【作者】 宋敏；

【导师】 滕月娥；

【作者基本信息】 中国医科大学 ， 肿瘤学， 2008， 硕士

【摘要】 前言肿瘤转移是恶性肿瘤的标志性生物学特征,也是恶性肿瘤相关死亡的首要原因。其中新生血管形成是肿瘤生长和转移的前提,血管的形成不仅为肿瘤生长提供了营养支持,同时也为肿瘤细胞进入循环系统提供主要途径。目前为止,关于乳腺癌的血行转移机制尚不清楚。进一步研究乳腺癌的血行转移机制,有效抑制肿瘤血管形成,可能会延长病人的生存期,降低病人的死亡率,为乳腺癌的治疗提供新的途径。近来有研究显示:环氧化酶-2(COX-2)与血管生成密切相关。其高表达可促进肿瘤细胞释放血管源性物质,对调节肿瘤新生血管的形成有重要的作用,但其发生机制尚不清楚。血管内皮生长因子(VEGF)是目前已知的最强的促肿瘤血管生成因子其能特异地作用于血管内皮细胞,在体内诱导血管形成。CD105(Endoglin)是一种与增生有关的内皮细胞的细胞膜抗原。其表达与细胞周期有关,能准确反映内皮细胞的增殖状态,目前被认为是一种非常好的检测内皮细胞的指示剂。有学者报导:COX-2可能通过上调VEGF的表达,促进肿瘤微血管的形成。但有关COX-2、VEGF在乳腺癌组织中的表达与肿瘤微血管密度的关系国内外鲜有报导。本研究采用免疫组化S-P法对乳腺癌组织COX-2、VEGF、CD105表达情况进行了检测,并且对其表达情况与疾病的病理生物学特征、患者的生存时间进行了分析,以判断其对预后的意义。实验方法1、病例与标本收集了本院外科1999年至2006年间行改良根治术及保乳术治疗的乳腺癌患者117例,发病年龄范围29～74岁,平均年龄51.4岁。所有患者术前未经任何抗癌治疗且术后均在我科接受化疗,随访资料完整。随访期限从4个月至70个月不等,平均随访日期为42.3个月。2、试剂兔抗人COX-2单克隆抗体、鼠抗人VEGF单克隆抗体、鼠抗人CD105单克隆抗体、SP试剂盒、DAB显色液,均购于福州迈新生物技术开发公司。二抗购于北京中杉公司。3、方法采用S-P法进行免疫组化测定,实验步骤严格按照说明书进行操作,并用PBS代替一抗作阴性对照,用已知的结、直肠癌COX-2、VEGF阳性片作阳性对照。4、结果判定CD105标记的MVD计数以呈现棕色单个皮细胞或内皮细胞群者,只要他们和邻近的微血管、肿瘤细胞或其它结缔组织分开,就把它们作为一个微血管。200×光镜下计数微血管的个数,取其平均值为微血管密度。COX-2、VEGF的判定以细胞浆和核膜出现棕黄色颗粒为阳性,以阳性细胞百分比与着色强度乘积值分为低表达、中度表达和高表达。阳性率以“中度+高度表达”计,阴性率以“低度表达”计。5、统计分析方法所得数据均值比较用t检验;生存率采用Kaplan-Meier法计算;生存期差别的比较用Log-Rank时序检验;两样本率的比较用x~2检验,以P＜0.05为差异有统计学意义。所有统计均在SPSS11.5统计软件包进行。实验结果1、乳腺癌组织中COX-2、VEGF的表达及与临床病理联系COX-2的阳性表达率为55.6%,其阳性表达在不同的临床分期(P=0.01、r=0.238)、淋巴结转移(P=0.01、r=0.238)、HER-2(P=0.025、r=0.218)、ER(P=0.026、r=-0.205)状态,有显著性差异,而与年龄、肿块大小、病理类型无显著性差异(P＞0.05)。VEGF的阳性表达率为66.7%,与淋巴结转移、临床分期有关(P＜0.05)。2、CD105标记MVD的表达情况及与COX-2、VEGF的关系以CD105为新生血管标记物测得的乳腺癌MVD值与患者的肿块大小、激素受体状况无关,而与淋巴结转移、TNM分期有关。并且其与COX-2及VEGF的阳性表达均呈正相关(P=0.029、r=0.201)(P=0.023、r=0.210)。3、乳腺癌复发与COX-2和VEGF表达的关系回顾术后乳腺癌复发患者的手术标本,其中COX-2、VEGF的阳性表达率较高,为(79.31%、86.21%)明显高于未复发组(47.73%%、60.23%)(P=0.003、P=0.01)。4、COX-2、VEGF表达与术后生存的关系经Kaplan-Meier生存曲线分析及Log-Rank时序检验可以看出COX-2与VEGF表达与无病生存率负相关(P=0.004、P=0.032)。且通过共检验得出二者共阳性组生存期最短(P=0.005).5、多因素分析结果显示:ER、PR、HER-2阳性表达及肿块大小、淋巴结转移情况以及COX-2阳性表达是乳腺癌的独立预后因素。结论1、乳腺癌组织中COX-2、VEGF均有高表达且二者表达呈正相关,COX-2的过表达可能是通过促进VEGF来参与肿瘤血管生成从而导致乳腺癌侵袭、转移的。2、COX-2、VEGF阴性表达病人预后好于阳性病人,且二者共阳性的病人预后最差,二者皆是预后不良指标。3、乳腺癌组织中CD105的表达是肿瘤新生血管的指示剂,其标记的微血管密度在临床可能更有意义。更多还原

【Abstract】 Tumor metastasis is the characteristic biological feature of malignant tumor, and the leading cause of malignant-tumor-related death. neovascularization is the precondition of tumor growth and tumor metastasis. The newly formed blood vessels not only provide nutritional support but also are the main pathway for the circulatory system. Until now,the mechanism of metastasis through blood in breast cancer is still not clear. Further research in mechanism of metastasis through blood will possiblely extend patients’ lifespan and decrease patients’ mortality rate by effectively inhibiting the tumor vascularization., which supports new pathway for the treatment of breast cancer.Recent studies demonstrate that there is a close relationship between Cyclooxygenase-2 (COX-2) and blood vessel formation. Its high expression may promote tumor cells to release vasogenic material and has a important role in the regulation of neovascularization. But the mechanism is unclear. Vascular endothelial growth factor (VEGF) is the strongest known to promote tumor angiogenesis factor, which can specifically act on the endothelial cells of blood vessel and in vivo induce blood vessel formation. Vascular endothelial growth factor (VEGF) is the strongest known to promote tumor angiogenesis factor, which can specifically act on the endothelial cells of blood vessel and in vivo induce blood vessel formation. but the relationship between the expression of COX-2 and VEGF in breast cancer tissue and tumor microcapillaries desity is hardly reported.This study used the S-P method of immunohistochemistry to examine COX-2, VEGF, and CD105 expression in breast cancer, the relationship between its expression and the biological character of breast cancer ,and the relationship between its expression and patients’ survival were analyzed to determine its significance on prognosis.Materials and Methods1. Patientsspecimen of 117 primary breast cancer patients undergoing modified radical mastectomy and conservative treatment was collected and the histomicroarray was formed using randomized allocation .the range of patient’s age is 29～74 years, with the average 51.4 years. All the patients didn’t recieive chemotherapy before surgery and received chemotherapy after chemotherapy in our department .the follow-up data was complete with the average follow-up duration 42.3 months ranging from 4 months to 70 months.2. AgentRabbit-anti-human COX-2 monoclonal antibody, Mouse-anti-human VEGF monoclonal antibody, Mouse-anti-human CD105 monoclonal antibody, SP examination kit, DAB colored solution were purchased from Fuzhou Mai xin bio-tech Developm- eent Company. The second antubodies was purchased from Beijing Zhongshan Goldenbrige Biotechnology.3. MethodsThe behavior leaMethods: the S-P method was used to determine the immumohistochemical results, the laboratory procedure were operated strictly according to the manufacture’s instruction and the primary antibody was replaced by PBS as negave control, with positive sides of Rectal Cancer COX-2 and VEGF as control group.4. AssessmentThe MVD count marked by CD105 shows brown single epitheilum or the endothelial cell group, neighboring. As long as they are separated form the neighboring capillaries ,tumor cells and other connective tissues,they are reguarded as microcapillaries. the number of the microcapillaries is counted 200×magnified light microscope. take the mean value for the MVD. when there are brown yellowish particles in cytoplasm and nuclear membrane,the COX-2、VEGF is determined as positive,which is divided nito hypoexpression ,moderate expression and hyperexpres-sion according to cross product of positive cell percentage and the coloring intensity. The positive rate was expressioned by"moderate degree expression+ high degree expression" or negative rate "low-level expression". 5. Statistical analysisThe average value is compared using T test, survival rate is calculated using Kaplan-Meier method, survival time is compared using Log-Rank test, take P<0.05 as having statisital significance for comparing the rate of two samples by x~2 test.All the statistical analyses is performed by using SPSS11.5 statistics software package.Results1. The expression of COX-2 VEGF in breast cancer tissue and clinical pathology: COX-2 positive expression rate was 55.6%, COX-2 positive expression in different clinical stages (P=0.01、r=0.238), lymph node metastasis (P=0.01、r=0.238), HER-2、ER status (P=0.025、r=0.218), is statistically different , but isn’t related to age, tumor size and pathological type(P>0.05). The VEGF positive expression is 66.7%, which is related to lymph node metastasis, clinical stage(P<0.05).2. MVD expression and COX-2, VEGF taking CD105 as a marker of neovscularizati- on ,breast cancer MVD value Isn’t related to tumor size and hormone receptor status ,but is positively related to lymph node metastasis and clinical stage (P=0.029, r=0.201). (P=0.023, r=0.210).3. Breast cancer relapse and COX-2 and VEGF expression: After reviewing surgery specimen of the breast cancer patients having relapse, COX-2, VEGF positive expression rate is (79.31%, 86.21%) is higher than nonrelapse patients (47.73%%, 60.23%).(P=0.003,P=0.01).4. COX-2、VEGF expression and postoperative survival relationship (Kaplan-Meier): The Kaplan-Meier survival curve analysis Log-Rank test shows COX-2, VEGF expression and disease free survival rate were negatively correlated(P=0.004、P=0.032) . It can be found that common positive suivival period acquired by common test is shortest (P=0.005) .5. Multivariate analysis showed that ER, PR, HER-2 positive expression and tumor size, lymph node metastasis and COX-2 expression was an independent prognostic factors in breast cancer.Conclusion1. The hyperexpression of COX-2 and VEGF in breast cancer is positive correlation, the expression of COX-2 leads to breast cancer invasion and metastasis possibly by promoting VEGF expression.2. COX-2 and VEGF negative expression patients surpass positive patients in the prognosis, and common positive patients were worst prognosis. Both are indicators of poor prognosis.3. CD105 expression in breast cancer is indicator of tumor new vessels , which marked capillary density is a possible value.更多还原