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1、上海交通大学硕士学位论文COX-2选择性抑制剂塞来昔布对人子宫内膜腺癌抑制作用的研究姓名：肖义涛申请学位级别：硕士专业：妇产科学指导教师：罗来敏20070501COX-2 选择性抑制剂塞来昔布对人子宫内膜腺癌抑制作用的研究中文摘要目的：探讨 COX-2 选择性抑制剂塞来昔布对人子宫内膜腺癌的体外及体内抑制作用及其相关机制，为治疗子宫内膜腺癌提供实验依据。方法：一体外试验以不同浓度的塞来昔布（25-200umol/L）分别以不同时间处理体外培养的 HEC-1B 细胞，用 MTT 法检测细胞增殖，用流式细胞术检测细胞周期和凋亡，用透射电镜观察细胞超微结构的改变，用 Transwell小室测定细胞侵
2、袭力，用 RT-PCR 检测 COX-2 mRNA 的表达。二体内试验130 只裸鼠皮下注射 HEC-1B 细胞，建立人子宫内膜癌裸鼠荷瘤模型。待成瘤后，裸鼠随机分为对照组与实验组，对照组予生理盐水口服 2 周,实验组分别予塞来昔布 4mg/d 和 2mg/d 口服 2 周。2从治疗开始每 3 天计算瘤体积一次，描绘肿瘤生长曲线；治疗结束后处死裸鼠，剥出瘤组织称重，计算抑瘤率，瘤组织标本分别用 RT-PCR 检测 COX-2 mRNA 的表达,用免疫组化法检测 COX-2 蛋白的表达和微血管密度（MVD）。结果：1MTT 结果显示塞来昔布对 HEC-1B 细胞的增殖具有抑制作用，且呈现时间和浓
3、度依赖性；流式细胞术结果显示塞来昔布能使 HEC-1B细胞 G0/G1期细胞比例增加，S 期和 G2/M 期细胞比例减少，细胞的凋亡率增加，与对照组相比差异有统计学意义（P0.05）；透射电镜结果发现塞来昔布能使细胞出现凋亡的特征性改变，低浓度处理的细胞呈现空泡化的早期凋亡改变，高浓度则出现晚期凋亡的凋亡小体；随着塞来昔布浓度的增加（50-200umol/L），Transwell 侵袭试验结果表明细胞穿膜数目逐渐减少， RT-PCR 结果显示 COX-2 mRNA 的表达逐渐减弱，与对照组相比均有显著差异（P0.05）。2塞来昔布对裸鼠皮下移植瘤的生长具有明显的抑制作用，且随着药物浓度的增加，
4、抑瘤作用逐渐增强（抑瘤率分别为 32.4和48.6）。同时随着药物浓度的增加，RT-PCR 结果显示移植瘤组织COX-2mRNA 的表达逐渐减弱，免疫组化结果显示移植瘤组织 COX-2 蛋白的表达及微血管密度（MVD）逐渐减少，实验组与对照组之间及各实验组之间的差异均有统计学意义（P0.05）。结论：COX-2 选择性抑制剂塞来昔布在体外及体内对人子宫内膜腺癌均具有明显的抑制作用，其机制可能与下调肿瘤细胞中 COX-2 的表达，减少组织中微血管密度 MVD 有关。关键词：环氧化酶2，子宫内膜癌，增殖，凋亡，侵袭，裸鼠Inhibitory Effects of Cyclooxygenase-2
5、selective inhibitorCelecoxib on Human Endometrium AdenocarcinomaABSTRACTObjective：To investigate the in-vitro and in-vivo inhibitory effect of Cyclooxygenase-2selective inhibitor Celecoxib on human endometrium adenocarcinoma and themechanism, providing theoretical basis for the treatment of human en
6、dometriumadenocarcinoma.Methods：1.The HEC-1B cells were treated with different concentrations of celecoxib（25-200umol/L）for different times,the cells proliferation was examined by MTTassay,the cell cycle and apoptosis were detected by flow cytometry,the change of cellsultramicrostructure were observ
7、ed by transmission electron microscope,the cellsinvasiveness were detected by transwell chamber,the expression of COX-2 mRNAwas analyzed by RT-PCR.2.Thirty nude mice were subcutaneously implanted with HEC-1B cells toestablish human endometrium adenocarcinoma xenografts in nude mice. When thediameter
8、 of tumor was about 5mm,they were divided randomely into threegroups,treated with normal sodium,low dose(2mg/d)of celecoxib and highdose(4mg/d) of celecoxib for two weeks.3.The volume of the tumors were calculated every 3 days and then describe thetumor growth curve；after finishing the treatment，the
9、 nude mice were killed and theweight of implanted tumors were measured,the tumor inhibition rates werecalculated.The expression of COX-2 mRNA in tumor samples was analyzed byRT-PCR,the expression of COX-2 protein and microvessel density in tumor sampleswere examined by immunohistochemistry.Results：1
10、.MTT results indicate that celecoxib can inhibit HEC-1B cells proliferation in atime- and dose-dependent manner, Flow cytometry results indicate that the cellpercent of G0/G1 phase increases, and the cell percent of S and G2/M phase decreases,cell apoptosis rate also increases, there is significant
11、difference contrast with controlgroup（P0.05）.Transmission electron microscope results indicate that there arecharacteristic apoptosis changes in HEC-1B cells. Transwell chamber assay indicatesthat the cells invasiveness gradually decrease with the increase of celecoxibdose(50-200umol/L)（P0.05）.RT-PC
12、R results indicate that the expression of COX-2mRNAgraduallydecreaseswiththeincreaseofcelecoxibdose(50-200umol/L)(P0.05).2.There is signifficant inhibitory effect of celecoxib on xenograftsin nudemice, with the increase of celecoxib dose the inhibitory rates also graduallyimprove(respectively 32.4%
13、and 48.6%). RT-PCR results indicate that the expressionof COX-2 mRNA gradually decreases with the increase of celecoxib dose.Immunohisto- chemistry results indicate that the expression of COX-2 protein andmicrovessel density in tumor samples gradually decrease with the increase ofcelecoxib dose. The
14、re is significant difference contrast between test and controlgroups, as well as between two test groups（P0.05）.Conclusions：Cyclooxygenase-2selectiveinhibitorCelecoxibcaninhibitHumanEndometrium Adenocarcinoma in vitro and in vivo, The mechanism may beassociated with down-regulation of COX-2 expression and decrease of microvesseldensity in tumor tissue.KEY WORDS：Cyclooxygenase-2；endometrium adenocarcinoma；proliferation；Apoptosis；invasiveness；nude mice符号说明英文缩写英文全称中文名称
