【作者】 张瑞山；

【导师】 路平；

【作者基本信息】 中国医科大学 ， 肿瘤学， 2010， 博士

【摘要】 前言越来越多的证据表明,乳腺癌起源于一部分数量很少,有着特殊表型的乳腺癌细胞群。这类细胞有自我更新的潜能,能够生成新的异质性肿瘤。这类肿瘤细胞代表了乳腺癌的无限制生长,侵袭和转移,免疫逃逸,放疗不敏感,化疗耐药等特点,这一类细胞群被称为肿瘤干细胞(CSC)。在实体肿瘤中,乳腺癌,胶质细胞瘤,直肠癌,前列腺癌,肺癌等几种恶性肿瘤中的肿瘤干细胞已经被成功分离并证明。乳腺癌中CD44+／CD24-表型的细胞被认为是富集乳腺癌干细胞的细胞群,它能够反映乳腺癌的生物学特点。从人乳腺癌转移淋巴结和癌性胸水中分离的乳腺癌CD44+／CD24-表型的细胞被确定具有干性。在乳腺癌肿瘤细胞中CD44+／CD24-表型的细胞亚群具有强大的成瘤能力,仅几百个这类细胞就可以在重症免疫缺陷小鼠中产生肿瘤,而注射100倍的不表达同样抗原的其他乳腺癌细胞亚群并不能形成肿瘤。越来越多的研究显示,乳腺癌干细胞是乳腺癌发生和发展的源泉,在乳腺癌的治疗中,完全杀灭和消除乳腺癌干细胞是彻底治愈乳腺癌的关键。本研究目的分离乳腺癌干细胞并验证其成瘤能力及其生物学行为特点,为乳腺癌的治疗提供依据。乳腺癌中CD44+／CD24-表型的细胞的分离培养,分化,成瘤等生理特征尚存在争论,其所占比率与乳腺癌临床病理学特点的关系鲜有报道,这样一群肿瘤细胞和乳腺癌临床指标的关系值得进一步探讨。Octamer-4 (Oct-4)是一种在胚胎干细胞和成体干细胞中表达的转录因子,他在干细胞和生殖细胞中与细胞的增殖分化,细胞的多能性和自我更新有关。迄今为止,Oct4在成体组织中的表达主要局限于具有干细胞特性的细胞,如皮肤基底细胞层中的个别细胞、乳腺干细胞和胃干细胞等。随着肿瘤干细胞理论不断深入,干细胞相关基因在恶性肿瘤中的作用越来越被学界所关注。目前Oct-4在乳腺癌干细胞的生物学行为中所起作用及机制的研究尚鲜有报道,且Oct-4蛋白的表达水平与乳腺癌生物学行为及临床病理特征的关系尚不清楚。我们应用基因芯和免疫组化方法对Oct-4蛋白的表达水平与乳腺癌生物学行为及临床病理特征的关系进行研究,以期为乳腺癌的治疗提供理论依据。实验材料与方法一、乳腺癌CD44+／CD24-表型细胞的分离以及其成瘤能力的鉴定应用流式细胞仪分选技术,从乳腺癌手术标本中,分离出乳腺癌CD44+／CD24-表型的肿瘤细胞亚群,分析这一类细胞在整个乳腺癌肿瘤细胞中所占的比率以及其与临床生物学行为的关系。在免疫缺陷小鼠体内验证乳腺癌CD44+／CD24-表型的肿瘤细胞与其它亚群细胞相比成瘤能力明显增强。二、干细胞相关基因Oct-4表达的检测用基因芯片方法检测Oct-4等肿瘤相关基因在乳腺癌中的表达情况。通过免疫组化方法分析Oct-4表达与乳腺癌临床病理指标的关系。三、统计方法所有数据用SPSS13.0统计学软件进行分析。组问比较用χ2检验,生存率分析用Kaplan-Meier法计算,生存率比较用Log rank检验,采用COX模型进行预后多因素分析。结果一、乳腺癌CD44+／CD24-表型细胞在病灶中的比率与临床病理学指标的关系以及其成瘤能力的特点乳腺癌CD44+／CD24-细胞在病灶中的比率3.75%-33.11%,平均含量(13.67+9.71%)。其比率与N分期有关(10.37%VS 16.16%P=0.037),与ER,Her-2受体表达状态密切相关(11.05%VSl6.27 P=0.043：9.47%VS 17.52%P=0.013)。用流式细胞仪分离出CD44+／CD24-标记亚群细胞5000个细胞接种糖尿病重症免疫缺陷鼠(SCID／NOD鼠)中可生成肿瘤,其余细胞接种105个细胞无肿物生成。用未经过分选的原代肿瘤细胞106个细胞接种免疫缺陷小鼠可以成瘤。说明乳腺癌中CD44+／CD24-细胞亚群与其他细胞相比,有更强大的成瘤能力,成瘤能力是未分选肿瘤细胞的100倍以上。二、干细胞相关基因Oct-4表达的临床意义1、CD44+／CD24-的乳腺癌肿瘤细胞与非CD44+／CD24-肿瘤细胞的基因表达谱差异：以下基因在实验组中表达明显高于对照组：干细胞分化相关因子CD44,Oct4,nestin,(145.82,64.28,49.17);细胞周期调节因子：APC,CDC2(4.79,33.0)；生长因子：HGF,TGF(12.82,37.38)。2、Oct-4在乳腺癌中的表达及与临床病理特征的关系：Oct-4基因在肿瘤组织表达明显高于癌旁,与组织学类型、有无淋巴结转移和乳腺癌的分子分型显著相关。Oct-4蛋白表达阳性患者术后生存率与Oct-4蛋白表达阴性患者术后生存率有统计学差异(P=0.001)。通过COX回归模型进行分析发现肿瘤大小,组织学类型,病期,淋巴结转移,Her-2,Oct4是乳腺癌独立的预后因素(P=0.031；0.012；0.001；0.002：0.030；0.003)。结论1、乳腺癌病灶中存在CD44+／CD24-表型的细胞亚群,不同病例中的CD44+／CD24-细胞在乳腺癌中的比率有很大差别。乳腺癌中CD44+／CD24-细胞比率与乳腺癌的进展有一定关系,与乳腺癌雌激素受体表达成正相关,与Her-2受体表达负相关。2、乳腺癌中CD44+／CD24-表型肿瘤细胞在体内实验中有很强的成瘤能力,证明这一细胞群具有干细胞的特征。3、通过本研究提示Oct-4在乳腺癌CD44+／CD24-表型细胞中高表达,不但与乳腺癌的生物学行为密切相关,而且是乳腺癌的独立预后因素。更多还原

【Abstract】 Emerging evidence have been carried out that the breast cancer cells originate from a rare population of tumor cells, which maintain the property of stem cells. These cells have enhanced self-renewal capacity, tumorigenic, and construct the whole population of tumor cells. These cells represent some main character of breast cancer such as unlimited growth pattern, invasion and metastasis, immunal released, irresponsable to radiation, chemotherapy resistance. Cancer stem cells were successfully isolated and identified in some solid tumors such as breast cancer, brain cancer, rectal cancer and prostate cancer. The population of breast cancer cells with the phenotype of CD44+/CD24- was considered to be enriched for breast cancer stem cells, and this population of tumor cells reflects some biology characteristics of breast cancer. Breast cancer cells with the phenotype of CD44+/CD24- isolated from metastatic pleural effussion show enhanced tumorigenesis and less to 100 cells with this type can form tumor in SCID/NOD mouse, further more other subpopulation of tumor cells 100 fold more fail to form tumor in vivo. Resent studies show that the breast cancer stem cells are the resource of the breast cancers. It is the key procedure to eliminate the breast cancer stem cells in treating breast cancers. The objective of this project is to isolate the population of CD44+/CD24- phenotype of breast cancer cells and identify its tumorigenic capacity and the correlation between the ratio of these cells in breast cancer and the pathological character of breast cancers.Octamer-4 (Oct-4) is a transcription factor in ESCs, it is correlated with the pluripotency, proliferative potential and self-renew capacity in embryonic stem cells and germ cells. Up to the present the research on Oct-4 is limited in cells with stem property, such as stem cells of skin basal layers, breast stem cells and gastric cells. With the profound investigation in cancer stem cells, role of stem related genes are more concerned. There is rare research about the mechanism of Oct-4 in breast cancer cells properties. We focus on the expression of Oct-4 in breast cancers with gene chip arrays and immunochemical analysis and illuminate its relationship with the clinical pathological character of breast cancer, in order to provide more evidence for breast cancer therapy.Materials and Methods一.Isolation of CD44+/CD24- phenotype breast cancer cells and identify its tumorigenic capacityThe flow cytometry(FCM) technique is performed to isolate the cell population with CD44+/CD24- phenotype from breast cancer specimens, and analysis the ratio of these cells in breast cancer and investigate its significant comparing with the breast cancer clinical parameters. Also we test the tumorigenic capacity of this population of cancer cells in vivo.二.Test of stem cell associate gene Oct-4Gene chip array and immunochemical analysis are used to prove the relationship between the expression of Oct-4 and breast cancer clinical parameters, immunochemical methods are also used to test the expression of ER, PR and Her-2 gene expression in breast cancer research.三.Statistic methodsDatas are analysis by SPSS13.0 statistic software. T-test and X2 test were used to analysis the relationship of these parameters. Kaplan-Meier method and Log rank method are used to analysis the survival rate and COX model is used to test prognosis factors.Results一.Ratio of CD44+/CD24- phenotype of breast cancer cells indicate clinicopathological characters and its tumorigenic capacityThe cell population of CD44+/CD24- phenotype take up the ratio of cancer cells between 3.75%-33.11%, the mean ratio is 13.67%. Compared with the NO stage of breast cancer, cases with positive lymph node metastasis, N1N2, exhibit an accelerated CD44+/CD24-cancer cell ratio (10.37% VS 16.16% P=0.037). In respect with the hormone receptor expression, CD44+/CD24-cancer cells have more enrichment in ER- cases than that in ER+ breast cancers (11.05% VS 16.27% P=0.043). Her-2+ breast cancers have higher CD44+/CD24- cancer cell ratio than Her-2- ones (9.47% VS 17.52% P=0.013). Breast cancer cells with phenotype of CD44+/CD24- isolated by FCM were injected in SCID/NOD mouse together with other subpopulation of cancer cells as negative a control, as little as 5000 CD44+/CD24- tumor cells can develop new tumor in mouse, nevertheless 105 other phenotype cancer cells fail to develop tumor, which indicate the enhanced tumorigenic facility in CD44+/CD24- subpopulation of breast cancer cells.二.Expression of Oct-4 in CD44+/CD24- breast cancer cells and its clinical implicationsThe difference of gene expression profile between CD44+/CD24- tumor cells and CD44+/CD24- negative tumor cells:the list of genes which are highly expressed:stem cells associated factors CD44, Oct4, nestin,(145.82,64.28,49.17);cell cycle regulators:APC,CDC2(4.79,33.0); growth factors:HGF, TGF (12.82,37.38). Oct-4 is highly expressed in breast cancer tissues compared with the adjacent normal tissues.Oct-4 positive breast cancer patients own shorter survival rate (P=0.001). COX model indicate that tumor size, histological type, stage of cancer, lymph node metastasis, Her-2 and Oct-4 expression are the independent prognosis factors of breast cancer (P=0.031;0.012; 0.001; 0.002; 0.030; 0.003).Conclusions1. Breast cancer cells contain a subpopulation of CD44+/CD24- tumor cells. CD44+/CD24- tumor cell ratio is correlated with N stage of breast cancer, and is with relationship to ER, Her-2 expression.2. The subpopulation of CD44+/CD24- phenotype of tumor cells are enriched for breast cancer stem cells and exhibit enhanced tumorigenicity which reflect its stem cell properties. 3. Oct-4 gene is highly expressed in CD44+/CD24- breast cancer cells. Its expression is correlated with clinicopathological character of breast cancer, and is an independent factor for the survival rate.更多还原