摘要
论文题目：p27、PCNA、CyclinD1等因子的表达和乳腺癌早期浸润的关系
专业：肿瘤学
研究生：李会平
指导教师：邢鲁奇教授
摘要
背景与目的乳腺癌是女性最常见的恶性肿瘤之一，其中浸润性导管癌约占乳腺癌的80%。微浸润是指在大量导管内癌的背景下，乳腺间质中，存在一处或几处明确的、离散的、小的镜下灶性浸润。乳腺导管肌上皮的缺失成为微浸润乳腺导管癌发展的关键，因此明确癌细胞突破肌上皮向间质浸润的作用机理有着十分重要的理论和临床意义，可为研究肿瘤的浸润转移机制和临床靶向提供理论依据。本实验采用p27、PCNA、CyclinD1等肿瘤增殖相关标记物检测微浸润乳腺导管癌中位于肌上皮缺失处及其附近的癌细胞的表达情况，并与同一导管内的其它癌细胞进行比较，探讨它们在乳腺导管癌浸润发展中的作用及与相关临床病理指标的关系，从而探索微浸润的发病机理，为乳腺导管癌的早期防治提供理论依据，并寻在微浸润中有特殊意义的因子标记物。
材料与方法选取乳腺微浸润性导管癌50例。采用双重染法，用SMA单克隆抗体标记肌上皮细胞，定位乳腺导管，然后检测p27、PCNA、CyclinD1蛋白在微浸润处和导管内的癌细胞中的表达，探讨它们与ER、PR、C-erbB-2的关系；分析不同生物指标在癌细胞向基质浸润中的作用，并观察微浸润癌细胞的病理形态学特征。对20例导管内癌、20例微浸润导管癌和40例浸润性导管癌，用免疫组织化学法（S-P法）检测p27、CyclinD1和Ki-67蛋白在各组中的表达，并进行统计学分析。
结果位于肌上皮缺失处的微浸润癌细胞缺乏肿瘤增殖相关指标p27、PCNA 和CyclinD1的表达；缺乏肿瘤侵袭相关指标MMP-9和TIMP-1表达；缺乏抑制凋亡因子bcl-2和抑癌基因p16表达；另一方面，乳腺导管癌中微浸润的癌细胞高表达原癌基因c-erb-B2。同一导管内其它的癌细胞明显的表达上述p27、PCNA和CyclinD1等指标。
p27、CyclinD1和Ki-67在20例导管内癌中高表达率分别为85%、10%、10%；在20例微浸润导管癌中分别为60%、25%、30%；在40例浸润性导管癌中分别为45%、55%、67.5%；p27，CyclinD1和Ki-67分别在导管内癌、微浸润导
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proliferation河南科技大学硕士学位论文
II 管癌和浸润性导管癌中的表达进行比较，P 值分别为0.012、0.001、0.000，P  <0.05有统计学意义；乳腺导管癌中p27与CyclinD1、p27与Ki-67的表达均呈负相关（1r =−，P  <0.01）。
结论 (1) 乳腺导管癌中微浸润的癌细胞与导管内其它的癌细胞相比，具有明显不同的形态学和免疫组化特征，它们缺乏某些细胞增殖、肿瘤侵袭等相关因子的表达，这些细胞可能具有更强的潜在增殖和浸润能力，为较早反映肿瘤微环境中多种病理改变的标志之一。
(2) 随着乳腺导管癌的浸润和进展，p27蛋白的表达率呈递减趋势，CyclinD1和Ki-67的表达率呈递增趋势。p27在乳腺导管癌的发生发展中可能具有明显的抑制作用，联合检测p27与CyclinD1/Ki-67对反映乳腺导管癌的恶性程度以及预后可能更为客观。
关 键 词：乳腺导管癌， 微浸润， 乳腺肿瘤细胞， 免疫组化
论文类型：基础研究
Abstract
Subject:  The relationship between the expression of p27, PCNA,  CyclinD1 (etc.) and breast tumor micro-invasion        Specialty:  Oncology                                          Name: Li hui-ping
Supervisor: P rofessor Xing lu-qi
ABSTRACT
Background and objective: The breast carcinoma is one of the most common malignant tumors, and 80% of them are invasive breast ductal carcinoma. Micro-invasive lesions refer to existence of one or several specific, discrete, small focal infiltration of the microscopic among the breast stroma and in the context of a large number of ductal carcinoma in situ. The focal ME cell layer disruption is a critical factor of the progression of the micro-invasive ductal breast cancer. Therefore, it has the great scientific and clinical significance to understand the intrinsic mechanism that ductal tumor cells penetrate the ME cell layer and reach the stroma for invasion or metastasis. In addition, it can provide a theoretical basis to study tumor invasion mechanisms and clinical targeted therapy. With p27, PCNA and CyclinD1 etc., some proliferation related markers, our experiments have examined that a subset of cell clusters overlying FDMCL, compared with adjacent cells within the same ducts, and discussed their effects in the development of breast ductal carcinoma and relations of related markers. Thereby we explored the intrinsic mechanism of the micro-invasive lesions, provide a theoretical basis for the early prevention and treatment of breast ductal carcinoma, and identified the specific markers of the micro-invasive lesions.
Materials and methods: Fifty cases micro-invasive breast ductal carcinoma  were selected. The doub
le immunostained method was adopted, ME was marked by the SMA single cloned antibody, and the orientation of breast duct was obtained. Then the expression of p27, PCNA and CyclinD1 in the micro-invasion and ductal tumor cells were checked, and the relationships between these cell clusters and ER, PR and C-erbB-2 were discussed. The different biomarkers’ roles of the stromal invasion were analyzed, and the pathological morphological profiles of the micro-invasive tumor cells were observed. Otherwise, immnuohistochemical S-P method was used to examine the expression of p27, CyclinD1 and Ki-67 in 20 cases of DCIS, 20 cases of micro-invasive breast ductal carcinoma, and 40 cases of invasive breast ductal
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河南科技大学硕士学位论文
IV carcinoma. Moreover, our results were statistically analyzed.
Results: The micro-invasive tumor cells overlying FDMCL lacked expression of tumor proliferation related markers: p27, PCNA and CyclinD1. They lacked expression of tumor progression related markers, including MMP-9 and TIMP-1. They also lacked expression of p16 (tumor suppressor gene) and bcl-2 (apoptosis inhibitory factor). On the other hand, these cell clusters showed a higher expres
sion of c-erb-B2. Adjacent cells within the same ducts were strongly positive expression for p27, PCNA and CyclinD1 etc.
In 20 cases of DCIS, the high expression rate of the cell molecules p27, CyclinD1 and Ki-67 were 85%, 10% and 10%, respectively. In 20 cases of micro-invasive ductal carcinomas, the high expression rate of them were 60%, 25% and 30%. In 40 cases of invasive ductal carcinomas, the high expression rate of them were 45%, 55% and 67.5%. The high expression rate of p27, CyclinD1 and Ki-67 in DCIS, micro-invasive ductal carcinomas and invasive ductal carcinomas had significant differences, P =0.012, 0.001, and 0.000, respectively, and had significance of statistics (P <0.05). The high-expression rates of p27 and CyclinD1/Ki-67 protein were inversely correlated in different stages of breast ductal carcinomas(1r =−, P  <0.01).
Conclusions: (1) Comparing with tumor cells within the duct, the micro-invasive tumor cells in the breast ductal carcinoma show obviously different morphological and immnuohistochemical character, and they lacked expression of cell proliferation, progression related markers. These cell clusters are likely to have a stronger potential proliferation and invasive ability, and they may be one of the signs which reflect many pathological changes in tumor microenvironment much earlier.
(2) With the malignant extent progression of the breast ductal carcinoma, the high-expression rates of p27 were gradually decreased, and the high-expression rates of CyclinD1 and Ki-67 were gradually increased. p27 may play an suppressive role in initiation and development of breast ductal carcinoma. The combined analysis of p27 and CyclinD1/Ki-67 expression is considered to be more reliable for assessing the malignant extent and prognosis of breast ductal carcinoma.
KEY WORDS : Breast ductal carcinomas, Micro-invasive lesions, Breast tumor cell
clusters, Immunohistochemistry
Dissertation Type : Fundermental research
第1章综述
第1章综述
浸润是恶性肿瘤最重要的生物学特征之一，乳腺导管的腺上皮被肌上皮层和基底膜与基质间隔开，肌上皮和基底膜的断裂成为肿瘤浸润的必需先决条件。传统理论认为从原位癌到浸润癌的发展，是由于肿瘤细胞和基质细胞产生了过多的蛋白酶，这些酶可以降解基底膜，使导管内的癌细胞可以通过基底膜的缺口浸入到基质[1]。然而，临床上应用基质金属蛋白酶抑制剂的效果却不尽人意，说明这种蛋白
酶为中心的理论不能完全反映肿瘤浸润的分子机制[2]。研究报道[3,4]，在有肌上皮断裂的乳腺微浸润肿瘤中，覆盖于断裂处的肿瘤细胞其基因和免疫组化性质均发生了改变，如它们缺乏ER的表达，可以高表达细胞增殖、血管生成和肿瘤浸润相关的基因等。结果使浸入基质的癌细胞的侵袭力更强，进而可以侵入脉管和淋巴管，但事实情况究竟是否如此也尚未定论。因此，充分了解这些微浸润的癌细胞的生物学性质，以及肿瘤微浸润局部微环境的改变与癌细胞浸润的关系，有助于我们进一步探索肿瘤浸润的机理。
另外，肌上皮细胞的生物学特征和结构的完整性与相邻肿瘤细胞的免疫组化性质的相互关系，也对肿瘤的浸润产生着影响，现对相关生物指标与浸润的关系，以及浸润的基本原理概括并讨论如下。
1.1 肌上皮细胞的正常分布、形态和功能
正常人类乳腺由两部分组成：上皮细胞和基质。上皮细胞包括两种类型，肌上皮和腺上皮，这些细胞组成具有分泌功能的小叶和导管结构。肌上皮位于腺细胞和基底膜之间，通过细胞桥粒和由半桥粒连接的基底膜附着在腺上皮细胞周围[5]。基底膜是由层粘蛋白、IV型胶原、硫酸乙酰肝素蛋白多糖、粘多糖和其它成分组成，围绕肌上皮细胞形成一个完整的层[6]。肌上皮细胞由粘附分子和细胞间的连接相连，环绕于整个导管系统，并间断地或像篮子一样半绕着绝大多数终级导管和小叶单位。
在正常乳腺组织，肌上皮细胞与腺上皮细胞的数量近似相等，肌上皮细胞一般呈立方形或梭形，有界
线清晰的细胞质和细胞核。然而，肌上皮的数量和形态在肿瘤发展的不同阶段会有很大的改变。在增生和原位癌，肌上皮细胞层经常是被削弱的，细胞质减少甚或几乎没有，形态上很像小血管的平滑肌细胞和纤维母细胞。在浸润损害和少数良性病变中，如乳腺腺病，肌上皮细胞消失或偶尔发现残留在这些损害的组织里。
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