【作者】 龙飞；

【导师】 邱雪杉；

【作者基本信息】 中国医科大学 ， 病理与病理生理学， 2005， 硕士

【摘要】 前言 乳腺癌是女性高发恶性肿瘤,其发病率及死亡率呈逐年上升趋势,在我国己跃居女性恶性肿瘤的第二位。侵袭和转移是乳腺癌的重要生物学特征之一,也是决定预后的关键因素。因此,对乳腺癌的侵袭和转移中发挥作用的因子的进一步研究将有助于乳腺癌的评估及临床治疗策略的制定。目前,探讨肿瘤相关的分子生物学标志物成为肿瘤研究的热点。催乳素(PRL)是由垂体前叶合成和分泌的一种多肽类激素,近来的研究发现其在乳腺癌中存在自/旁分泌环,推测它在乳腺癌的发生发展中也有一定的作用,其可通过刺激细胞增殖、抑制细胞凋亡、增强细胞的运动性和影响血管形成来改变乳腺癌细胞的生物学行为。本研究采用免疫组化SP法检测乳腺癌组织中PRL-3的表达情况和MVD,探讨PRL-3的表达与乳腺癌血管生成及其生物学行为之间的关系,为临床预测乳腺癌的转移潜能和预后、指导治疗提供客观有效的指标。 方法 1.材料:47例乳腺癌组织来源于辽宁省肿瘤医院1995年4月至2002年6月外科手术切除标本,其中有家族病史者9人;浸润性导管癌43人,浸润性小叶癌2人,腺癌1人,黏液癌1人;有淋巴结转移者39人;47例乳腺癌组织按国际抗癌联盟(UICC)1997年修订的乳腺癌TNM分期标准分为Ⅰ期2例,Ⅱ期21例,Ⅲ期23例,Ⅳ期1例。 2.方法:免疫组化SP染色方法,PRL-3多克隆抗体购自Santa Cruz公司,鼠抗人CD34、C-erBb-2、ER和PR抗体、S-P超敏试剂盒均购自福州迈新生物技术开发公司。免疫组化染色操作如下:石蜡切片常规脱蜡至水,3%过氧化氢-甲醇孵育30分钟,以消除内源性过氧化物酶活性。0.1%的更多还原

【Abstract】 IntroductionBreast cancer is one of malignant tumors of female, with increasing incidence and mortality rate for years. It has been on the second place among the malignant tumors of female in China. Invasion and metastasis is one of the most important biological features of breast carcinoma and the key factor for survival of patients. Therefore, study on the factors acting in the invasion and metastasis course will be contributed to value breast cancer and make out therapy strategy. Up to now, finding out some distinguishing molecular markers of tumor has been the hotspots in this field. Prolactin is a kind of hormone which was produced and secreted by the foreside of the pituitary. It is reported recently that there is a au-to/paracrine loop in breast cancer, which may be play a role in generation and progression of breast cancer. PRL can change the biological behaviors of breast cancer cell by promoting proliferation, restraining apoptosis, increasing motility and influencing angiogenesis. Using immumochistochemistry S - P method, the expression of PRL - 3 was tested in breast carcinoma and para - carcinoma, and count the microvessels on the section with positive expression of CD34, then the interaction relationship between the expression of PRL - 3 and angiogenesis and clinical - pathololgical features in breast cancer was investigated to apply object and useful guideline to determinate the potential of metastasis and prognosis and to guide clinical therapy.Materials and methodsImmunohistochemistry S - P method was used to test the expression of PRL- 3 and CD34 in breast carcinoma and para - carcinoma, as well as ER, PR, C -erBb -2. All tissue samples were collected from Liao Ning Tumor Hospital. 47 cases of primary breast carcinoma and 10 para - carcinoma were selected, including 43 invasive ductal carcinoma, 2 invasive lobular carcinoma, 1 adenocarcinoma and 1 mucinous cacinoma. Among all of the patients, 9 have family disease history and 39 have lymph node metastases. The pathological stages were classified as stage I in 2 patients, Ⅱ in 21, Ⅲ in 23 and Ⅳ in 1 according to the P - TNM classification of UICC ( 1977). For immunostainy, streptavidin peroxidase method was used, the first antibody PRL - 3 was produced in Santa Cruz Co. , the others and S - P kit were purchased from Fuzhou Maixin Biocom-pany. The sections were deparaffinized, water processed , then treated by 0. 1% trytsin to expose antigen and 3% hydrogen peroxide for 30 minutes to quench the endogenous peroxidase activity. After that, all were incubated for 30 minutes with normal non - immuneserum to eliminate nonspecific staining, and then incubated with primary antibody at 4℃ overnight. This was followed by incubation with biotin - labelled condary antibody and streptavition - peroxidase complex for 30 minutes. The color was developed with diaminobenzidine and the section were couterstained with harmatoxylin. The blank control were carried out by replacing the primary antibody with PBS.The expression of PRL - 3 by IHC staining was graded for both the extentand intensity of label. The extent of staining was evaluated on a scale of 0 -2:0<50% ; 1 =50 - 75% ; 2 >75% , and the intensity of staining was evaluatedon a scale of 0 - 2: no yellow was 0, buff was 1, yellow was 2. The case whichthe product of the two score was 2 or over 2 were positive ones.MVD including lymphatic was determined by the expression of CD34 in endothelial cell with blind method. Two doctors counted the vessels which shows positive expression of CD34. Chose three hot spots to count and then calculated the mean as MVD.In this study, IHC results were scored positive for ER and PR if immunore-activity showed greater than 5% nuclear staining, C - erBb -2 if showed greater than 5% plasm staining.The statistical method were according to X2 test、 t - test and ANOVA inSPSS 10.0.Results1. The expression of PRL - 3 in breast carcinoma and para - carcinoma. The expression of PRL - 3 was localized in cytoplasm or cell membrane,most in cytoplasm, only a little in nuclear. The positive expression rate in carcinoma was 80.9% , that in para - carcinoma was 40% , and they were significant different in statistics ( P = 0. 008 ).2. The relationship between the expression of PRL -3 and pathological features.The expression of PRL -3 was correlated closely with clinic stage (P =0. 001) , lymph node metastasis( P = 0.015) and tumor size( P = 0.031) , but had no relationship with age, gender, family disease history, and the positive rates of ER, PR and C - erBb -2.3. The relationship between MVD and clinic pathological features.MVD in breast carcinoma was higher than that in para - carcinoma(P =0. 031) and was correlated with clinic stage(P =0. 023) and lymph node metastasis (P = 0.000) , but had no relationship with age, gender, family disease history, tumor size and positive rates of ER, PR and C — erBb -2.4. The relationship between the expression of PRL -3 and MVD.The MVD mean of the group with positive expression of PRL - 3 was higher than that with negative expression of PRL - 3 , and they were significant different in statistics (P =0.000).Discussion1. The relationship between MVD and breast carcinoma.The growth of tumor depends on vessels to supply nutrition and pack off the waste. Breast cancer grows quickly, which may be attributed to its high MVD. Because of lacking of perfect structure, the new grown vessels supply chances for tumor to invade and metastasize, which results in poor prognosis. Nathansonand his colleagues found that MVD in breast cancer was associated with the metastasis into lymph node in armpit, which was an important factor that influenced prognosis. Our research showed MVD was positively correlated with lymph node metastasis and clinic stage, which suggest mammary tumorigenesis is the crucial factor in breast cancer metastasis.2. The relationship between the expression of PRL -3 and invasion and metastasis of breast cancer.The recent research found that PRLs of 23 KD was a newly small protein tyrosine phosphatases ( PTPs ) , which may be play a key role in the course of tumor invasion and metastasis. PRL regulates biological behavior of tumor by stimulating cell proliferation, inhibiting cell apoptosis and influencing angiogen-esis. It was reported that the PRL - 3 expression rates of metastases were significantly higher than those of primary colorectal cancers and normal colorectal epi-thelia. Upik also found the overexpression in gastric carcinoma and confirmed it ’s correlation with the metastasis in lymph node. Our experiment showed that the expression level of PRL - 3 was higher in breast carcinoma than that in para -carcinoma and its overexpression was associated with lymph node metastasis, clinic stage and tumor size, which implies that PRL - 3 can promote the growth, invasion and metastasis of breast cancer cell.3. The relationship between the expression of PRL -3 and MVD.PRL - 3 can not only directly effect on mammary epithelial cells themselves , but also influence mammary carcinogenesis . PRL - 3 can act as circulating hormones and as paracrine/autocrine factors to stimulate various stages of the formation and remodeling of new blood vessels, including endothelial cell proliferation, migration, protease production and apoptosis. Some research reveal that PRL - 3 gene was expressed by tumor endothelium, but not normal endothelium. Our results showed that PRL - 3 protein was not expressed by endothelial in breast cancer and the MVD mean of the group with positive expression of PRL - 3 was higher than that with negative expression of PRL -3 (t =5.718, P=0.000). It implies that the expression of PRL -3 is closely correlated with mammary tumorigenesis, which may accelerate growth and metastasis of breast cancer by promoting tumorigenesis.更多还原