【作者】 石晶；

【导师】 刘云鹏；

【作者基本信息】 中国医科大学 ， 内科学， 2007， 硕士

【摘要】 前言肿瘤的多药耐药是化疗失败的主要原因，耐药泵的产生是形成多药耐药的重要机制之一，其中，乳腺癌耐药相关蛋白(Breast cancer resistant protein BCRPABCG2)是较晚发现的药泵蛋白。蒽环类药物是ABCG2的底物，而蒽环类药物恰恰是乳腺癌治疗中的常用药，所以研究ABCG2与蒽环类药物应用的关系对于乳腺癌的治疗意义重大。缺氧诱导因子-1(Hypoxia inducible factor-1，HIF-1)与多药耐药关系非常密切。已有研究证实，HIF-1可以上调P-gp，MRP的表达，而且，与P-gp相似，在ABCG2启动子中也存在HIF-1的结合基序(binding motifs)，这强烈提示了HIF-1可能调节ABCG2的表达。本研究采用免疫组织化学SP法对乳腺癌组织HIF-1α，ABCG2的表达进行检测，并结合完整的临床、随访资料，分析它们之间的关系及HIF-1α，ABCG2与临床特点，无病生存的关系。实验方法1、病例和标本122例乳腺癌组织石蜡标本来自于中国医科大学附属第一临床学院接受乳腺癌手术治疗的病人。患者术前均未接受放疗或化疗，术后106例接受了以蒽环类化疗药为主的辅助化疗，入选病例均为女性，平均年龄50.2岁，肿块≤2cm者32例(26.2％)，发生淋巴结转移66例(54.1％)，临床TNM分期Ⅲ期患者34例(27.9％)，病理类型为浸润性导管癌88例(72.1％)。从手术日期到复发转移或随访截止日期(2006年12月)确定为患者无病生存期(DFS)，为6～59个月，平均32.2个月，共有32例(26.2％)复发转移病例。122例乳腺癌癌组织取15例正常乳腺组织作为对照。2、试剂ABCG2单克隆抗体、SP检测试剂盒和DAB显色剂购自福建迈新生物技术有限公司，HIF-1α多克隆抗体购自武汉博士德生物技术有限公司。3、方法乳腺癌组织芯片制作。采用免疫组化SP法，按照SP检测试剂盒说明书所示步骤进行，切片经柠檬酸缓冲液(PH=6.0)高压热修复预处理。4、结果判定ABCG2的判定以胞膜上出现棕黄色颗粒为显色，选取5个高倍镜视野(×400)作阳性细胞计数，以阳性细胞数≤10％为阴性，＞10％为阳性；HIF-1α以胞核或胞浆内有棕黄色或棕褐色细颗粒为阳性表达，阳性细胞数≤10％为阴性，＞10％为阳性。5、统计分析方法采用SPSS13.0软件进行数据分析，率的比较采用卡方检验；ABCG2和HIF-1α表达的相关性采用Spearman秩和相关分析；无病生存数据分析采用Kaplan-Meier法，组间比较采用Log rank检验；P＜0.05有统计学意义，所有检验和P值均为双侧。实验结果1、ABCG2、HIF-1α在乳腺癌组织中的表达及相关性122例乳腺癌组织ABCG2、HIF-1α的阳性表达率分别为40.2％、59.0％，15例正常乳腺组织未检出ABCG2、HIF-1α表达。ABCG2与HIF-1α的表达呈正相关(r=0.21，p=0.03)。2、ABCG2、HIF-1α表达与乳腺癌临床病理特征的关系经统计学分析，ABCG2表达与肿块大小(r=0.21，p=0.02)呈正相关；HIF-1α表达与肿块大小(r=0.33，p＜0.01)、淋巴结转移(r=0.25，p＜0.01)、临床分期(r=0.21，p=0.022)呈正相关。3、乳腺癌组织ABCG2、HIF-1α表达与无病生存期的关系经Kaplan-Meier生存曲线分析，ABCG2表达与122例患者的无病生存期有负相关趋势，无统计学意义(p=0.052)，与106例接受了以蒽环类化疗药为主的辅助化疗患者的无病生存期呈负相关，有统计学意义(p=0.033)。HIF-1α表达与122例患者的无病生存期呈负相关，有统计学意义(p=0.030)。HIF-1α与ABCG2共表达与122例患者的无病生存期呈负相关，有明显统计学意义(p=0.004)。结论1、ABCG2表达阳性是接受了以蒽环类化疗药为主的辅助化疗的乳腺癌患者的预后不良指标2、HIF-1α与ABCG2表达明显相关3、HIF-1α与ABCG2共检验可以更好的判断乳腺癌患者预后更多还原

【Abstract】 IntroductionMultidrug resistance(MDR) is one of the main reasons resulting in failure of chemotherapy in patients with breast cancer. The over expression of multidruge resistance associated protein (MRP) play a more important role in MDR, and MRP includes Breast cancer resistant protein (BCRP ABCG2), which discovered lately. Anthracycline is substrates of ABCG2, and properly anthracycline was commonly used in treatment of breast cancer . It is important to investigate the expression of BCRP in breast cancer and the correlation with anthracycline.Hypoxia inducible factor-1(HIF-1) has tightly correlation with MDR, and can up-regulate the expression of P-gp and MRP, which have binding motifs of HIF-1 in promoter field as well as ABCG2. We think that HIF-1 also can up-regulate the expression of ABCG2.In this study, we detected the expression of ABCG2 and HIF-1αusing immunohistochemical SP methods to investigate the relationship between them, as well as the correlate with clinicopathological parameters and Disease Free Survival Time in human breast cancer.Methods1. Patients122 breast cancer patients underwent completely resected which were enrolled from the first affiliated hospital, China Medical University (CMU) . None of them underwent chemotherapy or radiotherapy before operation. But 22 patients (18.0%) received chemotherapy of taxane and 106 patients (86.9%) received chemotherapy of anthtacycline after operation .All the cases were female with median age of 50.2 years,. Among of them there were 32 cases (26.2%) of the tumor size≤2cm, 66 cases (54.1 %) of lymph node metastasis, 88 cases (72.1%) of infitrating ductal carcinoma and 34 (27.9%) advanced cases. 32 patients (26.2%) occurrenced relapse and matastasis .The Diseas Free Survival Time (DFS) ranged from 6 to 59 months. Normal breast tissue of 15 cases was selected as controls.2. MaterialsThe monoclonal antibodies against ABCG2 and SP immunohistochemical kits and DAB substrate solution were purchased from Fujian Maixin Biotechology, Inc; the polyclonal antibodies against HIF-1αwere purchased from Wuhan Boster Biotechnology, Inc.3. MethodsImmunohistochemical staining SP method was performed. All slides were treated with 0.01 mol/L citric acid buffer (PH=6.0) and recovered the antigen activity by autoclave.4. AssessmentOutcome definition of ABCG2 were stained palm yellow particle in cell membrane, positive cells≤10% as negative , >10% as positive , at high magnification(400×). Outcome definition of HIF-1αwere stained palm yellow particle in cytosol or nucelus, positive cells≤10% as negative cells , >10% as positive, at high magnification(400×).5. Statistical analysisStatistical methods x~2 test, Spearman rank correlation test and Kaplan-Meier survival analysis were performed by SPSS software version 13.0. Results1. Expression of ABCG2 and HIF-1αin breast cancer patientsPositive expression of ABCG2、HIF-1αin 122 cases was 40.2%、59.0%、respectively, however, was negative in 15 cases normal breast tissue.2. The relationship between expression of ABCG2 and HIF-1αandclinicopathologic parameters in breast cancer patientsExpression of ABCG2 was positive correlated with tumour size (r=0.21 p=0.02), but not correlated with lymph node metastasis(r=0.04, p>0.05) and clinical stage (r=0.46 P>0.05) . Expression of HIF-1αwas positive correlated with tumour size (r=0.33 p<0.01), lymph node metastasis (r=0.25, p<0.01) and clinical stage (r=0.21, P=0.022) .3.The relationship between expression of ABCG2 and HIF-1αExpression of ABCG2 was positive correlated with HIF-1α(r=0.206, p=0.03).4.The relationship between expression of ABCG2、HIF-1αandDisease Free Survival Time in patients with breast cancerIn 106 patients received chemotherapy of anthtacycline after operation, ABCG2 expression was correlated with DFS by Kaplan-Meier survival analysis (the value of p was 0.033). In 122 patients HIF-1αexpression was correlated with DFS by Kaplan-Meier survival analysis (the value of p was 0.030). The both expression of ABCG2 and HIF-1αwas also correlated with DFS by Kaplan-Meier survival analysis (the value of p was 0.004).Conclusion1 Expression of ABCG2 was bad predictor for patients received chemotherapy of anthtacycline after operation. 2 Expression of ABCG2 was positive correlated with expression of HIF-1α..3 Detection of ABCG2 / HIF-1αin bresat cancer tissues might be significant in prognosis prediction.更多还原