001）.3 Flow cytometric analysis:the MG132 combined with cisplatin group induced apoptosis of HeLa cells more effectively than the control,cisplatin and MG132 groups,and there were statistically significant differences among experiment groups（P＜0.001）.Cell cycle arrested at S phase significantly.4 Western blotting:compared with the control group,the expression of p65 in cisplatin group was obviously up-regulated （P＜0.05）.Compared with the control group,expression of p65 in MG132 and th MG132 combined with cisplatin groups was obviously down-regulated（P＜0.05）.But there was no statistically significant differences between MG132 and MG132 combined with cisplatin groups. （p＞0.05）.5 Immunocytochemistry:compared with the control group and the cisplatin group,the expression of GST-πshowed a clear decrease among the MG132 combined with cisplatin groups（P＜0.001）。6 Comparison of the weight and volum of xenograft:when the nude mice were sacrificed,the weight of the xenografts of every group are:control group:0.86±0.12g,MG132 group:0.74±0.13g,cisplatin group:0.49±0.08g,the MG132 with cisplatin group:0.27±0.04g.The volume of the xenografts of every group are:control group:1004±239.83mm³,MG132 group:863±192.52mm³,cisplatin group:551±143.60mm³,the MG132 with cisplatin group were 236±71.64mm³ （P＜0.001）.The analysis of variance was used to examine the differences between the four groups on the volume and the weight of the xenografts on the nude mice.There were statistically significant differences.（p＜0.05）7 Tumor inhibiting rate and inhibiting rate of the weight of xenograf:comparing with the control group,the tumor inhibiting rates of MG132 group,cisplatin group and the MG132 with cisplatin group were 15%,48%,81%,the inhibiting rates of the weight of the xenografts were 9%,43%,69%respectively.Conclusion 1.Proteasome inhibitor MG132 can enhance the sensitivity of cisplatin to HeLa cell in vitro.2.The effect of enhancement of MG132 on sensitivity of cisplatin to Hela cells was proposed to be followed by the down-regulation of p65 and GST-π3.MG132 can inhibit the xenografts of Hela cells on the nude mice and can increase the inhibition effect of cisplatin to them. Part ThreeObjectives To investigate the effect of the UPP inhibitor MG132 and cisplatin on proliferation and apoptosis of human cervical carcinoma cell line HCE1 monolayer and MCS and to compare the difference between the two cell culture models.To observe whether HCE1/MCS is resist to the treatment of cisplatin and to probe if MG132 can reverse the HCE1/MCS resistance to cispaltin,as well as the relationship between the effect and the expression of NF-κB、bcl-2.Methods 1 An HCE1 multicellular spheroids model was establish using liquid overlay technique and rotating culture technique.To observe the growth and histological structure of HCE1/MCS and draw the growth curve.2 HCE1 monolayer cells were cultured and treated with MG132 （1.5umol/L、2.0umol/L、2.5 umol/L、5.0 umol/L、10.0 umol/L、20.0 umol/L） and cispaltin（5.0ug/mL、10.0ug/mL、15.0ug/mL） for 48 hours respectively and cell viability was detected by trypan blue exclusion assay with IC₁₀ and IC₅₀ calculated respectively.3 Drug treatment and experiment:（1）.Grouping:according to the combinations of drugs,4 groups were established.Control group:cells were treated with the same concentration of DMSO;MG132 group:cells were treated with MG132 （2.0umol/L）;Cisplatin（DDP） group:Cells were treated with cisplatin （6.0ug/mL）;MG132 and cisplatin group:cells were treated with cisplatin 6.0ug/mL + MG132 2.0umol/L.（2）.HCE1/monolayer and HCE1/MCS were cultured and treated with the 4 groups of drugs mentioned above for 48 hours,1) cell growth was observed by light microscope.2) cell viability were measured by trypan blue exclusion assay.3) cell cycle and apoptosis were detected by flow cytometer.（3）.HCE1/monolayer and HCE1/MCS were cultured and treated with 4 groups of drugs mentioned above for 24 hours,1) The expression of NF-κB（p65） was detected by western-blot assay.2) bcl-2 expression was detected by immunohistochemistry.Results 1 Establishment of HCE1 multicellular spheroid model:（1）. Growth state of HCE1 monolayer cells:in a good condition and after rotating for 24h,cells began to aggregate into multicellular spheroids and became more and more compact and larger in size with the culture time prolonged.（2）.Histological structure of HCE1/MCS:central necrosis core and cell outgrowth around the MCS were observed when MCS got adherence.Non-structure red-stain region was observed using HE stain. （3）.Growth curve of HCE1/MCS:the growth of HCE1/MCS turned into platform after 10 days.The double time of HCE1/MCS cells was 4.43±0.47 days. 2 Growth inhibition rate of HCE1 induced by MG132 and cisplatin tested by trypan blue exclusion assay.（1）HCE1/monolayer cells were cultured and treated with MG132 1.5umol/L、2.0umol/L、2.5 umol/L、5 umol/L、10 umol/L、20 umol/L for 48 hours,growth inhibition rates were:5.89%±0.48%,10.00%±0.33%,20.33%±1.21%, 50.00%±1.76%,61.67%±5.00%,78.56%±4.16%respectively,IC₁₀ was 2.0umol/L.（2）HCE1/monolayer cells were cultured and treated with DDP 5.0ug/mL、10.0 ug/mL、15.0 ug/mL for 48 hours,cell viability were 39.22%±6.20%,80.34%±1.53%,93.89%±3.47%respectively.IC50 is 6.1mg/L.3 Drug treatment and experiment:（1）.After treated with 4 groups of drugs for 48 hours.1)Morphology viewing:①control group:HCE1/monolayer cells grew in good condition, adherent to irregular diagonal corniform.HCE1/MCS were also in good condition,cells were round,adhered to each other.②MG132 group: several cells shrinkage,turned round,and detached which were observed in monolayer cells.A few of multicellular spheroids’ disaggregation and shrinkage were observed in HCE1/MCS③Cisplatin group:A lot of cells shrinkage,turned round,and detached which were observed in monolayer cells.No more change were observed in HCE1/MCS.④MG132 and cisplatin group:The majority of cells shrinkage,turned round and detachment were observed in monolayer cells.Disaggregation and shrinkage were observed in HCE1/MCS.2) After treated with 4 groups of drugs for 48 hours:①control group:cell inhibition rate were 1.00%±0.00%both in monolayer and MCS groups.②MG132 group:cell inhibition rate of HCE1/monolayer and HCE1/MCS were 11.67%±2.34%and 10.78%±1.17%respectively.There was no significant difference between the tow groups（P＞0.05）③Cisplatin group:cell inhibition rate of HCE1/monolayer and HCE1/MCS were 45.00%±7.44%and 9.45%±5.98%respectively.There were significant differences.（P＜0.05）④MG132 and cisplatin group:Cell inhibition rate of HCE1/monolayer were 92.67%±2.52%、91.33%±2.18%respectively.There was no significant difference between them.（P＞0.05）3) Cell cycle and apoptosis in experiment groups were detected by flow cytometer.①control group:HCE1/MCS had a much higher G1/G0 stage cell （P＜0.05） and less S stage（P＜0.05） than HCE1/monolayer.There was no significant difference in G2/M stage cells between HCE1/MCS and HCE1/monolayer（P=0.21）.②MG132 group:apoptosis and G2/M stage block were observed in both HCE1/monolayer and HCE1/MCS.③Cisplatin group:neither apoptosis nor S stage block were observed of HCE1/MCS.④MG132 and DDP group:Apoptosis were observed in both HCE1/monolayer and HCE1/MCS,with apoptosis rate of 99.0%and 95.2%respectively.（2）.After treated with 4 groups of drugs for 24 hours.1) NF-κB expression was detected by western-blot assay:①HCE1/MCS cells expressed higher NF-κB than HCE1/monolayer.（P＜0.05）②In multicelluar spheroids:（A） MG132 group:NF-κB expression was significantly lower that of the control group.（P＜0.05）（B） Cisplatin group:NF-κB expression was significantly higher than that of the control group.（P＞0.05）（C） MG132 and cisplatin group:NF-κB expression was significantly lower than that of the control group.（P＜0.05）,there was no significant change after treated with MG132 and cisplatin+ MG132.2) bcl-2expression was analysed by immunohistochemistry:①bcl-2 expression was stronger in HCE1/MCS than in HCE1/monolayer.②（A） MG132 group:bcl-2 expression was significantly lower than that of the control group.（P＜0.05）（B） DDP group:bcl-2 expression was significantly higher than that of control group.（P＞0.05）（C） MG132 and cisplatin group:bcl-2 expression was significantly lower than that of the control group.（P＜0.05）,and there was no significant difference between the groups exposed by MG132 and cisplatin + MG132（P＞0.05）.Conclusions 1 HCE1/MCS present natural resistance to cisplatin and it may become a good model for the study of tumor chemoresistance. 2.MG132 can induce inhibition and apoptosis of HCE1/MCS cells, up-regulate the sensitivity to cisplatin of HCE1 cells and partially reverse the natural resistance of HCE1/MCS to cisplatin.3.The partially reversion of the natural resistance to cisplatin induced by MG132 in HCE1/MCS were proposed to be in relation to the up-regulation of NF-κB、bcl-2 expression.更多还原