【作者】 李鹏；

【导师】 胡川闽；

【作者基本信息】 第三军医大学 ， 临床检验诊断学， 2009， 博士

【摘要】 乳腺癌已经成为仅次于肺癌的威胁人类健康的主要恶性疾病之一。尽管随着医学的进步乳腺癌患者5年和10年生存率分别提高到70%和50%,但仍有约40%左右的患者死于肿瘤复发或转移。最近的研究发现,乳腺癌细胞转移的首要条件是形成称为“树突状伪足”的突起,这种“突起”中具备含有蛋白酶的囊性结构,通过这种囊性结构与肿瘤细胞膜的融合,释放蛋白酶降解包围它们的细胞外基质蛋白,导致肿瘤细胞自原发灶逃脱。IBP是在人免疫系统中大量表达并具有重要功能的蛋白质,它参与TCR信号刺激下T细胞免疫突触的形成;与Th2细胞的分化关系密切;通过抑制IRF-4的转录因子活性调控介素17与介素21的产生,影响Th17细胞的功能;影响MAPKs与NF-κB参与TLRs传导通路;对维持免疫内环境的稳定发挥重要作用,IBP knockout小鼠表现为自身免疫免疫性疾病的症状并对致死性LPS产生抵抗。除此之外,IBP具有鸟苷酸交换因子(GEF)的作用,能够激活Rho GTPase家族的部分成员。所有这些研究结果均提示IBP可以参与细胞骨架的重构、细胞极化、细胞间的信息传递等多种重要生理过程,然而,由于缺乏特异性抗IBP抗体,目前对IBP在人体其它组织(尤其是肿瘤)中的表达及功能的研究较少。本课题以乳腺癌为研究对象,对IBP在乳腺癌中的表达及其可能的临床意义进行研究,并通过体外试验证实IBP在乳腺癌中的功能及其可能的机制,研究主要从三个方面进行:一、特异性抗IBP单克隆抗体的制备及鉴定如前所述,目前对IBP异位表达和功能研究较少,文献中缺少相关的研究线索,为此,我们通过生物信息学的方法对IBP的蛋白结构进行分析,选择其抗原性强的特异区域进行重组表达,免疫BALB/C小鼠,经细胞融合及筛选成功获得多株抗IBP单克隆抗体。进一步通过ELISA、WB、IHC、ICC等实验,确定能够满足多种实验需要的表位特异性抗IBP单克隆抗体。二、IBP在乳腺癌中表达及其临床意义的研究1.利用MCF-7、MDA-MB-231、MDA-MB-435、MDA-MB-453、MDA-MB-468、SK-BR-3等多株乳腺癌细胞株通过WB及RT-PCR技术分析IBP的表达情况;2.通过双重免疫荧光染色确定IBP在乳腺癌中的表达模式主要位于胞膜及胞浆;3.收集了107例不同临床分级的临床乳腺癌标本构建组织芯片;4.通过免疫组化染色对IBP在临床样本中的表达情况进行分析,与临床样本的其它病理学指标进行统计学分析确定其临床意义。三、IBP在乳腺癌中的功能及其机制的研究1.利用IBP表达阴性的MDA-MB-435细胞,通过质粒转染构建IBP过表达细胞模型,利用IBP表达阳性的MDA-MB-231细胞,通过RNAi的方法构建IBP低表达细胞模型;2.通过MTT法检测各组细胞增殖情况的差异,判断IBP对细胞生长的影响;3.利用Transwell试验,经穿膜细胞染色计数研究各组细胞侵袭能力,确定IBP对细胞侵袭能力的影响;4.以临床常用的乳腺癌治疗药物紫杉醇为诱导剂,观察IBP的表达对细胞耐受紫杉醇杀伤能力的影响;5.通过细胞骨架染色观察IBP与细胞骨架的关系,通过免疫荧光确定IBP与粘着斑形成的关系;6.通过双重免疫荧光染色与IP确定IBP与Rac1b间的关系。主要研究结果如下:1.经过生物信息学分析,明确了IBP蛋白的一些可能的分子特征:非分泌蛋白;具有与胞膜及Actin结合的潜能;具备多个磷酸化位点;除已经明确具有重要功能的PH及DH结构域外其N端及C端还存在可能与其功能关系密切的结构域。进一步通过对IBP蛋白二级及三级结构的分析,确认IBP410-488符合抗体制备中对表位的要求,通过重组表达该片段经融合和对融合后的杂交瘤细胞进行ELISA及WB筛选,获得7株(A2, A9, C6, D5, F5, G3,与G4)分泌特异性抗IBP单克隆抗体的杂交瘤细胞,经IHC分析,其中5株(A2, A9, C6, D5与F5)细胞所分泌的抗体可应用于IHC对IBP的检测,而A9, C6与D5三株抗体IHC染色最强;2.WB与RT-PCR证实IBP选择性表达于MCF-7、MDA-MB-231及SK-BR-3细胞,经IBP与DiI双重染色确定乳腺癌中的IBP主要表达与胞膜与胞浆; 3.收集了107例临床乳腺癌手术组织(73例无淋巴结转移,34例存在淋巴结转移),利用组织芯片技术进行免疫组化检测,通过分析,我们在正常乳腺及乳腺良性病变中未见IBP表达,而有57例乳腺癌患者病理切片中IBP表达阳性(阳性率53.3%),经进一步与其它指标进行统计学分析,我们发现IBP的表达与乳腺癌的病理分级(p=0.005<0.01)及淋巴结转移正相关(p=0.003<0.01) ,与P53的表达负相关(p=0.026<0.05);4.经MTT、Transwell等试验证实IBP有促进乳腺癌增殖、侵袭的作用,在一定程度上还具有紫杉醇抗药性。5.IBP过表达MDA-MB-435细胞形态发生明显变化,伪足生成明显增多,而RNAi致IBP低表达MDA-MB-231细胞伪足生成减少。通过细胞骨架染色发现过表达IBP与微管及微丝均存在共定位而与后者共定位程度更为明显,其定位位置主要位于粘着斑,IBP的过表达可诱导中间丝的成分之一波形蛋白的表达上调,而波形蛋白本身即是肿瘤恶性度及转移性上升的标志。6.双重免疫荧光染色及IP证实IBP与Rac1b具有协同作用,能够促进Rac1b所致的上皮-基质转化现象(波形蛋白上调)的发生。综上所述,除免疫系统外,IBP在乳腺癌中也存在相当程度的表达,该表达与乳腺癌的转移密切相关;乳腺癌中IBP的表达具有促进乳腺癌细胞增殖及侵袭的作用,而其促进乳腺癌细胞侵袭转移的可能机制在于IBP不但可以促进细胞伪足的形成,参与粘着斑的构成提高细胞的迁移能力,还能通过与Rac1b的协同作用促进细胞内活性氧的产生,从而使细胞能够突破基底层实现转移。本课题尝试把乳腺癌转移的两个首要条件伪足形成与基底膜破坏联系起来进行研究,发现IBP与这二者均具有密切关系,可能是二者的桥梁。上述研究成果对进一步完善乳腺癌发生、转移机制的认识,为临床乳腺癌的早期诊治提供新的靶点及思路。更多还原

【Abstract】 Nowadays breast cancer has ranked second after lung cancer and is harmful to people’s health. The survival rates for breast cancer patients suffering 5 years and 10 years have increased to 70% and 50% respectisely, but about 40% of them still die of the cancer recurrence or metastasis. Recent research has found that the first requirement for the matastasis of breast cancer cells is the process of forming "invadopodia", which are actin-driven, finger-like membrane protrusions. The extracellular matrix proteins around it will be degraded by protease, which is released through the integration between cystic cancer and the cell membrane structure. And then it will allow the cancer cells to escape from the primary cancer. IBP (IRF-4 binding protein) is one of the proteins expressed in the immune system with important functions. IBP is involved in the formation of T cell-mediated immunity synapses after TCR signal stimulation. It is closely related with Th2 cell differentiation, affecting the function of Th17 cells by inhibiting IRF-4 transcription factor activity to regulate the production of IL-17 and IL-21. IBP played an important role in the maintenance of the immune environment stability. Loss of IBP in mice resulted in the development of systemic autoimmunity and developmental defects at the earliest stage of thymocyte differentiation. In addition, IBP plays a role of as a GEF, guanine nucleotide-exchange factor; IBP can activate some members of the Rho GTPase family. All these results indicate that IBP can take part in the reconstruction of the cytoskeleton, cell polarization, the signal transduction and many other important physiological processes. However, there is little research about the IBP expression and function presented within the body because of the lack of specific anti-IBP antibody.In this study, the IBP expressions in breast cancer and its possible clinical significances have been studied, and the function of IBP in breast cancer has been confirmed by in-vitro study. We studied the following three aspects: 1. The preparation and characterization of specific anti-IBP antibodiesFirst of all, the structure of IBP protein was analyzed by the bioinformatics method. The region with strong antigenicity was chosen for recombinant expression. The anti-IBP antibody was prepared by immunization BALB / C mouse, fusion and screening. In addition, the specific anti- IBP monoclonal antibody was determined by a variety of tests: ELISA, WB, IHC, ICC, and so on.2. The research of IBP expression in breast cancer and Its clinical significance(1) The study of IBP expression by WB and RT-PCR was based on breast cancer cell line of MCF-7, MDA-MB-231, MDA-MB-435, MDA-MB-453, MDA-MB-468, SK-BR-3.(2) The determination of IBP expression patterns in breast cancer was carried out by double immunofluorescence staining. The results showed that IBP expression patterns were primarily in the membrane and cytoplasm.(3) The construction of breast cancer tissue microarray was accomplished by collecting 107 cases of clinical breast cancer samples with different pathological classification.(4) The analysis of IBP expression in clinical samples was based on immunohistochemical staining, and the statistical analysis on IBP expression and other pathological indications to determine its clinical significance.3. The study of IBP function and mechanism in breast cancer(1) The construction of IBP over-expression model by plasmid transfection was based on MDA-MB-435 cells with IBP negative expression and IBP low-expression model made by RNAi method based on MDA-MB-231 cells with IBP positive expression.(2) The determination of the effects on cell growth was based on cell proliferation in each group, by the MTT assay.(3) The ability of cell invasion was based on analysis of stained-cells penetrating study by trans-well experiments; determining the effects of IBP on the ability of cell invasion.(4) The effects of IBP expression in breast cancer cells on the resistance to paclitaxel, which is commonly used in clinical treatment of breast cancer, was determined by MTT assay.(5) The relationship among IBP expression and cytoskeleton was based on the cytoskeleton staining, the relationship assay between IBP expression and the focal adhesion formation was based on immunofluorescence staining. (6) The determination of the relationship between IBP expression and Rac1b was based on double immunofluorescent staining.The results are as follow:1. We obtained 7 strains of hybridoma cells (A2, A9, C6, D5, F5, G3, and G4) excreting anti-IBP monoclonal antibody after ELISA and WB screening The mAb from five of them (A2, A9, C6, D5 and F5) could be applied to the IHC staining of IBP. Three antibodies (A9, C6 and D5) showed the strongest IHC staining.2. IBP expression was studied in five human breast cancer cell lines and was found positinely in MCF-7, MDA-MB-231 and SKBR3 cell lines at both the mRNA and protein levels. In addition, we confirmed that IBP located on the membrane of MCF-7 cells by A9 mAb and DiI double staining .3. Among the tissue samples analyzed here, 20 normal breast tissue samples, and 20 benign breast lesion samples were negative for IBP immunocytochemical staining with mAb A9. In contrast, the IBP protein was detected in the cytoplasm of many breast cancer cells, as indicated by the 57 IBP-positive samples among the 107 breast cancer tissue samples that were tested (53.3%). Based on the statistics analysis, we found that there was a significant positive correlation (P < 0.01) between the IBP expression level and the tumor grade as well as the presence of lymph node metastasis and also, there was a negative correlation between IBP and p53 protein levels (P< 0.05). Our results demonstrate that IBP is over-expressed in a considerable proportion of breast cancer cell lines and cancer tissues, and, therefore, IBP may be involved in breast cancer progression.4. Comparison of the growth rates of the tumor cells over a 9-day culture period revealed the MDA-MB-231 breast cancer cells with lower IBP expression grew more slowly (P<0.05) than cells that were transfected with the control siRNA construct. The IBP-over-expressing breast cancer cells grew much faster than their corresponding parental cells. To analyze the role of IBP in the invasiveness of breast cancer cells, a matrigel cell invasion assay system was used. This assay revealed that IBP-positive breast cancer cells exhibited greater penetration of extra matrix proteins when compared with IBP-negative tumor cells.5. With IBP over-expression the cell morphology of MDA-MB-435 changed significantly and invadopodia formation increased significantly. In contrast, with RNAi-induced IBP lower-expression, the cell invadopodia were greatly reduced. Further more, it has been found that IBP co-localized with microfilament. The co-localization was mainly situated on adhesion plaque, but the mainly situated was on larger peripheral focal adhesions with IBP low-expression.6. It was confirmed that there was synergistic effect between IBP and Rac1b based on double immunofluorescence staining.IBP was expressed not only in the immune system but also in breast cancer. IBP expression was closely related to the progression of breast cancer: IBP promotes the proliferation and invasion of breast cancer cells, the mechanisms of that may be that IBP would enhance the formation of cell invadopodia, participate in the construction of cell adhesion plaque to increase cell migration, and promote the generation of intracellular reactive oxygen species based on the synergistic effects with Rac1b, which allow the basal cells to achieve the transfer from the breakthrough. In our work, we tried to study the two primary requirements for the breast cancer’s metastasis (invadopodia formation and basal lamina damage) by linking them to each other. It has been found that IBP was closely related to invadopodia formation and basal lamina damage, and it may be a bridge between both of them. The results of this study may improve the understanding of the occurrence and transfusion mechanisms of breast cancer and provide new targets and ideas for the clinical diagnosis and treatment early.更多还原