乳腺癌干细胞的体外培养、分离、鉴定和乳腺癌相关基因表达及意义的研究
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摘要
背景与目的：乳腺癌(breast cancer)是女性常见的恶性肿瘤。在西欧和北美高发区,是严重威胁妇女健康的常见病和多发病。进入21世纪,全世界每年约有120万妇女患乳腺癌,50万人死于乳腺癌。美国为乳腺癌的高发国家,乳腺癌的发病率呈明显的上升趋势,1973-1976年美国每年估计有10万人患乳腺癌,每年有3万人死于该病；1990年全年约有15万新发病例,有4.4万人死亡,依据发病率变化趋势估计,21世纪美国将每8名妇女中就有1人一生中将患乳腺癌。我国为乳腺癌的低发国,但近年来的临床观察提示,我国乳腺癌发病率在不断的上升,每年约增长3%,在京、津、沪等大中城市,乳腺癌已跃居女性恶性肿瘤之首年龄有明显的提前趋势,且我国临床诊断的乳腺癌病期偏晚。为此,乳腺癌的研究日益引起人们的关注。
肿瘤干细胞理论认为,肿瘤细胞内存在一群具有干细胞性质的细胞(称肿瘤干细胞或肿瘤起始细胞,cancer stem cells or cancer initiating cells, CSCs or CICs),不受外界调控的恶性增殖、多向分化,从而形成肿瘤。越来越多的研究证实在脑胶质瘤、乳腺癌、前列腺癌和恶性黑色素瘤等实体瘤中存在肿瘤干细胞。
Hedgehog家族成员包括SHH、PTCH、GIi-1和SMOH。Hedgehog信号通路激活的具体机制目前尚不完全清楚,多数学者认为主要包括配体依赖的激活和配体非依赖的激活。配体依赖的激活是指配体如SHH、IHH(Indian Hedgehog)与受体PTCH结合,诱导原癌基因Smo的激活,引起Hedgehog信号通路的激活,这已在小细胞肺癌、肝癌、乳腺癌、脑恶性肿瘤和消化道癌中得到证实；配体非依赖的激活主要指信号通路成员如PTCH或SMOH突变所启动的Hedgehog信号通路的激活,这已经在Gorlin-相关肿瘤(如基底细胞癌和横纹肌肉瘤等)中得到证实。GIi-1基因的高表达是Hedgehog信号通路活化目前比较公认的标志之一。多项研究证实Hedgehog信号通路在多发骨髓瘤、乳腺癌和恶性胶质瘤等的干细胞中高表达,并在其自我更新中起重要作用。敲除SHH信号可导致小鼠神经干祖细胞明显减少,在体外培养几乎不能形成神经球；相反,激活SHH途径,使成年小鼠终脑的干祖细胞数量明显增加。IHH突变可减少肠道干细胞的增殖和分化。
细胞角蛋白(cytokeratin, CK)是分布于上皮细胞的中间纤维丝主要表达于上皮细胞,在人类上皮组织中共发现至少20个成员,其表达具有很强的组织特异性和分化特异性。每种上皮组织在生长分化的不同阶段都表达不同的CK,其表达受细胞分化调节,如腺上皮主要表达I型CK(包括CK7, CK8, CK18, CK19, CK20),而鳞状上皮主要表达Ⅱ型CK(包括CK1, CK5/6, CK14)。因此,对不同CK的定位研究和观察不同状态下CK的表达,对了解上皮细胞的增殖和分化状态以及疾病的发生和发展有一定的意义。
研究提示,CK5是基层的标志角蛋白,在乳腺普通增生细胞中,CK5常阳性,而在多数浸润性导管癌及导管原位癌中,CK5常阴性,所以CK5常作为浸润性和导管原位癌与普通型增生鉴别的依据之一。CK8是单纯上皮的标志角蛋白,它能够穿透乳腺癌细胞的外表面,并以一种可溶的杂多聚体的形式从癌细胞中释放出来,CK8存在于许多单纯上皮细胞和癌中,也用于乳腺癌的鉴别诊断。乳腺癌耐药蛋白(breast cancer resistance protein, BCRP)是一个位于细胞膜上的ABC半转运蛋白,有研究提示它可能是乳腺上皮组织低分化的标志,在异常增生的组织中BCRP高表达,是细胞未分化成熟的表现BCRP的存在可能与肿瘤细胞耐药并引起肿瘤复发和治疗失败有关。
本课题以乳腺癌细胞系MCF-7和人乳腺癌组织为研究对象,通过体外培养、分选乳腺癌干细胞,并采用实时定量PCR技术及免疫组织化学方法分别检测Hedghehog通路在MCF-7细胞系CD44+/CD24-/low干细胞亚群中的表达情况以及Hedghehog通路的重要蛋白,细胞角蛋白在人乳腺癌组织中的表达情况。分析它们与不同乳腺病变之间可能存在的相关性,探讨乳腺癌的发生机制,将为乳腺癌的早期诊断、预后评价和基因治疗指标的筛选提供有价值的实验依据。
材料与方法：乳腺癌细胞系MCF-7和人乳腺病变组织均选自大连医科大学中日临床病理中心,2004-2009年人乳腺组织标本164例,其中正常乳腺组织23例、乳腺增生组织16例、乳腺纤维腺瘤组织26例、导管上皮非典型增生12例、导管原位癌6例和浸润性导管癌81例。患者年龄28-75岁,平均年龄45岁。所收集的标本均为手术切除送检标本,术前均未接受放化疗治疗,术后获得明确病理诊断并得到患者及其家属的知情同意。所有切片均经2位高年资病理医师复诊,其中正常乳腺组织为13例癌旁乳腺组织,距肿瘤5cm。本研究采用免疫组织化学(IHC)、免疫细胞化学(ICC)、实时定量聚合酶链式反应(Realtime-PCR)和细胞培养、磁珠分选等方法,检测Hedgehog通路在MCF-7细胞系CD44+／CD24-／low干细胞亚群中的表达情况,Hedghehog通路的重要蛋白,细胞角蛋白在人乳腺癌组织中的表达情况和分布特点。实验数据采用SPSS 11.5软件包,应用Kruskal-Wallis和Mamm Whitney的检验方法,对各因子在不同乳腺病变组织中的分布进行统计学差异分析。应用Spearmen相关性检验判断相关因素之间的统计学关系
结果：
一、乳腺癌细胞系MCF-7中CD44+／CD24-／low亚群比例为(8.90+1.73)%。乳腺癌细胞系MCF-7的CD44+／CD24-／low亚群中Hedgehog信号通路处于明显活化状态,其中Hedgehog信号通路中重要下游信号分子GIi-1在MCF-7细胞系CD44+／CD24-／low细胞亚群中的表达要明显高于非CD44+／CD24-／low细胞亚群(P为0.007)。
二、PATH 1和Gli-1在正常乳腺组织、乳腺瘤组织、及乳腺癌中的阳性表达率呈现逐渐升高的趋势,PATH1表达率分别为10.0%,46.1%和96.2%,Gli-1表达率分别为20.0%,69.2%和98.1%。随着乳腺病变组织分化程度的下降,PATH1和Gli-1的阳性表达率明显增加,呈强阳性表达。浸润性导管癌组织中的表达明显高于正常乳腺组织,乳腺癌组织中的表达明显高于乳腺瘤组织(P<0.05)。
三、CK5和CK8在正常乳腺组织、乳腺增生、非典型增生及乳腺癌中的阳性表达率依次下降。CK5在正常乳腺组织、乳腺增生、导管上皮非典型增生、导管原位癌和浸润性导管癌组织的阳性表达率分别为84.6%,62.5%,41.7%,33.3%,19.0%。CK8在正常乳腺组织、乳腺增生、导管上皮非典型增生、导管原位癌和浸润性导管癌组织的阳性表达率分别为100.0%,100.0%,75%,66.7%,66.7%。绝大部分浸润性导管癌呈现CK5-/CK8+,28例(66.7%),只有8例(16.7%)呈现CK5+／CK8-,8例呈现CK5+／CK8+,14例(29.2%)呈现CK5-／CK8-。随着乳腺病变组织分化程度的下降,BCRP的阳性表达率明显增加,在正常乳腺组织、乳腺增生、导管上皮非典型增生、导管原位癌和浸润性导管癌组织的阳性表达率分别为15.4%,250%,25%,33.3%,42.9%。浸润性导管癌组织中的表达明显高于正常乳腺组织,乳腺癌组织中的表达明显高于乳腺增生组织(P<0.05)。
结论：
一、乳腺癌细胞系MCF-7中存在CD44+／CD24-／low干细胞标志细胞亚群,其中Hedgehog信号通路处于明显活化状态。
二、PATH1和Gli-1在乳腺不同病变组织中的表达和分布存在差异,分化程度越低表达越强。
三、不同分化程度的乳腺导管癌中CK5和CK8的表达不同,分化程度越高CK8的表达越弱、CK5的表达相对较强,分化程度越低CK8的表达越强、CK5的表达相对较弱甚至缺失。BCRP在乳腺不同病变组织中的表达和分布存在差异,提示BCRP可能在乳腺肿瘤细胞分化方面有一定的作用。CK5.CK8和BCRP在不同乳腺病变组织中的相关性,三者联合检测有助于乳腺病变的病理学诊断和判断其分化程度及预后。
综上所述,免疫磁珠法分选乳腺癌干细胞是一种简便可靠的方法,分选得到的CD44+／CD24-／low干细胞标志细胞亚群中,Hedgehog信号通路处于明显活化状态。几种肿瘤干细胞标志物可用于乳腺病变的病理学诊断。
Backgrouds and Objectives:Breast cancer is a common female malignant tumor. It is common disease and frequently-occurring disease with a high incidence in Western Europe and North America, and is a serious threat to women's health. Into the 21st century, about 120 million women worldwide each year suffer from breast cancer,and 500,000 people die from breast cancer. The United States as countries with high incidence of breast cancer, showed a clear upward trend of breast cancer incidence from 1973 to 1976, the U.S. An estimated 10 million people suffering from breast cancer each year and 3 million people die of the disease. In 1990, of 15 million new cases, there are 44000 people were killed, according to the estimated incidence rate trends, in the 21st century in the United States, every eight women will have a person's life with breast cancer. Low incidence of breast cancer is showed in China, but in recent years, clinical observation suggest that breast cancer incidence rates in China continue to rise, growing at about 3 percent per year, in Beijing, Tianjin, Shanghai and other major cities, breast cancer has leapt the first female malignancy, age, significantly ahead of the trend, and the clinical diagnosis of breast cancer in our side late stage of disease. For this reason, breast cancer research reaches growing concern.
Cancer stem cell theory concern that tumor cells exist in a group of cells with stem cell properties(called cancer stem cells or tumor cells, or cancer initiating cells, CSCs or CICs), subject to external regulation and control of malignant proliferation of multi-differentiation to form tumors. Growing number of studies confirmed the brain glioma, breast cancer, prostate cancer and malignant melanoma and other solid tumors exist in cancer stem cells.
Hedgehog family members are including SHH, PTCH, GIi-1, and SMOH. Specific mechanism of activation of Hedgehog signaling pathway is currently not entirely clear, most scholars believe mechanisms including ligand-dependent activation and ligand-independent activation. Ligand-dependent activation is that ligand, such as SHH, IHH (IndianHedgehog) combined with the receptor PTCH, Smo-induced activation of proto-oncogene, causing the activation of Hedgehog signaling pathway, which has been confirmed in small cell lung cancer, liver cancer, breast cancer, brain cancer and digestive tract cancer. Ligand-independent signaling pathway activated mainly refers to members such as PTCH or SMOH mutations in the Hedgehog signaling pathway initiated by the activation, which has already in the Gor-lin-associated tumors (such as basal cell carcinoma, into the neural tube cell carcinoma and rhabdomyosarcoma, etc.) been confirmed.GIi-1 gene's highly expression is relatively recognized a marker of activation of the Hedgehog signaling pathway. A number of studies confirmed the Hedgehog signaling pathway in stem cells of multiple myeloma, breast cancer and malignant glioma and other cancers that highly expressed Hedgehog signaling pathway in stem cells, and the Hedgehog signaling pathway is confirmerd play an important role in their self-renewal. SHH signaling knock-out mice can lead to significantly reduced neural stem progenitor cells, so that in vitro almost can not form neurospheres. On the contrary, activating SHH ways can make the final adult mouse brain's stem progenitor cells increased significantly. IHH mutation can reduce the intestinal stem cell proliferation and differentiation
Cytokeratin (cytokeratin, CK) is distributed in the epithelial cells of the intermediate filament silk, mainly expressed in the epithelial cells, were found in human epithelial tissue of at least 20 members, its expression is highly tissue-specific and differentiation-specific. Each type of epithelial tissue in different stages of growth and differentiation express different CK, its expression by regulating cell differentiation, such as the main expression of typeⅠepithelial CK (including CK7, CK8, CK18, CK19, CK20), while the main expression in squamous cellⅡ-type CK (including CK1, CK5/6, CK14). Therefore, the positioning of different CK research and observation CK expression under different conditions, is certainly significant to understand epithelial cell proliferation and differentiation status, disease occurrence and development.
Research suggests that, CK5 is a sign of grass-roots keratin in proliferative cells in normal breast, CK5 always positive, but in the majority of invasive ductal carcinoma and ductal carcinoma in situ, CK5 often negative, so CK5 is often use to identify invasive and duct in situ cancer and common type of hyperplasia. CK8 is simply a sign of epithelial keratin, it can penetrate the outer surface of breast cancer cells, and in a soluble form of hybrid polymer is released from the cancer cells, CK8 present in many simple epithelial cells and carcinoma, but also for the differential diagnosis of breast cancer. Breast cancer resistance protein (breast cancer resistance protein, BCRP) is located in the membrane of the ABC half-transporter proteins, there are researches suggesting that it may be a sign of poorly differentiated breast epithelial tissue, in the abnormal proliferation of tissue high expression of BCRP is a cell undifferentiated maturity, BCRP may be related to the presence of drug-resistant tumor cells and cause tumor recurrence and treatment failure.
In this report,breast cancer cell line MCF-7 and human breast cancer tissues were studied by real-time quantitative PCR technology and immunohistochemistry, in order to detect Hedghehog pathway in MCF-7 cells CD44+/CD24-/low stem cell subsets in the Hedghehog path expression, as well as some important proteins, cytokeratin, chromogranin proteins in human breast cancer tissue expression. We analyze the correlation of their different breast lesions, so that provide a new way of thinking for breast cancer prevention and improving the diagnosis and treatment of breast cancer. Proved for these academic problems will provide valuable experimental evidence for early diagnosis of breast cancer, prognosis evaluation and screening of gene therapy targets.
Materials and Methods:Breast cancer cell line MCE-7 and human breast lesions were selected from the Dalian Medical University and China-Japan Center for Clinical Pathology. Of total 89 human breast tissue samples from 2004 to 2008, there were 13 cases of normal breast tissues,16 cases of breast hyperplasia,12 cases of atypical ductal hyperplasia,6 cases of ductal carcinoma in situ and 42 cases of invasive ductal carcinoma. Of total 75 human breast tissue samples from 2008 to 2009, there were 10 cases of normal breast tissues,26 cases of breast fibroadenoma tissues,39 cases of invasive ductal carcinoma. Patients aged 28 to 75 years with an average age of 45 years. Samples were collected from specimens resected, had not received preoperative radiotherapy and chemotherapy, all patients were clearly pathological diagnosed and got informed consent of patients and their families, access to the Dalian Medical University Ethics Committee. All sections were confirmed by two pathologists referral of high qualification, of which 13 cases of normal breast tissue adjacent to breast tissue from the tumor 5cm. In this study, immunohistochemistry (IHC), immunocytochemistry (ICC), real-time quantitative polymerase chain reaction (Realtime-PCR) and cell culture methods and magnetic cell separation system(MACS) were used to detect the expression of Hedghehog pathway in MCF-7 cells CD44+/CD24-/low stem cell subsets, and to detect expression and distribution of Hedghehog pathway important protein, cytokeratin, chromogranin proteins in human breast cancer tissue. SPSS 11.5 package was use for statistics analysis with the experimental data, distribution of all factors in different pathological were analysed significantly by Mamm Whitney and Kruskal-Wallis test methods, and Spearmen correlation test was applied to judge the statistical relationship between relevant factors.
Results:1 CD44+/CD24-/low subpopulation ratio (8.90±1.73)% in the breast cancer cell line MCF-7. Hedgehog signaling pathway of the CD44 +/CD24-/low subgroups in breast cancer cell line MCF-7 is significantly activated, including Hedgehog signaling pathway important downstream signaling molecules GIi-1 in MCF-7 cells CD44+/CD24-/low cells subgroup expression is significantly higher than non-CD44+/CD24-/low cell subsets (P= 0.007).
2 Positive expression rate of PATH 1 and Gli-1 in normal breast tissue, breast tumor tissue, and breast cancer showed gradually increased, PATH1 expression rates were 10.0%,46.1% and 96.2%, Gli-1 expression rates were 20.0%,69.2% and 98.1%. With the differentiation of breast lesions decreased, PATH1 and Gli-1 positive expression rate was increased, showed strong positive expression. Their expresss in invasive ductal carcinoma tissues was significantly higher than normal breast tissue, was significantly higher than that of fibroadenoma tissue (P<0.05).
3 Positive rate of CK5 and CK8 in the normal breast tissue, breast hyperplasia, atypical hyperplasia and breast cancer showed descending. Positive expression rates of CK5 in normal breast tissue, breast hyperplasia, atypical ductal hyperplasia, ductal carcinoma in situ and invasive ductal carcinoma were 84.6%,62.5%,41.7%,33.3%,19.0%. Positive expression rates of CK8 in the normal breast tissue, breast hyperplasia, atypical ductal hyperplasia, ductal carcinoma in situ and invasive ductal carcinoma positive expression rates were 100.0%,100.0%,75%,66.7%,66.7%. Most of the 28 cases of invasive ductal carcinoma (66.7%) were CK5-/CK8+, only 8 cases (16.7%) were CK5+/CK8-,8 cases presented CK5+/CK8+,14 cases (29.2%) showed CK5-/CK8-. With the decline of differentiation of breast lesions, BCRP positive expression was significantly increased in normal breast tissue, breast hyperplasia, ductal atypical hyperplasia, ductal carcinoma in situ and invasive ductal carcinoma, the positive expression rates were 15.4%,250%,25%,33.3%,42.9%. Positive expression in invasive ductal carcinoma tissues was significantly higher than in normal breast tissue, was significantly higher than breast hyperplasia (P<0.05).
Conclusions:1 Hedgehog signaling pathway of CD44+/CD24-/low signs stem cell subsets in breast cancer cell line MCF-7 is significantly activated.
2 Distribution and expression degree of PATH1 and Gli-1 in different breast diseases were significantly different, the lower differentiation, the stronger the expression,which was contribute to the pathological diagnosis of breast lesions.
3 The expression of CK5 and CK8 were different in different degrees of breast ductal carcinoma. In different degree of differentiation, the higher differentiation, the weaker the expression of CK8, but CK5 expression was stronger, on the contrary, the lower degree of differentiation, the stronger the expression of CK8, expression of CK5 relatively weak or absent. Distribution and expression of BCRP in different breast diseases tissues were significantly different, suggesting that BCRP may have a role in breast cancer cells differentiation. Different expressions of CK5, CK8 and BCRP in different breast tissue may be contribute to the pathological diagnosis of breast lesions.
In conclusion, immunomagnetic beads sorting of breast cancer stem cells is a simple and reliable method of sorting the CD44+/CD24-/low stem cell mark cell subsets, Hedgehog signaling pathway is significantly activated. Several cancer stem cell markers can be used for the pathological diagnosis of breast lesions
肿瘤干细胞理论认为,肿瘤细胞内存在一群具有干细胞性质的细胞(称肿瘤干细胞或肿瘤起始细胞,cancer stem cells or cancer initiating cells, CSCs or CICs),不受外界调控的恶性增殖、多向分化,从而形成肿瘤。越来越多的研究证实在脑胶质瘤、乳腺癌、前列腺癌和恶性黑色素瘤等实体瘤中存在肿瘤干细胞。
Hedgehog家族成员包括SHH、PTCH、GIi-1和SMOH。Hedgehog信号通路激活的具体机制目前尚不完全清楚,多数学者认为主要包括配体依赖的激活和配体非依赖的激活。配体依赖的激活是指配体如SHH、IHH(Indian Hedgehog)与受体PTCH结合,诱导原癌基因Smo的激活,引起Hedgehog信号通路的激活,这已在小细胞肺癌、肝癌、乳腺癌、脑恶性肿瘤和消化道癌中得到证实；配体非依赖的激活主要指信号通路成员如PTCH或SMOH突变所启动的Hedgehog信号通路的激活,这已经在Gorlin-相关肿瘤(如基底细胞癌和横纹肌肉瘤等)中得到证实。GIi-1基因的高表达是Hedgehog信号通路活化目前比较公认的标志之一。多项研究证实Hedgehog信号通路在多发骨髓瘤、乳腺癌和恶性胶质瘤等的干细胞中高表达,并在其自我更新中起重要作用。敲除SHH信号可导致小鼠神经干祖细胞明显减少,在体外培养几乎不能形成神经球；相反,激活SHH途径,使成年小鼠终脑的干祖细胞数量明显增加。IHH突变可减少肠道干细胞的增殖和分化。
细胞角蛋白(cytokeratin, CK)是分布于上皮细胞的中间纤维丝主要表达于上皮细胞,在人类上皮组织中共发现至少20个成员,其表达具有很强的组织特异性和分化特异性。每种上皮组织在生长分化的不同阶段都表达不同的CK,其表达受细胞分化调节,如腺上皮主要表达I型CK(包括CK7, CK8, CK18, CK19, CK20),而鳞状上皮主要表达Ⅱ型CK(包括CK1, CK5/6, CK14)。因此,对不同CK的定位研究和观察不同状态下CK的表达,对了解上皮细胞的增殖和分化状态以及疾病的发生和发展有一定的意义。
研究提示,CK5是基层的标志角蛋白,在乳腺普通增生细胞中,CK5常阳性,而在多数浸润性导管癌及导管原位癌中,CK5常阴性,所以CK5常作为浸润性和导管原位癌与普通型增生鉴别的依据之一。CK8是单纯上皮的标志角蛋白,它能够穿透乳腺癌细胞的外表面,并以一种可溶的杂多聚体的形式从癌细胞中释放出来,CK8存在于许多单纯上皮细胞和癌中,也用于乳腺癌的鉴别诊断。乳腺癌耐药蛋白(breast cancer resistance protein, BCRP)是一个位于细胞膜上的ABC半转运蛋白,有研究提示它可能是乳腺上皮组织低分化的标志,在异常增生的组织中BCRP高表达,是细胞未分化成熟的表现BCRP的存在可能与肿瘤细胞耐药并引起肿瘤复发和治疗失败有关。
本课题以乳腺癌细胞系MCF-7和人乳腺癌组织为研究对象,通过体外培养、分选乳腺癌干细胞,并采用实时定量PCR技术及免疫组织化学方法分别检测Hedghehog通路在MCF-7细胞系CD44+/CD24-/low干细胞亚群中的表达情况以及Hedghehog通路的重要蛋白,细胞角蛋白在人乳腺癌组织中的表达情况。分析它们与不同乳腺病变之间可能存在的相关性,探讨乳腺癌的发生机制,将为乳腺癌的早期诊断、预后评价和基因治疗指标的筛选提供有价值的实验依据。
材料与方法：乳腺癌细胞系MCF-7和人乳腺病变组织均选自大连医科大学中日临床病理中心,2004-2009年人乳腺组织标本164例,其中正常乳腺组织23例、乳腺增生组织16例、乳腺纤维腺瘤组织26例、导管上皮非典型增生12例、导管原位癌6例和浸润性导管癌81例。患者年龄28-75岁,平均年龄45岁。所收集的标本均为手术切除送检标本,术前均未接受放化疗治疗,术后获得明确病理诊断并得到患者及其家属的知情同意。所有切片均经2位高年资病理医师复诊,其中正常乳腺组织为13例癌旁乳腺组织,距肿瘤5cm。本研究采用免疫组织化学(IHC)、免疫细胞化学(ICC)、实时定量聚合酶链式反应(Realtime-PCR)和细胞培养、磁珠分选等方法,检测Hedgehog通路在MCF-7细胞系CD44+／CD24-／low干细胞亚群中的表达情况,Hedghehog通路的重要蛋白,细胞角蛋白在人乳腺癌组织中的表达情况和分布特点。实验数据采用SPSS 11.5软件包,应用Kruskal-Wallis和Mamm Whitney的检验方法,对各因子在不同乳腺病变组织中的分布进行统计学差异分析。应用Spearmen相关性检验判断相关因素之间的统计学关系
结果：
一、乳腺癌细胞系MCF-7中CD44+／CD24-／low亚群比例为(8.90+1.73)%。乳腺癌细胞系MCF-7的CD44+／CD24-／low亚群中Hedgehog信号通路处于明显活化状态,其中Hedgehog信号通路中重要下游信号分子GIi-1在MCF-7细胞系CD44+／CD24-／low细胞亚群中的表达要明显高于非CD44+／CD24-／low细胞亚群(P为0.007)。
二、PATH 1和Gli-1在正常乳腺组织、乳腺瘤组织、及乳腺癌中的阳性表达率呈现逐渐升高的趋势,PATH1表达率分别为10.0%,46.1%和96.2%,Gli-1表达率分别为20.0%,69.2%和98.1%。随着乳腺病变组织分化程度的下降,PATH1和Gli-1的阳性表达率明显增加,呈强阳性表达。浸润性导管癌组织中的表达明显高于正常乳腺组织,乳腺癌组织中的表达明显高于乳腺瘤组织(P<0.05)。
三、CK5和CK8在正常乳腺组织、乳腺增生、非典型增生及乳腺癌中的阳性表达率依次下降。CK5在正常乳腺组织、乳腺增生、导管上皮非典型增生、导管原位癌和浸润性导管癌组织的阳性表达率分别为84.6%,62.5%,41.7%,33.3%,19.0%。CK8在正常乳腺组织、乳腺增生、导管上皮非典型增生、导管原位癌和浸润性导管癌组织的阳性表达率分别为100.0%,100.0%,75%,66.7%,66.7%。绝大部分浸润性导管癌呈现CK5-/CK8+,28例(66.7%),只有8例(16.7%)呈现CK5+／CK8-,8例呈现CK5+／CK8+,14例(29.2%)呈现CK5-／CK8-。随着乳腺病变组织分化程度的下降,BCRP的阳性表达率明显增加,在正常乳腺组织、乳腺增生、导管上皮非典型增生、导管原位癌和浸润性导管癌组织的阳性表达率分别为15.4%,250%,25%,33.3%,42.9%。浸润性导管癌组织中的表达明显高于正常乳腺组织,乳腺癌组织中的表达明显高于乳腺增生组织(P<0.05)。
结论：
一、乳腺癌细胞系MCF-7中存在CD44+／CD24-／low干细胞标志细胞亚群,其中Hedgehog信号通路处于明显活化状态。
二、PATH1和Gli-1在乳腺不同病变组织中的表达和分布存在差异,分化程度越低表达越强。
三、不同分化程度的乳腺导管癌中CK5和CK8的表达不同,分化程度越高CK8的表达越弱、CK5的表达相对较强,分化程度越低CK8的表达越强、CK5的表达相对较弱甚至缺失。BCRP在乳腺不同病变组织中的表达和分布存在差异,提示BCRP可能在乳腺肿瘤细胞分化方面有一定的作用。CK5.CK8和BCRP在不同乳腺病变组织中的相关性,三者联合检测有助于乳腺病变的病理学诊断和判断其分化程度及预后。
综上所述,免疫磁珠法分选乳腺癌干细胞是一种简便可靠的方法,分选得到的CD44+／CD24-／low干细胞标志细胞亚群中,Hedgehog信号通路处于明显活化状态。几种肿瘤干细胞标志物可用于乳腺病变的病理学诊断。
Backgrouds and Objectives:Breast cancer is a common female malignant tumor. It is common disease and frequently-occurring disease with a high incidence in Western Europe and North America, and is a serious threat to women's health. Into the 21st century, about 120 million women worldwide each year suffer from breast cancer,and 500,000 people die from breast cancer. The United States as countries with high incidence of breast cancer, showed a clear upward trend of breast cancer incidence from 1973 to 1976, the U.S. An estimated 10 million people suffering from breast cancer each year and 3 million people die of the disease. In 1990, of 15 million new cases, there are 44000 people were killed, according to the estimated incidence rate trends, in the 21st century in the United States, every eight women will have a person's life with breast cancer. Low incidence of breast cancer is showed in China, but in recent years, clinical observation suggest that breast cancer incidence rates in China continue to rise, growing at about 3 percent per year, in Beijing, Tianjin, Shanghai and other major cities, breast cancer has leapt the first female malignancy, age, significantly ahead of the trend, and the clinical diagnosis of breast cancer in our side late stage of disease. For this reason, breast cancer research reaches growing concern.
Cancer stem cell theory concern that tumor cells exist in a group of cells with stem cell properties(called cancer stem cells or tumor cells, or cancer initiating cells, CSCs or CICs), subject to external regulation and control of malignant proliferation of multi-differentiation to form tumors. Growing number of studies confirmed the brain glioma, breast cancer, prostate cancer and malignant melanoma and other solid tumors exist in cancer stem cells.
Hedgehog family members are including SHH, PTCH, GIi-1, and SMOH. Specific mechanism of activation of Hedgehog signaling pathway is currently not entirely clear, most scholars believe mechanisms including ligand-dependent activation and ligand-independent activation. Ligand-dependent activation is that ligand, such as SHH, IHH (IndianHedgehog) combined with the receptor PTCH, Smo-induced activation of proto-oncogene, causing the activation of Hedgehog signaling pathway, which has been confirmed in small cell lung cancer, liver cancer, breast cancer, brain cancer and digestive tract cancer. Ligand-independent signaling pathway activated mainly refers to members such as PTCH or SMOH mutations in the Hedgehog signaling pathway initiated by the activation, which has already in the Gor-lin-associated tumors (such as basal cell carcinoma, into the neural tube cell carcinoma and rhabdomyosarcoma, etc.) been confirmed.GIi-1 gene's highly expression is relatively recognized a marker of activation of the Hedgehog signaling pathway. A number of studies confirmed the Hedgehog signaling pathway in stem cells of multiple myeloma, breast cancer and malignant glioma and other cancers that highly expressed Hedgehog signaling pathway in stem cells, and the Hedgehog signaling pathway is confirmerd play an important role in their self-renewal. SHH signaling knock-out mice can lead to significantly reduced neural stem progenitor cells, so that in vitro almost can not form neurospheres. On the contrary, activating SHH ways can make the final adult mouse brain's stem progenitor cells increased significantly. IHH mutation can reduce the intestinal stem cell proliferation and differentiation
Cytokeratin (cytokeratin, CK) is distributed in the epithelial cells of the intermediate filament silk, mainly expressed in the epithelial cells, were found in human epithelial tissue of at least 20 members, its expression is highly tissue-specific and differentiation-specific. Each type of epithelial tissue in different stages of growth and differentiation express different CK, its expression by regulating cell differentiation, such as the main expression of typeⅠepithelial CK (including CK7, CK8, CK18, CK19, CK20), while the main expression in squamous cellⅡ-type CK (including CK1, CK5/6, CK14). Therefore, the positioning of different CK research and observation CK expression under different conditions, is certainly significant to understand epithelial cell proliferation and differentiation status, disease occurrence and development.
Research suggests that, CK5 is a sign of grass-roots keratin in proliferative cells in normal breast, CK5 always positive, but in the majority of invasive ductal carcinoma and ductal carcinoma in situ, CK5 often negative, so CK5 is often use to identify invasive and duct in situ cancer and common type of hyperplasia. CK8 is simply a sign of epithelial keratin, it can penetrate the outer surface of breast cancer cells, and in a soluble form of hybrid polymer is released from the cancer cells, CK8 present in many simple epithelial cells and carcinoma, but also for the differential diagnosis of breast cancer. Breast cancer resistance protein (breast cancer resistance protein, BCRP) is located in the membrane of the ABC half-transporter proteins, there are researches suggesting that it may be a sign of poorly differentiated breast epithelial tissue, in the abnormal proliferation of tissue high expression of BCRP is a cell undifferentiated maturity, BCRP may be related to the presence of drug-resistant tumor cells and cause tumor recurrence and treatment failure.
In this report,breast cancer cell line MCF-7 and human breast cancer tissues were studied by real-time quantitative PCR technology and immunohistochemistry, in order to detect Hedghehog pathway in MCF-7 cells CD44+/CD24-/low stem cell subsets in the Hedghehog path expression, as well as some important proteins, cytokeratin, chromogranin proteins in human breast cancer tissue expression. We analyze the correlation of their different breast lesions, so that provide a new way of thinking for breast cancer prevention and improving the diagnosis and treatment of breast cancer. Proved for these academic problems will provide valuable experimental evidence for early diagnosis of breast cancer, prognosis evaluation and screening of gene therapy targets.
Materials and Methods:Breast cancer cell line MCE-7 and human breast lesions were selected from the Dalian Medical University and China-Japan Center for Clinical Pathology. Of total 89 human breast tissue samples from 2004 to 2008, there were 13 cases of normal breast tissues,16 cases of breast hyperplasia,12 cases of atypical ductal hyperplasia,6 cases of ductal carcinoma in situ and 42 cases of invasive ductal carcinoma. Of total 75 human breast tissue samples from 2008 to 2009, there were 10 cases of normal breast tissues,26 cases of breast fibroadenoma tissues,39 cases of invasive ductal carcinoma. Patients aged 28 to 75 years with an average age of 45 years. Samples were collected from specimens resected, had not received preoperative radiotherapy and chemotherapy, all patients were clearly pathological diagnosed and got informed consent of patients and their families, access to the Dalian Medical University Ethics Committee. All sections were confirmed by two pathologists referral of high qualification, of which 13 cases of normal breast tissue adjacent to breast tissue from the tumor 5cm. In this study, immunohistochemistry (IHC), immunocytochemistry (ICC), real-time quantitative polymerase chain reaction (Realtime-PCR) and cell culture methods and magnetic cell separation system(MACS) were used to detect the expression of Hedghehog pathway in MCF-7 cells CD44+/CD24-/low stem cell subsets, and to detect expression and distribution of Hedghehog pathway important protein, cytokeratin, chromogranin proteins in human breast cancer tissue. SPSS 11.5 package was use for statistics analysis with the experimental data, distribution of all factors in different pathological were analysed significantly by Mamm Whitney and Kruskal-Wallis test methods, and Spearmen correlation test was applied to judge the statistical relationship between relevant factors.
Results:1 CD44+/CD24-/low subpopulation ratio (8.90±1.73)% in the breast cancer cell line MCF-7. Hedgehog signaling pathway of the CD44 +/CD24-/low subgroups in breast cancer cell line MCF-7 is significantly activated, including Hedgehog signaling pathway important downstream signaling molecules GIi-1 in MCF-7 cells CD44+/CD24-/low cells subgroup expression is significantly higher than non-CD44+/CD24-/low cell subsets (P= 0.007).
2 Positive expression rate of PATH 1 and Gli-1 in normal breast tissue, breast tumor tissue, and breast cancer showed gradually increased, PATH1 expression rates were 10.0%,46.1% and 96.2%, Gli-1 expression rates were 20.0%,69.2% and 98.1%. With the differentiation of breast lesions decreased, PATH1 and Gli-1 positive expression rate was increased, showed strong positive expression. Their expresss in invasive ductal carcinoma tissues was significantly higher than normal breast tissue, was significantly higher than that of fibroadenoma tissue (P<0.05).
3 Positive rate of CK5 and CK8 in the normal breast tissue, breast hyperplasia, atypical hyperplasia and breast cancer showed descending. Positive expression rates of CK5 in normal breast tissue, breast hyperplasia, atypical ductal hyperplasia, ductal carcinoma in situ and invasive ductal carcinoma were 84.6%,62.5%,41.7%,33.3%,19.0%. Positive expression rates of CK8 in the normal breast tissue, breast hyperplasia, atypical ductal hyperplasia, ductal carcinoma in situ and invasive ductal carcinoma positive expression rates were 100.0%,100.0%,75%,66.7%,66.7%. Most of the 28 cases of invasive ductal carcinoma (66.7%) were CK5-/CK8+, only 8 cases (16.7%) were CK5+/CK8-,8 cases presented CK5+/CK8+,14 cases (29.2%) showed CK5-/CK8-. With the decline of differentiation of breast lesions, BCRP positive expression was significantly increased in normal breast tissue, breast hyperplasia, ductal atypical hyperplasia, ductal carcinoma in situ and invasive ductal carcinoma, the positive expression rates were 15.4%,250%,25%,33.3%,42.9%. Positive expression in invasive ductal carcinoma tissues was significantly higher than in normal breast tissue, was significantly higher than breast hyperplasia (P<0.05).
Conclusions:1 Hedgehog signaling pathway of CD44+/CD24-/low signs stem cell subsets in breast cancer cell line MCF-7 is significantly activated.
2 Distribution and expression degree of PATH1 and Gli-1 in different breast diseases were significantly different, the lower differentiation, the stronger the expression,which was contribute to the pathological diagnosis of breast lesions.
3 The expression of CK5 and CK8 were different in different degrees of breast ductal carcinoma. In different degree of differentiation, the higher differentiation, the weaker the expression of CK8, but CK5 expression was stronger, on the contrary, the lower degree of differentiation, the stronger the expression of CK8, expression of CK5 relatively weak or absent. Distribution and expression of BCRP in different breast diseases tissues were significantly different, suggesting that BCRP may have a role in breast cancer cells differentiation. Different expressions of CK5, CK8 and BCRP in different breast tissue may be contribute to the pathological diagnosis of breast lesions.
In conclusion, immunomagnetic beads sorting of breast cancer stem cells is a simple and reliable method of sorting the CD44+/CD24-/low stem cell mark cell subsets, Hedgehog signaling pathway is significantly activated. Several cancer stem cell markers can be used for the pathological diagnosis of breast lesions
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