【作者】 薛金慧；

【导师】 吕新全； 李惠翔；

【作者基本信息】 郑州大学 ， 病理学与病理生理学， 2012， 硕士

【摘要】 背景及目的乳腺癌是女性最常见的恶性肿瘤之一,严重影响了妇女的身心健康甚至危及生命。目前乳腺癌的发生机制尚未完全阐明,国内外多项研究认为乳腺癌是一种肿瘤干细胞疾病。乳腺癌干细胞(breast cancer stem cells,BCSCs)可能是乳腺癌发生及治疗后复发和转移的根源。因此,彻底去除乳腺癌干细胞有可能彻底治愈乳腺癌。乙醛脱氢酶1(aldehyde dehydrogenase1,ALDH1)已经被证实为乳腺癌干细胞的一个可靠的标记物。ALDH1属于乙醛脱氢酶家族中的一员,其表达与乳腺癌的发生、发展、预后和转移密切相关,且在乳腺癌干细胞表面高表达。ALDH1是一种能氧化细胞内的醛类的解毒酶,在干细胞的分化早期氧化视黄醇而形成视黄酸。越来越多的证据表明,肿瘤干细胞在粘附、迁移和侵袭能力等方面具有特征性。与分化子代细胞相比,肿瘤干细胞过表达多种侵袭相关的因子,包括CXC趋化因子受体4(CXC chemokine receptor4,CXCR4). CD44、MMP2等,提示肿瘤干细胞可能在恶性肿瘤的侵袭转移中扮演重要的角色。MMPs家族是一个高度保守的内源性蛋白水解酶家族,是细胞外基质的主要调节物质,几乎能够降解细胞外基质的所有成分,其中MMP-2是家族中的主要成员之一,主要作用于基底膜上的Ⅳ型胶原,在肿瘤细胞突破基底膜屏障,发生侵袭转移的过程中发挥重要作用。CXCR4属于G蛋白耦联7次跨膜受体,是CXC趋化因子家族成员基质细胞衍生因子1(stromal cell derived factor-1,SDF-1)/CXCL12的受体,与其配体SDF-1构成的SDF-1-CXCR4生物学轴,可能在多种恶性肿瘤细胞移动、浸润、转移和增殖中发挥重要作用。有关联合检测ALDH1、MMP2、XCR4三者在乳腺癌浸润性导管癌(invasive ductal carcinoma,IDC)原发灶及淋巴结转移灶中的表达及其与乳腺癌浸润及淋巴结转移关系的研究,至今尚未见有报道。且对ALDH1的生物学功能研究不多,对其作用机理也知之甚少。为了探讨ALDH1标记的乳腺癌干细胞在乳腺癌原发灶及淋巴结转移灶中的表达以及与乳腺癌浸润转移的关系和可能的机制,本实验采用免疫组织化学SP法及原位杂交技术分别检测ALDH1、MMP2、CXCR4蛋白及ALDH1mRNA在90例乳腺癌原发灶及相应的60例淋巴结转移灶中的表达情况,旨在探讨三者之间的相互关系及可能的作用机制,为乳腺癌浸润转移的治疗开辟新的思路。方法1.采用免疫组化SP法检测90例IDC原发灶组织和相应的60例淋巴结转移灶组织中ALDH1、MMP2、CXCR4蛋白的表达。2.采用原位杂交技术检测90例IDC原发灶组织和相应的60例淋巴结转移灶组织中ALDH1mRNA的表达。3.统计学处理：应用SPSS17.0统计学软件,率的比较采用χ2检验(chi-square),相关性分析采用Spearman相关分析,检验水准α=0.05。结果1.在90例乳腺IDC原发灶中,ALDH1蛋白、ALDHlmRNA、MMP2蛋白、CXCR4蛋白的阳性表达率分别为27.8%(25/90),30%(27/90),70.0%(63/90),76.7%(69/90)。在60例原发灶及相应的淋巴结转移灶中,ALDH1蛋白(mRNA)的阳性率分别为28.3%(31.7%)、35%(35%),且在两个部位的表达相互呈正相关,且ALDH1蛋白及1nRNA在原发灶及相应的淋巴结转移灶的表达分布特征相似,均为散在或小簇状分布；MMP2蛋白的阳性率分别为81.7%、75.0%,在两个部位的表达无相关性；CXCR4蛋白的阳性率分别为85.0%、80.0%,在两个部位的表达无相关性。2.在90例乳腺IDC原发灶中,ALDH1蛋白及mRNA的表达均与较高组织学分级,ER阴性表达,PR阴性表达正相关(P<0.05)；MMP2蛋白与CXCR4蛋白的表达均与组织学分级和淋巴结转移正相关(P<0.05)。在60例淋巴结转移灶中,ALDH1蛋白及mRNA的表达均与ER阴性表达,PR阴性表达正相关(P<0.05)；MMP2蛋白与CXCR4蛋白的表达与乳腺IDC的临床病理特征均无关(P>0.05)。3.在90例乳腺IDC原发灶中,ALDH1蛋白和MMP-2蛋白的表达呈正相关(r=0.298,P<0.05)；ALDH1蛋白和CXCR4蛋白的表达呈正相关(r=0.283,P<0.05)；MMP-2蛋白和CXCR4蛋白的表达呈正相关(r=0.556,P<0.05),且MMP2与CXCR4蛋白在ALDH1表达阳性组的阳性率显著高于表达阴性组的阳性率,差异有统计学意义(P<0.05)。在60例乳腺IDC淋巴结转移灶中,ALDH1蛋白和MMP-2蛋白的表达呈正相关(r=0.343,P<0.05)；ALDH1蛋白和CXCR4蛋白的表达呈正相关(r=0.280,P<0.05)；MMP-2蛋白和CXCR4蛋白的表达呈正相关(r=0.481,P<0.05),且MMP2与CXCR4蛋白在ALDH1表达阳性组的阳性率显著高于表达阴性组的阳性率,差异有统计学意义(P<0.05)。4.在90例乳腺IDC原发灶中,ALDH1与MMP2蛋白共阳性的23例癌组织中,表达ALDH1的乳腺癌细胞同时表达MMP2蛋白的有20例(87.0%)；ALDH1与CXCR4蛋白共阳性的24例癌组织中,表达ALDH1的乳腺癌细胞同时表达CXCR4蛋白的有23例(95.8%)。在60例淋巴结转移灶中,ALDHl与MMP2蛋白共阳性的20例癌组织中,表达ALDH1的乳腺癌细胞同时表达MMP2蛋白的有18例(90%);ALDH1与CXCR4蛋白共阳性的20例癌组织中,表达ALDH1的乳腺癌细胞同时表达CXCR4蛋白的有19例(95%)。结论1ALDH1蛋白及mRNA在乳腺IDC原发灶与转移灶中均有表达,且主要表达在ER阴性、PR阴性、低分化癌组织中。2.在乳腺IDC中,ALDH1的表达可能促进了肿瘤的浸润与转移,其机制可能与ALDH1阳性细胞同时表达MMP2及CXCR4有关。更多还原

【Abstract】 Background and objectiveBreast cancer is one of the most frequent malignant tumors of women, which has a strong impact on their physical and psychological health, and what is even more serious is that it can threaten their lives. At present, the pathogenesis of breast cancer has not elucidated completely. Several domestic and abroad studies suggest that its occurrence and development is a progressional process in which many factors participate. At present, breast cancer stem cell becomes a hot topic of research.lt may initiate the growth of breast cancer and it may be the root cause of its recurrence and far metastasis after neoadjuvant chemotherapy. As a result, exterminating the breast cancer stem cell thoroughly may cure breast cancer completely.Aldehyde dehydrogenase1(ALDH1) has been identified as a reliable marker of breast cancer stem cells. ALDH1belongs to a member of the aldehyde dehydrogenase family, wich is closely related to occurrence, development, prognosis and metastasis of breast cancer and highly expressed on the surface of breast cancer stem cells. ALDH1, a detoxifying enzyme responsible for the oxidation of intracellular aldehydes, was shown to play a role in the early differentiation of stem cells, through its role in oxidizing retinol to retinoic acid. Growing evidence suggests that cancer stem cells possess characteristic in terms of adhesion, migration and attack ability. Compared to differentiated offspring cells, cancer stem cells can overexpress a variety of invasion-related factors including CXCR4, CD44, MMP2and so on. It prompts that cancer stem cells maybe paly an important role in the invasion and metastasis of malignant tumor.MMPs family is a highly conserved family of endogenous proteolytic enzymes and is a major regulator of extracellular matrix material, which can degrade almost all components of the extracellular matrix. MMP-2is one of the main members of the family and plays an important role in the process of tumor’s invasion and metastasis, because it mainly acts on the basement membrane collagen type IV and promotes tumor cells break through the basement membrane barrier.CXC chemokine receptor4belongs to the G protein-coupled receptors superfamily and is one of CXC-chemokine receptors, which is the specific receptor of Stromal cell derived factor-1(SDF-1). The CXCR4-SDF-1biology axis maybe paly an important role in the migration, invasion, metastasis and proliferation of breast cancer.The researches about combined detection of ALDH1, MMP2, CXCR4expressions in both primary carcinomas and lymph node metastases of invasive ductal carcinoma and their relationship with breast cancer invasion and lymph node metastasis have not been reported. In addition, fewer studies have been used for the biological function of ALDHl,and the mechanism of action of ALDH1is also poorly understood.To investigate the expressions of breast cancer cells which are marked with ALDH1in primary carcinomas and lymph node metastases of breast cancer and their relationship with breast cancer invasion and metastasis and the possible mechanism, the present study detects the expressions of ALDH1,MMP2,CXCR4proteins by immunohistochemistry and ALDH1mRNA by in situ hybridization in90cases of primary carcinomas and60cases of lymph node metastases of invasive ductal carcinoma,and explore the relationship among ALDH1,MMP2,CXCR4and the probable mechanism.It is helpful for researchers to find out new ideas for the therapy of breast cancer invasion and metastasis.Methods1. Expressions of ALDH1, MMP2, CXCR4proteins were detected by immunohistochemical staining of SP method in90cases of primary carcinomas and60cases of lymph node metastases of invasive ductal carcinoma.2. Expression of ALDHlmRNA was detected by the technique of in situ hybridization in90cases of primary carcinomas and60cases of lymph node metastases of invasive ductal carcinoma.3. Statistical analysis:All the dates were analyzed by SPSS17.0statistical software package. The Chi-square was used for the comparison of positive rates; the Spearman’s correlation analysis was used for analyzing the correlation of two variables. The level of significant difference is a=0.05.Results1. The positive rates of ALDH1protein, ALDHl mRNA, MMP2protein and CXCR4protein were27.8%(25/90),30%(27/90),70.0%(63/90) and76.7%(69/90) respectively in90cases of primary carcinomas of invasive ductal carcinoma. The positive rates of ALDH1protein (mRNA) were28.3%(31.7%) and35%(35%) respectively in60cases of primary carcinomas and corresponding lymph node metastases of invasive ductal carcinoma, and the expressions in two parts were positively related to each other. And the expression characteristics of lymph node metastases that ALDH1protein and mRNA are both scattered or small cluster-like distribution were similar with the primary cancers.The positive rates of MMP2protein were81.7%and75.0%respectively in60cases of primary carcinomas and corresponding lymph node metastases of invasive ductal carcinoma, but there was no correlation in the two parts. The positive rates of CXCR4protein were85.0%and80.0%respectively in60cases of primary carcinomas and corresponding lymph node metastases of invasive ductal carcinoma, but there was also no correlation in the two parts.2. In90cases of primary carcinomas of invasive ductal carcinoma, the expressions of ALDH1protein and mRNA were both positive correlated with high histological grade, ER negativity, PR negativity(P<0.05); the expressions of MMP2protein and CXCR4protein were both positive correlated with high histological grade and lymph node metastasis(P<0.05). In60cases of lymph node metastases of invasive ductal carcinoma, the expressions of ALDH1protein and mRNA were both positive correlated with ER negativity, PR negativity(P<0.05); the expressions of MMP2protein and CXCR4protein were both not correlated with any clinicopathological parameter(P>0.05).3. In90cases of primary carcinomas of invasive ductal carcinoma, there was a positive correlation between the expression of ALDH1protein and MMP-2protein (r=0.298, P<0.05); there was a positive correlation between the expression ofALDHlprotein and CXCR4protein (r=0.283, P<0.05); there was a positive correlation between the expression of MMP-2protein and CXCR4protein (r=0.556, P<0.05); moreover, the positive rates of MMP-2protein and CXCR4protein in ALDH1-positive group were significantly higher than that in ALDH1-negative group, and the difference was statistically significant (P<0.05). In60cases of lymph node metastases of invasive ductal carcinoma, there was a positive correlation between the expression ofALDHl protein and MMP-2protein (r=0.343, P<0.05); there was a positive correlation between the expression ofALDHl protein and CXCR4protein (r=0.280, P<0.05); there was a positive correlation between the expression of MMP-2protein and CXCR4protein (r=0.481, P<0.05); moreover, the positive rates of MMP-2protein and CXCR4protein in ALDH1-positive group were significantly higher than that in ALDH1-negative group, and the difference was statistically significant (P<0.05).4. In90cases of primary carcinomas of invasive ductal carcinoma, ALDH1and MMP2proteins were both positive expressions in23cases of carcinoma tissues,20cases(87.0%) of which were found ALDH1-positive cells expressed MMP2protein at the same time; ALDH1and CXCR4proteins were both positive expressions in24 cases of carcinoma tissues,23cases(95.8%) of which were found ALDH1-positive cells expressed CXCR4protein at the same time.In60cases of lymph node metastases, ALDH1and MMP2proteins were both positive expressions in20cases of carcinoma tissues,18cases(90.0%) of which were found ALDH1-positive cells expressed MMP2protein at the same time; ALDH1and CXCR4proteins were both positive expressions in20cases of carcinoma tissues,19cases(95%) of which were found ALDH1-positive cells expressed CXCR4protein at the same time.Conclusions1. ALDHl protein and mRNA were expressed both in primary carcinomas and lymph node metastases, and were mainly expressed in the ER negative、PR negative、 poorly differentiated cancer tissue.2. In breast infiltrative ductal carcinoma tissues, ALDH1-positive expression may play an important role in promoting invasion and metastasis of breast cancer, and the mechanism may be related to expressions of MMP-2and CXCR4of ALDH1-positive breast cancer cells.更多还原