【作者】 蒲军；

【导师】 李少林；

【作者基本信息】 重庆医科大学 ， 肿瘤学， 2007， 硕士

【摘要】 目的：研究体外低氧对乳腺癌细胞和体内塞来昔布对乳腺癌组织低氧区uPA、uPAR和HIF-1α表达的影响及机制；以及研究塞来昔布对乳腺癌组织低氧区的细胞凋亡和血管生成的影响，探讨塞来昔布治疗低氧肿瘤的可行性。方法：采用CoCl₂模拟体外低氧模型，人乳腺癌MDA-MB-231细胞用0、50、100、150、200μmol／L CoCl₂分别处理16h，用于观察量效关系；用100μmol／L CoCl₂分别处理0、8、16、24、32h，用于观察时效关系。采用RT-PCR检测uPA mRNA和uPAR mRNA的表达，免疫细胞化学法检测uPA、uPAR和HIF-1α的蛋白表达。建立人乳腺癌裸鼠皮下移植瘤模型，给予选择性COX-2抑制剂塞来昔布（10mg／kg／d）灌胃，共30天，采用免疫组化法检测移植瘤低氧区uPA、uPAR和HIF-1α蛋白以及MVD的表达。采用TUNEL法检测移植瘤低氧区细胞的凋亡指数（AI）。结果：在量效组中，各处理组（50、100、150、200μmol／L CoCl₂，16h）的uPA mRNA和uPAR mRNA的表达高于对照组（0μmol／LCoCl₂，16h）（P＜0.05）。在时效组中，各处理组（100μmol／L CoCl₂，8、16、24、32h）的uPA mRNA和uPAR mRNA的表达高于对照组（100μmol／LCoCl₂，0h）（P＜0.05）。在MB-231细胞中uPA mRNA和uPAR mRNA的表达随CoCl₂浓度的增加或处理的时间延长逐渐增强（P＜0.05），在量效组中100μmol／L时高表达，在时效组中16h时高表达，然后两组的表达都逐渐减弱（P＜0.05）。uPA、uPAR和HIF-1α蛋白的表达也具有类似效应。在体内，塞来昔布组的uPA、uPAR和HIF-1α蛋白的表达分别为0.207±0.049、0.203±0.048和0.181±0.058；对照组的uPA、uPAR和HIF-1α蛋白表达分别为0.298±0.071、0.289±0.068和0.281±0.075。塞来昔布能够抑制移植瘤低氧区uPA、uPAR和HIF-1α蛋白的表达（P＜0.05）。塞来昔布组的MVD和AI分别为19.80±2.59和5.616±0.892，对照组的MVD和AI分别为26.80±3.42和3.089±0.609，塞来昔布能够促进移植瘤低氧区MB-231细胞的凋亡和抑制其血管生成（P＜0.01）。结论：①CoCl₂模拟体外低氧能够诱导乳腺癌MB-231细胞uPA、uPAR和HIF-1α的表达上调，高表达需要适宜的低氧条件，HIF-1α可能参与了uPA、uPAR表达的调控。②塞来昔布不仅能够诱导乳腺癌移植瘤低氧区细胞凋亡，还能抑制其血管的生成，其中可能与塞来昔布能够下调uPA、uPAR和HIF-1α的表达有关。③塞来昔布有望成为低氧肿瘤治疗的新选择。更多还原

【Abstract】 OBJECTIVE: To study the effect ofhypoxia on the breast cancer cellsexpressing uPA, uPAR and HIF-la and to investigate its potentialmechanism in vitro. To study the contribution of celecoxib to the hypoxicbreast cancer tissues expressing uPA, uPAR and HIF-1a and to studyinfluence of apoptosis and angiogenesis of celecoxib to the hypoxic breastcancer in vivo,and further to explore feasibility that celecoxib was used totreat hypoxic tumors.METHODS:The hypoxic model was simulated by COCl₂ in vitro.Thehuman breast cancer MDA-MB-231 cells were exposed to the differentconcentration COCl₂（0, 50, 100, 150, 200μmol/L）for 16h respectively,to investigate the dose-efficiency relation.Meanwhile MB-231 cells wasexposed to CoCl₂（100μmol/L）for different time（0, 8, 16, 24,32h）respectively to investigate the time-effcicency relation.The uPA mRNA and uPAR mRNA levels were analyzed using RT-PCR.The uPA, uPAR and HIF-1αprotein were deteced through the immuno-cytochemical technique. MB-231 human breast cancer xenografts in nudemice were constructed successfully. These bearing tumor mice wereadministered with celecoxib（10mg/kg/d） which was a selective COX-2 inhibitor for 30 days. The expression ofuPA, uPAR and HIF-1αproteinwas detected by the immunohistochemical technique in the hypoxictissues of the xenografts and MVD was also assessed by theimmunohistochemical technique. Apoptosis index（AI）was detected by theTUNEL technique in the hypoxic tissues of the xenografts.RESULTS: In the dose-efficiency relation group, the expression ofuPA mRNA and uPAR mRNA in MB-231 cells treated with COC12（50, 100,150, 200μmol/L for 16h）was higher than that of control group （0μmol/LCOCl₂,16h） （P＜0.05）. In the time-efficiency relation group, the expressionof uPA mRNA and uPAR mRNA in MB-231 cells treated with COCl₂（100μmol/L for 8, 16, 24, 32h） was higher than that of controlgroup（100μmol/L CoCl₂, Oh） （P＜0.05）. The data indicated that expressionof uPA mRNA and uPAR mRNA up-regulated with increasing concentra-tions of CoCl₂ or the prolongation of treated time （P＜0.05）.And the highexpression of uPA mRNA and uPAR mRNA was observed with 100μmol/Lin dose-dependent manner and for 16h in time-dependent manner, thendown-regulated gradually in the both dose-dependent and time-dependentmanners （P＜0.05）.The similar effects of uPA, uPAR and HIF-1αwerealso observed in protein level. The expression of uPA, uPAR and HIF-1αof celecoxib treatment group in protein level was 0.207±0.049,0.203±0.048 and 0.181±0.058 respectively in vivo, the expression of uPA,uPAR and HIF-1αof control group in protein level was 0.298±0.071, 0.289±0.068 and 0.281±0.075 respectively in vivo. The expression ofuPA,uPAR and HIF-1αin protein level was significantly decreased in thehypoxic tissues of celecoxiib treatment group（P＜0.05）.The MVD and AIof the celecoxiib treatment group was 19.80±2.59 and 5.616±0.892respectively and the MVD and AI of control was 26.80±3.42 and3.089±0.609 respectively.Celecoxib induced apoptosis of MB-231 cells andsuppressed angiogenesis of MB-231 cells in the hypoxic tissues of thexenografts （P＜0.01）.CONCLUSION:①The up-regulation of uPA,uPAR and HIF-1a is induced by hypoxiain the breast cancer MB-231 cells simulated by COCl₂ and the optimalcondiction is required for high expression of uPA,uPAR and HIF-1a in vitro.HIF-1a is presumed to be involved in the regulation of the uPA and uPARof MB-231 cells.②The celecoxib not only could induce apoptosis of breast cancerMB-231 cells but also suppress angiogenesis in the hypoxic tissues ofxenografts, which is probably correlated with the down-regulation ofuPA,uPAR and HIF-1a treated with celecoxib.③Celecoxib is hopefully a new choice in treatment hypoxic tumors.更多还原