【作者】 郎哲；

【导师】 陈刚；

【作者基本信息】 河北医科大学 ， 内科学， 2010， 硕士

【摘要】 目的:肺癌是世界上最常见的恶性肿瘤之一,近年来我国肺癌的发病率和死亡率明显升高。由于肺癌早期症状不典型,人们的保健意识缺乏等原因,临床确诊时多已发展到晚期,此时肺癌已经发生转移,所以预后差。肿瘤的侵袭和转移是多基因调控的复杂过程,这些基因之间的相互作用目前成为了国内外研究的热点。近期研究结果表明环氧化酶-2 (cyclooxygenase-2, COX-2)在肿瘤形成过程中有重要作用,其不仅促进肿瘤细胞增生,抑制肿瘤细胞凋亡,还与肿瘤的侵袭和转移有关。血管内皮生长因子-C(vascular endothelial growth factor-C, VEGF-C)是VEGF家族的成员,表达于乳腺癌、宫颈癌、结肠癌、肺癌、前列腺癌等多种肿瘤细胞。肿瘤细胞分泌的VEGF-C通过自分泌机制与其受体-血管内皮生长因子受体-3(vascular endothelial growth factor receptor-3, VEGFR-3)结合,是调控淋巴管生成最重要的信号通路。生存素(Survivin)是凋亡抑制蛋白(inhibitor of apoptosis proteins, IAP)基因家族的成员,它抑制细胞凋亡,参与细胞周期调控,是目前发现的最强的凋亡抑制因子,与肿瘤的发生、发展和预后密切相关。β-连环素(β-catenin)可介导细胞粘附,亦具有信号传导的功能,其异常表达可促进肿瘤的转移。研究表明COX-2与VEGF-C,VEGFR-3,Survivin,β-catenin的表达有协同上升趋势。尼美舒利(nimesulide)是COX-2选择性抑制剂,动物实验证实,尼美舒利不但可以预防肿瘤的发生,还可以抑制肿瘤的生长和转移,并且具有增强化疗和放疗的作用。奥沙利铂(oxaliplatin)是第三代铂类广谱抗癌药,通过产生烷化结合物作用于DNA,形成链内和链间交联,从而抑制DNA的合成及复制。本实验通过建立裸鼠皮下肺癌移植瘤模型,观察COX-2抑制剂尼美舒利联合化疗药物奥沙利铂对肿瘤的生长以及对COX-2,VEGF-C,VEGFR-3,Survivin,β-catenin表达的影响,进一步探讨肿瘤转移的分子机制,以及这两种药物联合应用可能产生的协同作用。方法:选4~5周龄BALB/c雄性裸小鼠26只,体重20~24克,在IVC环境中以无菌饲料及灭菌纯净水饲养,饲养环境无特定病原体,环境温度、湿度适宜。使用含10%小牛血清、100U/ml青霉素和100U/ml链霉素的DMEM培养基,在37℃含5%C02培养箱中培养人肺癌A549细胞,细胞贴壁生长,至70%~80%融合时用0.25%胰蛋白酶消化传代。取处于对数生长期的A549细胞,以0.25%的胰酶液进行消化,PBS洗涤,用不含血清的DMEM培养基稀释,以血细胞计数板进行计数,调整细胞密度为1×107/ml。裸鼠皮肤消毒后用1ml注射器在右侧腋部皮下注射上述细胞悬液0.2ml,建立肺癌裸鼠移植瘤模型。其中25只裸鼠成瘤,游标卡尺测量肿瘤结节的长径(a)、短径(b),按公式V=ab2/2,估算肿瘤近似体积。待移植瘤平均直径约4mm后,剔除肿瘤体积最小的裸鼠,将裸鼠随机分为对照组、尼美舒利组、奥沙利铂组、尼美舒利/奥沙利铂联合组,每组6只。奥沙利铂组裸鼠采用腹腔注射奥沙利铂甘露醇注射液,剂量为10mg/kg/次,每4天给药1次,同时口服灭菌蒸馏水;尼美舒利组采用灌胃给药,剂量为20mg/kg/d,同时腹腔注射等量生理盐水;尼美舒利/奥沙利铂联合组给予尼美舒利灌胃和奥沙利铂腹腔注射,用量与两药单用组相同;对照组口服灭菌蒸馏水,腹腔注射等量生理盐水。观察裸鼠生长情况,每5天测量肿瘤体积1次。给药30天后,处死裸鼠,切取移植瘤组织,免疫组织化学法检测肿瘤组织中COX-2, VEGF-C, VEGFR-3, Survivin,β-catenin蛋白的表达,以北航真彩色病理图像分析系统计算平均积分光密度值。荧光实时定量PCR法检测肿瘤组织中COX-2, VEGF-C, VEGFR-3, Survivin,β-cateninmRNA的表达。结果:接种裸鼠26只,25只裸鼠成瘤,成瘤率96.2%,成瘤时间为细胞种植后第5~8天。肿瘤细胞种植后第12天,肿瘤直径平均达4 mm。治疗结束后,各组移植瘤体积分别为:对照组1498.83±429.11 mm3;尼美舒利组857.37±113.73 mm3;奥沙利铂组748.43±42.22 mm3;联合用药组442.53±103.81 mm3。尼美舒利组、奥沙利铂组和联合用药组抑瘤率分别为44.34%、51.60%和73.00%。方差分析组间比较显示,尼美舒利组、奥沙利铂组和联合用药组肿瘤生长较对照组明显缓慢( P < 0.05 )。联合用药组抑瘤率较尼美舒利组和奥沙利铂组抑瘤率明显提高( P < 0.05 )。尼美舒利组和奥沙利铂组对移植瘤生长抑制作用无显著性差异( P > 0.05 )。以图像分析系统对COX-2, VEGF-C, VEGFR-3, Survivin,β-catenin蛋白免疫组织化学染色切片进行积分光密度测定,方差分析结果显示,与对照组比较,尼美舒利组COX-2, VEGF-C, VEGFR-3, Survivin,β-catenin蛋白表达水平均明显降低(分别P < 0.05 );与对照组比较,奥沙利铂组COX-2, VEGF-C, VEGFR-3蛋白表达水平升高(分别P < 0.05 ),而Survivin,β-catenin蛋白表达水平降低(分别P < 0.05 );与对照组比较,联合用药组COX-2, VEGF-C, VEGFR-3, Survivin,β-catenin蛋白表达水平均明显降低(分别P < 0.05 )。荧光定量RT-PCR法测定COX-2, VEGF-C, VEGFR-3, Survivin,β-cateninmRNA表达,方差分析结果显示,与对照组比较,尼美舒利组COX-2, VEGF-C, VEGFR-3, Survivin,β-cateninmRNA表达水平均明显降低(分别P < 0.05 );与对照组比较,奥沙利铂组COX-2, VEGF-C, VEGFR-3mRNA表达水平升高(分别P < 0.05 ),而Survivin,β-cateninmRNA表达水平降低(分别P < 0.05 );与对照组比较,联合用药组COX-2, VEGF-C, VEGFR-3, Survivin,β-cateninmRNA表达水平均明显降低(分别P < 0.05 )。结论:尼美舒利单药或联合奥沙利铂均能明显抑制人肺癌裸鼠移植瘤的生长和COX-2, VEGF-C, VEGFR-3, Survivin,β-catenin的表达。奥沙利铂能明显抑制人肺癌裸鼠移植瘤的生长和Survivin,β-catenin的表达。尼美舒利与奥沙利铂联合应用提高了奥沙利铂的抗肿瘤效果。更多还原

【Abstract】 Objective: Lung cancer is one of the most common malignant tumors in the world, in recent years the incidence rate and the mortality rate of lung cancer in our country are on the rise obviously. Because the incipient symptoms of lung cancer are atypical, the lack of health care consciousness and so on, the lung cancer may have been developed into the advanced stage when clinical diagnosis. At this time the lung cancer have already metastased, therefore the survival rate is low, the prognosis is bad. The tumor invasion and metastasis is a polygenes regulation complex processes, the research of the interaction between these gene has become hot spot at present both at home and abroad. The recent studies show cyclooxygenase-2(COX-2) plays an imporant role on the formation of cancer. COX-2 promote the hyperplasia of tumor cells, inhibit tumor cell apoptosis, it also promote the invasion and metastasis. Vascular endothelial growth factor-C(VEGF-C) is a member of VEGF family, it express in breast cancer, cervical cancer, colon cancer, lung cancer, prostate cancer and so on, vascular endothelial growth factor receptor-3(VEGFR-3)is its receptor. The VEGF-C and VEGFR-3 is the most important signal way of controlling the form of lymphangiogenesis. Survivin, a member of IAP family, is the strongest factor that can control the apoptosis of cells. There are very closely relationship between Survivin and tumor occurrence, development and prognosis.β-catenin can induce cell adhesion, it also has the signal conduction function. Its exceptionally expresses may promote the tumor metastasis. Many studies show that the expression of COX-2, VEGF-C, VEGFR-3, Survivin andβ-catenin can increase together. Nimesulide is the selective COX-2 inhibitors. The animal experimentation confirmed that Nimesulide not only prevent tumor occurrence, but also inhibit tumor growth and metastasis, more importantly, it can enhance the effectiveness of chemotherapy and the radiotherapy. Oaliplatin is the third generation platinum chemotherapy medicine. It can inhibit DNA synthesis and replication. This study is designed to evaluate the effects of nimesulide in combination with oxaliplatin, on tumor growth, COX-2, VEGF-C, VEGFR-3, Survivin andβ-catenin expression in lung cancer xenograft in nude mice. To discussion the molecular mechanism of tumor metastasis, as well as the synergistic effect that nimesulide in combination with oxaliplatin may produce.Methods: 26 nude mice, 4-5-week-old , BALB / c , male, weighing 20 ~ 24 grams, fed with sterile feed and sterile purified water in a rearing environment with no specific pathogens and ambient temperature and suitable humidity. With DMEM medium containing 10% newborn bovine serum, 100U/ml penicillin and 100U/ml streptomycin, human lung cancer A549 cells are cultivated in an incubator of 37℃with 5% C02 and cells are digested and passed with 0.25% trypsin when cells are growed adherently to 70% ~ 80% confluence. Digest the A549 cells in logarithmic growth phase with 0.25% trypsin and dilute them with serum-free DMEM dilution medium to a cell density of 1×107/ml after counting with CBC board. The human lung cancer A549 cell suspension were injected subcutaneously in nude mice to establish axillary tumor model. 25 nude mice formed tumor, after the tumor diameter reached an average of 4mm ,remove the nude mice which have the smallest tumor volume and then randomly divided them into four groups, 6 nude mice each group: control group, nimesulide-treated group, oxaliplatin-treated group, and nimesulide combined with oxaliplatin-treated group. Medecine were administered respectively. Have a 5 day measurement of tumor volume and execute nude mice after administering 30 days, cut and transplant tumor tissue, immunohistochemistry detection of COX-2, VEGF-C, VEGFR-3, Survivin,β-catenin protein expression, RT-PCR assay Tumor COX-2, VEGF-C, VEGFR-3, Survivin,β-cateninmRNA expression.Results: 25 nude mice formed tumor. The tumor formation rate was 96.2%. The tumor diameter reached an average of 4mm on the 12th days after planting tumor cells.After the treatment was finished, the tumor volume of each group was as follows: control group, 1498.83±429.11mm3; nimesulide-treated group, 857.37±113.73mm3; oxaliplatin-treated group, 748.43±42.22mm3; nimesulide combined with oxaliplatin-treated group, 442.53±103.81mm3. Tumor inhibition rates of nimesulide-treated group, oxaliplatin-treated group, and nimesulide combined with oxaliplatin-treated group were 44.34%, 51.60% and 73.00%, respectively. Compared with the control group, analysis of variance between the two groups showed that tumor growth in nimesulide-treated group, oxaliplatin-treated group, and nimesulide combined with oxaliplatin-treated group is relatively slow, and the difference was statistically significant (respectively, P <0.05). The inhibition rate of nimesulide combined with oxaliplatin-treated group was significantly increased (P <0.05) than that of nimesulide-treated group and oxaliplatin-treated group. The inhibition rate shows no evident differences between nimesulide-treated group and oxaliplatin-treated group , while the difference was not statistically significant ( P> 0.05).According to the integral optical density measurement by image analysis system for COX-2, VEGF-C, VEGFR-3, Survivin,β-catenin protein in immunohistochemical staining sections. Compared with the control group, statistical analysis of variance showed that the expression levels of COX-2, VEGF-C, VEGFR-3, Survivin,β-catenin protein of nimesulide-treated group were significantly reduced (respectively, P <0.05). Compared with the control group, statistical analysis of variance showed that the expression levels of COX-2, VEGF-C, VEGFR-3 protein of oxaliplatin-treated group were significantly increased, the expression levels of Survivin,β-catenin protein of oxaliplatin-treated group were significantly reduced (respectively, P <0.05). Compared with the control group, statistical analysis of variance showed that the expression levels of COX-2, VEGF-C, VEGFR-3, Survivin,β-catenin protein of nimesulide combined with oxaliplatin-treated group were significantly reduced (respectively, P <0.05). According to the real-time fluorescence quantitative analysis of COX-2, VEGF-C, VEGFR-3, Survivin,β-cateninmRNA, compared with the control group, statistical analysis of variance showed that the expression levels of COX-2, VEGF-C, VEGFR-3, Survivin,β-cateninmRNA of nimesulide-treated group were significantly reduced(respectively, P <0.05). Compared with the control group, statistical analysis of variance showed that the expression levels of COX-2, VEGF-C, VEGFR-3mRNA of oxaliplatin-treated group were significantly increased, the expression levels of Survivin,β-cateninmRNA of oxaliplatin-treated group were significantly reduced (respectively, P <0.05). Compared with the control group, statistical analysis of variance showed that the expression levels of COX-2, VEGF-C, VEGFR-3, Survivin,β-cateninmRNA of nimesulide combined with oxaliplatin-treated group were significantly reduced (respectively, P <0.05).Conclusion: Using nimesulide alone or in combination with oxaliplatin were able to significantly inhibit the growth of lung cancer xenografts in nude mice, as well as the expression of COX-2, VEGF-C, VEGFR-3, Survivin,β-catenin. Oxaliplatin were able to significantly inhibit the growth of lung cancer xenografts in nude mice, as well as the expression of Survivin andβ-catenin. When nimesulide being combined with oxaliplatin, it can enhance the effect of oxaliplatin against tumor.更多还原